Microbial Physiology Genetics and Ecology

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MICROBIAL PHYSIOLOGY GENETICS AND ECOLOGY

Dr. Jyotsna Rathi

M MANGLAM PUBLICATIONS DELHI -110053 (INDIA)

Published by :

MANGLAM PUBLICATIONS L-21/1, Street No. 5, Shivaji Marg, Near Kali Mandir, J.P. Nagar, Kartar Nagar, West Ghonda, Delhi-53 Phone: 011-22945677, 9968367559,9868572512 E-mail: [email protected] manglam. [email protected]

Microbial Physiology Genetics and Ecology

©Reserved First Edition 2009 ISBN 978-81-907127-6-7

All rights reserved no part ofthis work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the Publisher.

PRINTED IN INDIA

Published by D.P. Yadav for Manglam Publications, Delhi. Laser Typeset by Amandeep (Graphic Era) Delhi-92. Printed at Sachin Printers, Maujpur, Delhi-53

PREFACE The present title "Microbial Physiology. Genetics and Ecology" is a fast expanding branch of science, and it is impossible to present all of it in a book of this size. Author has therefore tried to present selected portions of microbial physiology in sufficient detail that the student can understand them through reading the book. The present title covers the various physiological processes of microorganisms, the way of characters are transmitted and expressed, and the influence of climate conditi6ns on them. The aim in writing this book was to bring together the relevant aspects of the biology of microorganisms. Thus it offers a comprehensive and uptodate information on microbial science. Author has covered the vast information available on the subject in a concise form so as to cater to the needs of both undergraduate and postgraduate students. It will be equally useful as a reference book to the researchers and teachers as well. The author expresses her thanks to all those friends, colleagues, and research scholars whose continuous inspirations have initiated her to bring this title. The author wishes to thank the M/s. Manglam Publications, Delhi for bringing out this book. Constructive criticisms and suggestions for improvement of the book will be thankfully acknowledged. Author

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Contents 1. Introduction ........................................................ 1 1.1

1.2

Elements of Epidemiology ......................................... 1 1.1.1 Some Definitions ....................................... 1 1.1.2 Chain of Infection ...................................... 2 Pathogens and Parasites Found in Domestic Wastewater ............................................................... 8 1.2.1 Bacterial Pathogens .................................... 8 1.2.2 Viral Pathogens ........................................ 22 1.2.3 Protozoan Parasites .................................. 28 1.2.4 Helminth Parasites ................................... 39 1.2.5 Other Problem· Causing Microorganisms .... 40

2. Microbial Metabolism and Growth ................. 42 2.1

Metabolic Diversity of Freshwater Bacteria ............... 42 Key Metabolic Parameters ........................ 42 . 2.1.1 2.1.2 CO 2 Fixation ............................................ 43 2.1.3 Breakdown of Organic Matter in Aerobic and Anaerobic Environments .................... 44 2.1.4 Bacterial Adaptations to Low-nutrient Environments .......................................... 49 2.2 Photosynthetic Bacteria ............................................ 51 2.2.1 General Characteristics ............................. 52 2.2.2 Motility .................................................... 52 2.2.3 Ecology ................................................... 53 (i)

CONTENTS

(ii)

2.3

2.4

2.5

2.6

2.7

Bacteria and Inorganic Cycles ... ,.............................. 54 2.3.1 Bacterial Metabolism and the Sulphur Cycle ....................................................... 54 Bacterial Populations ................................................ 56 2.4.1 Techniques for Counting Bacterial Populations .............................................. 57 2.4.3 Biological Significance of Total and Viable Counts .................................................... 58 Bacterial Productivity ............................................... 59 2.5.1 Measurement of Productivity .................... 59 2.5.2 Regulation of Bacterial Populations and Biomass ................................................... 60 2.5.3 Primary and Secondary Productivity: Correlation Between Bacterial and Algal Populations .............................................. 61 2.5.4 Role of Dissolved Organic Carbon ........... 64 2.5.5 Bacterial Productivity and Aqua tic Food Webs ....................................................... 65 Microbial Growth .................................................... 66 Algal Blooms and Eutrophication .............. 66 2.6.1 2.6.2 Formation of colonial blue-green algal blooms .................................................... 68 2.6.3 Environn'ental Effects of Blue-green Blooms .................................................... 71 Metabolism of Starter Cultures ................................ 74 2.7.1 Carbohydrate Utilization by Lactic Acid Bacteria ................................................... 75 2.7.2 Protein Metabolism .................................. 89 2.7.3 Citrate Metabolism ................................... 96 2.7.4 Metabolism of Propioni Bacteria ............... 99 2.7.5 Metabolism of Molds and other Flavorcontributing Microorganisms .................. 100 2.7.6 Metabolic Engineering ........................... 102

3. Fermentation ................................................... 103 3.1

Introduction .......................................................... 103 3.1.1 Defined and Characterized ..................... 104 3.1.2 Lactic Acid Bacteria ............................... 105

CONTENTS

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3.1.3

3.2

3.3

3.4 3.5 3.6 3.7 3.8

3.9

3.10

Metabolic Pathways and Molar Growth Yields .................................................... 109 Dairy Products ...................................................... 110 3.2.1 Milk Biota .............................................. 110 3.2.2 Starter Cultures, Products ...................... 112 3.2.3 Cheeses ................................................. 116 Apparent Health Benefits of Fermented Milks ........ 118 3.3.1 Lactose Intolerance ................................ 118 3.3.2 Cholesterol ............................................ 120 3.3.3 Anticancer Effects .................................. 121 3.3.4 Probiotics .............................................. 121 Diseases Caused by Lactic Acid Bacteria ................. 122 Fermented Fruit and Vegetable Products ............... 122 Fresh and Frozen Vegetables ................................. 123 3.6.1 Spoilage ............................................... 124 Spoilage of Fruits .................................................. 129 Fresh-cut Produce ................................................. 130 3.8.1 Microbial Load ...................................... 130 3.8.2 Pathogens .............................................. 131 Fermented Products .............................................. 133 3.9.1 Breads ................................................... 133 3.9.2 Olives, Pickles, and Sauerkraut .............. 134 3.9.3 Beer, Ale, Wines, Cider, and Distilled Spirits Beer andAle ............................... 138 3.9.4 Cider ..................................................... 142 Miscellaneous Fermented Products ........................ 144

4. Microbial Genetics ......................................... 150 4.1 4.2 4.3 4.4 4.5

Genetics ................................................................ DNA Replication ................................................... 4.2.1 Chromosome Connection ...................... Protein Synthesis ................................................... 4.3.1 Genotype and Phenotype ....................... Controlling Genes ................................................. 4.4.1 Operon Model ............................................. Mutations .............................................................. 4.5.1 Mutation Rate ........................................

150 151 152 152 154 154 155 155 157

CONTENTS

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4.6

4.7

Genetic Interactions .............................................. 157 4.6.1 Genetic Diversity .................................... 158 4.6.2 Mechanisms for Gene Transfer in Freshwater Systems ............................... 162 4.6.3 Evidence for Gene Transfer in the Aquatic Environment .............................. 166 Recombinant DNA Technology .............................. 169 4.7.1 Genetic Engineering: Designer Genes ..... 169 4.7.2 Gene Therapy: Makes You Feel Better .. , 171 4.7.3 DNA Fingerprinting ............................... 172 4.7.4 Recombinant DNA Technology and Society .................................................. 173

5. Microbial Ecology .......................................... 175 5.1

5.2

5.3

5.4

General Introduction ............................................. 175 5.1.1 Aquatic Existence ................................... 175 5.1.2 Global Water Supply - Limnology and Oceanography ....................................... 176 5.1.3 Freshwater Systems: Some Terms and Definitions ............................................. 177 5.1.4 Biology of Freshwater Microorganisms .. , 179 Biodiversity of Microorganisms .............................. 179 5.2.1 Domains of Life ..................................... 179 5.2.2 Size Range ............................................ 180 5.2.3 Autotrophs and Heterotrophs ................. 182 5.2.4 Planktonic and Benthic Microorganisms ... 184 5.2.5 Metabolically Active and Inactive States .. 186 5.2.6 Evolutionary Strategies: r-selected and Kselected Organisms ................................ 187 Biodiversity in Ecosystems, Communities, and Species Populations ............................................... 190 5.3.1 Main Ecosystems ................................... 191 5.3.2 DiversitywithinSubsidiaryCommunities .. 191 5.3.3 Biodiversity within Single-species Populations ............................................ 192 Bjofilm Community: A Small-scale Freshwater Ecosystem ............................................................. 193 5.4.1 Interactions between Microorganisms ..... 194

CONTENTS

(v)

5.4.2 5.4.3

5.5

5.6

5.7 5.8

Biomass Formation and Transfer ............ 196 Maintenance of the Internal Environment .......................................... 196 5.4.4 Interactions with the External Environment .......................................... 197 Pelagic Ecosystem ................................................. 197 5.5.1 Interactions between Organisms ............. 198 5.5.2 Trophic Connections and Biomass Transfer ................................................. 199 5.5.3 Maintenance of the Internal Environment .......................................... 205 5.5.4 Interactions with the External Environment .......................................... 205 Homeostasis and Ecosystem Stability ..................... 207 5.6.1 Stress Factors ........................................ 207 5.6.2 General Theoretical Predictions: Community Response ............................ 208 5.6.3 Observed Stress Responses: From Molecules to Communities ..................... 209 5.6.4 Assessment of Ecosystem Stability .......... 210 5.6.5 Ecosystem Stability and Community Structure ............................................... 211 5.6.6 Biological Response Signatures ............... 213 Pelagic Food Webs ................................................ 213 Communities and Food Webs of Running Waters ... 214 5.8.1 Allochthonous Carbon ............................ 214 5.8.2 Pelagic and Benthic Communities ........... 216 5.8.3 Microbial Food Web .............................. 219

6. Light as Abiotic Factor .................................. 222 6.1

Light Environment ................................................ 6.1.1 Physical Properties of Light: Terms and Units of Measurement ............................ 6.1.2 Light Thresholds for Biological Activities ................................................ 6.1.3 Light Under Water: Refraction, Absorption, and Scattering .....................

223 223 224 225

CONTENTS

(vi)

6.1.4

6.2

6.3

.,

6.4

6.5

6.6

6.7

Light Energy Conversion: From Water Surface to Algal Biomass ........................ 228 Photosynthetic Processes in the Freshwater Environment ......................................................... 229 6.2.1 Light and Dark Reactions ...................... 229 6.2.2 Photosynthetic Microorganisms .............. 230 6.2.3 Measurement of Photosynthesis .............. 2.31 6.2.4 Photosynthetic Response to Varying Light Intensity ....................................... 232 Light as a Growth Resource .................................. 234 6.3.1 Strategies for Light Uptake and Utilization .............................................. 235 6.3.2 Light-Photosynthetic Response in DifferentAlgae ....................................... 235 6.3.3 Conservation of Energy .......................... 237 6.3.4 Diversity in Small Molecular Weight Solutes and Osmoregulation .................. 238 Algal Growth and Productivity ............................... 240 6.4.1 Primary Production: Concepts and Terms .................................................... 240 6.4.2 Primary Production and Algal Biomass ... 240 6.4.3 Field Measurements of Primary Productivity ........................................... 241 Photosynthetic Bacteria .......................................... 244 6.5.1 Major Groups ......... ~ .............................. 244 6.5.2 Photosynthetic Pigments ........................ 244 6.5.3 Bacterial Primary Productivity ................ 246 Photoadaptation: Responses of Aquatic Algae to Limited Supplies of Light Energy ....................... 246 6.6.1 Different Aspects of Light Limitation ...... 247 6.6.2 Variable Light Intensity: Light-Responsive Gene Expression .................................... 248 6.6.3 Photosynthetic Responses to Low Light Intensity ................................................ 250 6.6.4 Spectral Composition of Light: Changes in Pigment Composition ......................... 255 Carbon Uptake and Excretion by Algal Cells ........... 256 Changes in Environmental CO 2 and pH ... 256 6.7.1

(vii)

CONTENTS

6.7.2

6.8

6.9

6.10

Excretion of Dissolved Organic Carbon by Phytoplankton Cells ............................... 258 Competition for Light and Carbon Dioxide between Algae and Higher Plants ........................................ 262 6.8.1 Balance between Algae and Macrophytes in Different Aquatic Environments .......... 262 6.8.2 Physiological and Environmental Adaptations in the Competition between Algae and Macrophytes .......................................... 263 Damaging Effects of High Levels of Solar Radiation: Photoinhibition ...................................................... 267 6.9.1 Specific Mechanisms of Photoinhibition .... 268 6.9.2 General Effects of Photoinhibition .......... 270 6.9.3 Strategies for the Avoidance of Photoinhibition ...................................... 271 6.9.4 Photoinhibition and Cell Size .................. 273 6.9.5 Lack of Photo inhibition in Benthic Communities ......................................... 276 6.9.6 Photoinhibition in Extreme High-light Environments ........................................ 277 Periodic Effects of Light on Seasonal and Diurnal Activities of Freshwater Biota ................................. 279 6. 10.1 Seasonal Periodicity ............................... 279 6.10.2 Diurnal Changes .................................... 280 6.10.3 Circadian Rhythms in Blue-green Algae .. 281 6.10.4 Circadian Rhythms in Dinoflagellates ...... 283

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1 Introduction 1.1 ELEMENTS OF EPIDEMIOLOGY 1.1.1 Some Definitions Epidemiology is the study of the spread of infectious diseases in populations. Infectious diseases are those that can be spread from one host to another. Epidemiologists play an important role in the control of these diseases. Incidence of a disease is the number of individuals with the disease in a population, whereas prevalence is the percentage of individuals with the disease at a given time. A disease is epidemic when the incidence is high and endemic when the incidence is low. Pandemic refers to the spread of the disease across continents. Infection is the invasion of a host by an infectious microorganism. It involves the entry (e.g., through the gastrointestinal and respiratory tracts, skin) of the pathogen into the host and its multiplication and establishment inside the host. Inapparent infection (or covert infection) is a subclinical infection with no apparent symptoms (i.e., the host reaction is not clinically detectable). It does not cause disease symptoms but confers the same degree of immunity as an overt infection. For example, most enteric viruses cause inapparent infections. A person with inapparent infection is called a healthy carrier. Carriers constitute, however, a potential source of infection for others in the community. Nosocomial infections are hospitalacquired infections, which affect approximately 2.5 mi1lion patients annually in the United States. This represents approximately 5 percent of patients with a documented infection acquired in a hospital.

MICROBIAL PHYSIOLOGY, GENETICS AND ECOLOGY

2

Intensive care unit patients represent about 20 percent of hospitalacquired infections. In developing countries, the overall rates vary between 3 and 13.5 percent. The patients most at risk for nosocomial infections are elderly patients, patients with intravenous devices or urinary catheters, those on parenteral nutrition or antimicrobial chemotherapy, and HIV and cancer patients. Pathogenicity is the ability of an infectious agent to cause disease and injure the host. Pathogenic microorganisms may infect susceptible hosts, leading sometimes to overt disease, which results in the development of clinical symptoms that are easily detectable. The development of the 'disease depends on various factors, including infectious dose, pathogenicity, and host and environmental factors. Some organisms, however, are opportunistic pathogens that cause disease only in compromised individuals.

1.1:2 Chain of Infection The potential for a biological agent to cause infection in a susceptible host depends on the various factors described in the following.

1.1.2.1 Type of infectious agent Several infectious organisms may cause diseases in humans. These agents include bacteria, fungi, protozoa, metazoa (helminths), rickettsiae, and viruses. Evaluation of infectious agents is based on their virulence or their potential for causing diseases in humans. Virulence is related to the dose of infectious agent necessary for infecting the host and causing disease. The potential for causing illness also depends on the stability of the infectious agent in the environment. The minimal BACTERIA

VIRUSES

PROTOZOA

HELMINTHS

e.g. Salmonella Shigella Vibrio cholera

e.g. HAV Norwalk agent

e.g., Giardia Cryptosporidlum

e.g., Ascaris Taema

HUMAN

L -_ _ __

I

Figure 1.1 Categories of organism'> of public health significance.

INTRODUCTION

3

infective dose (MID) varies widely with the type of pathogen or parasite. For example, for Salmonella typhi or enteropathogenic E. coli, thousands to millions of organisms are necessary to establish infection, whereas the MID for Shigella can be as low as 10 cells. A few protozoan cysts or helminth eggs may be sufficient to establish infection. For some viruses, only one or a few particles are sufficient for infecting individuals. For example, 17 infectious particles of echovirus 12 are sufficient for establishing infection. Table 1.1 Minimal infective doses for some pathogens and parasites

Organism Salmonella spp. Shigella spp. Escherichia coli Escherichia coli 01 57: H 7 Vibrio cholerae Campylobacter jejuni Giardia lamblia Cryptosporidium Entamoeba coli Ascaris Hepatitis A virus

Minimal infective dose 10 1-102 106-108 2 mg/L free residual chlorine). This bacterium appears to be ubiquitous in the environment and has been isolated from waste-water, soil,

16


and natural aquatic environments including tropical waters. Its presence in wastewater has been linked, on one occasion at least, with increased levels of antibodies among wastewater irrigation workers. However, the epidemiological significance of this finding remains unclear. In the natural environment, this pathogen can thrive in association with other bacteria which may provide the L-cysteine required by Legionella, green and blue-green algae, amoeba (e.g., Acanthamoeba, Naegleria), and other protozoa (e.g., Hartmanella, Tetrahymena), or ciliates. Conversely, 16-32 percent of heterotrophic plate count bacteria isolated from chlorinated drinking water were found to inhibit Legionella species. Legionella association with protozoa provides increased resistance to biocides such as chlorine, low pH, and high temperatures. Protozoa are able to sustain the intracellular growth of L. pneumophila and have an effect on its virulence to mammalian cells. It was found that the same virulence genes are required to infect both protozoa and mammalian cells. Control of Legionella in water distribution systems includes thermal treatment (e.g., increase of water temperature to 60-70 C followed by flushing), treatment with bactericidal agents (e.g., copper, silver), and hyperchlorination up to 50 mg/L. However, because biofilms protect bacterial pathogens from inactivation by free chlorine, treatment with monochloramine provides a better control of Legionella in water distribution lines. An epidemiological study showed that 10 times fewer outbreaks of Legionnaires' disease occurred in hospitals having monochloramine as disinfectant residual than those using free chlorine as a residual. D

1.2.1.9 Bacteroides fragiis Bacteroides species are a major part of the microorganisms in the human colon and account for approximately 25 percent of all colonic isolates. This pathogen has been found in wastewater at levels ranging from 6.2 x 104 to 1.1 x 105 colony forming units/ mL, 9.3 percent of which were enterotoxigenic. Enterotoxinproducing strains of this anaerobic bacterium may be involved in causing diarrhea in humans.

1.2.1.10 Opportunistic bacterial pathogens This group includes heterotrophic gram-negative bacteria belonging to the following genera: Pseudomonas, Aeromonas, Klebsiella, Flavobacterium, Ellterobacter, Citrobacter, Serratia, Acinetobacter, Proteus and Prol'idencia, and nontubercular

INTRODUCTION

17

mycobacteria. Segments of the population particularly at risk of infection with opportunistic pathogens are newborn babies, and elderly and sick people. These organisms may occur in high numbers in institutional (e.g., hospital) drinking water and attach to water distribution pipes, and some of them may grow in finished drinking water. However, their public health significance with regard to the population at large is not well known. Pseudomonas aerugil10sa is ubiquitous in the environment and is frequently found in water, wastewater, soils and plants. Although it poses no risks in drinking water, it is responsible for 10-20 percent of nosocomial (i.e., hospital-acquired) infections. Other opportunistic pathogens are the nontubercular mycobacteria, which cause pulmonary infections and other diseases. The most frequently isolated nontubercular mycobacteria belong to the species of Mycobacterium avium complex (MAC) (i.e., M. avium and M. intracellulare), which infect humans (mostly AIDS and other immunocompromised patients) and animals (e.g., pigs). Another member of the MAC complex is M. avium subspecies paratuberculosis (MAP), which causes inflammation. There is now evidence that infection with MAP is one of the causes of Crohn' s disease, a chronic inflammation of the intestine affecting animals and humans. A new generation of antibiotics (rifabutin, clarithromycin, azithromycin) has been proposed to control or reduce MAP activity in the gastrointestinal tract. Mycobacteria are ubiquitous and are found in environmental waters, including drinking water, ice, and hospital hot water systems, soils, plants, air-water interface of aquatic environments, biofilms in water distribution systems, medical instruments, and aerosols. Owing to the hydrophobic nature of the cell surface of mycobacteria, a major route for their transmission is via aerosolization. This also explains their resistance to commonly used disinfectants and to antibiotics. Mycobacteria persist well and grow under environmental conditions. Potable water, particularly hospital water supplies, can support the growth of these bacteria, which may be linked to nosocomial infections. Their growth in water distribution systems was correlated with assimilable organic carbon and biodegradable organic carbon levels. Mycobacterium avium and M. il1tracellulare can be detected using cultural, biochemical, and molecular-based methods (commercially available DNA probes and PCR amplification).

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY

18

1.2.1.11 Helicobacter pylori Helicobacter pylori is a bacterial agent that is responsible for peptic ulcers (chronic gastritis), stomach cancer, lymphoma, and adenocarcinoma. In the United States alone, over 5 million people are diagnosed annually with peptic ulcers, and some 40,000 of them undergo surgery, sometimes leading to death in complicated cases. There are indications of person· to-person as well as waterborne and food-borne transmissions of this pathogen. It has been suggested that H. pylori may be transmitted via four routes: the fecal-oral route, oral-oral route (person-to-person transmission via saliva), gastric-oral route (e.g., contaminated vomit in children), and by endoscopic procedures in the hospital setting. This pathogen has been associated with an increased risk of gastric cancer among sewage workers. Helicobacter pylori infection is generally treated via administration of antibiotics such metronidazole, tetracycline, amoxycillin, clarithromycin, and azithromycin. The treatment is complicated by the appearance of antibiotic resistance in this pathogen. Helicobacter pylori has been detected in wastewater, seawater, and drinking water. When exposed to environmental conditions, H. pylori enters a viable but nonculturable (VBNC) state that allows its persistence in aquatic environments. Although Johnson and colleagues 5 4

• H. pylori

a

0

E. coli

3

c: 0

"" -52 ()

~

8'

~

1

0 -1 0.00

0.05

010

0.15

0.20

0.25

030

035

Chlorine (mg/L)

Figure 1.2 Effect of chlorine on Helicobacter pylori and Escherichia coh.

INTRODUCTION

19

(1997) reported that H. pylori was readily inactivated by free chlorine, it is, however, more resistant than E. coli to chorine. This higher resistance to disinfectants may allow this pathogen to persist in water distribution systems. Helicobacter pylori was detected in environmental samples, using culture-based methods as well as immunological, autoradiography, and molecular-based methods. Although no standard method is available for H. pylori detection, this pathogen was recently isolated from wastewater using fluorescent in situ hybridization (FISH), a combination of immunomagnetic separation OMS) and culturing techniques, and identified using PCR-based 16S rRNA sequences. Helicobacter DNA was also isolated from biofilms in municipal water distribution systems.

1.2.1.12 Antibiotic-resistant bacteria Antibiotics act on microorganisms by inhibiting peptidoglycan synthesis, protein synthesis, and nucleic acid synthesis (interruption of nucleotide metabolism, inhibitition of RNA polymerase or DNA gyrase) or by affecting the cell membrane integrity. Increased use of antibiotics is often associated with an increased resistance of bacteria to these chemicals, especially in the hospital setting. A global rise in antibiotic resistance has been reported. A comparison of preantibiotic era strains of E. coli and Salmonella enterica to contemporary strains showed that the former were susceptible to antibiotics, whereas 20 percent of the latter displayed resistance to at least one of the antibiotics. In the United States, 46 percent of Streptococcus pneumoniae isolates are now resistant to penicillin, and methicillin-resistant Staphylococcus aureus accounts for 30 percent of nosocomial infections with this pathogen. An investigation of the antibiotic resistance pattern of E. coli strains in a wastewater treatment plant in Austria showed that these strains were resistant to 16 of 24 tested antibiotics. Antibiotic-resistant strains numbers increase when the influent to the wastewater treatment plant is from a hospital source. Resistance to vancomycin has also been documented. Vancomycin-resistant enterococci (VRE) were isolated from 60 percent of raw wastewater and 36 percent of wastewater effluents. As regards resistance to vancomycin, the minimum inhibitory concentrations (MIC) of VRE from hospital wastewaters were found to be much higher than those from residential wastewaters. Using a real-time PCR assay, the antibiotic resistance genes vanA of VRE, and ampC (resistance gene for the synthesis

20


of ,B-Iactamase) of Enterobacteriaceae were detected in 20 and 78 percent of wastewater samples, respectively. Patients receiving antibiotic therapy harbor a large number of antibiotic-resistant bacteria in their intestinal tract. These bacteria are excreted in large numbers in feces and eventually reach the community wastewater treatment plant. The genes coding for antibiotic resistance are often located on plasmids (R factors) and, under appropriate conditions, can be transferred to other bacteria through conjugation that requires cell-to-cell contact, or through other. modes of recombination. If the recipient bacteria are potential pathogens, they may be of public health concern as a result of their acquisition of antibiotic resistance. Drug-resistant microorganisms produce nosocomial and community-acquired human infections, which can lead to increased morbidity, mortality, and disease incidence. Resistance to antibiotics, including quinolones (e.g., nalidixic acid, ciprofloxacin), can in turn complicate and increase the cost of therapy based on administration of antibiotics to patients exposed to pathogens of environmental origin. Patients infected with antibiotic-resistant bacterial strains are likely to require hospitalization, sometimes for long periods. Bacterial resistance to antibiotics has been demonstrated in terrestrial and aquatic environments, particularly those contaminated with wastes from hospitals. Gene transfer between microorganisms is known to occur in natural environments as well as in engineered systems such as wastewater treatment plants. Investigators have used survival chambers to demonstrate the transfer of R plasmids among bacteria in domestic wastewater. The mean transfer frequency in wastewater varied between 4.9 X 10--5 and 7.5 X 10-5• The highest transfer frequency (2.7 x 10-4 ) was observed between Salmonella enteritidis and E. coli. Nonconjugative plasmids (e.g., pBR plasmids) can also be transferred and this necessitates the presence of a mobilizing bacterial strain to mediate the transfer. Several indigenous mobilizing strains have been isolated from raw wastewater. Each of these strains is capable of aiding in the transfer of the plasmid pBR325 to a recipient E. coli strain. Under laboratory conditions, plasmid mobilization from genetically engineered bacteria to environmental strains was also demonstrated under low temperature and low nutrient conditions in drinking water. The occurrence of multiple-antibiotic resistant (MAR) indicator and pathogenic (e.g., Salmonella) bacteria in water and wastewater

21

INTRODUCTION

Total coliform

Total MAR colllform

5 4

3 2

6/84 Date Figure 1.3 Multiple-antibiotic resistant (MAR) bacleria in dOlllestic wastewater.

treatment plants has been documented. In untreated wastewater, the percentage of multiple-antibiotic resistant coliforms varies between less than 1 to about 5 percent of the total coliforms. Chlorination appears to select for resistance to antibiotics in wastewater treatment plants. However, others observed that chlorination increased the bacterial resistance to some antibiotics (e.g., ampicillin, tetracycline) but not to others (e.g., chloramphenicol, gentamicin). The proportion of bacteria carrying R factors seems to increase after water and wastewater treatment. For example, in one study, MAR was expressed by 18.6 percent of heterotrophic plate count bacteria in untreated water as compared to 67.8 percent for bacteria in the distribution system. Similarly, in a water treatment plant in Oregon, the percentage of MAR bacteria rose from 15.8 percent in untreated (river) water to 57.1 percent in treated water. Multiple-antibiotic resistance is furthermore associated with resistance to heavy metals (e.g., Cu 2 +, Pb 2 +, Zn2+). This phenomenon was observed both in drinking water and wastewater. The public health significance of this phenomenon deserves further study. Strategies for tackling the serious problem of drug resistance include the reduced use of antibiotics in humans and animals, preventive measures for the transmission of infectious diseases, and increased efforts by the scientific community to better understand the mechanisms of drug resistance in microorganisms.

22


1.2.2 Viral Pathogens Water and wastewater may become contaminated by approximately 140 types of enteric viruses. These viruses enter into the human body orally, multiply in the gastrointestinal tract, and are excreted in large numbers in the feces of infected individuals. Many of the enteric viruses cause non apparent infections that are difficult to detect. They are responsible for a broad spectrum of diseases ranging from skin rash, fever, respiratory infections, and conjunctivitis to gastroenteritis and paralysis. It was estimated that nonpolio enteroviruses cause 10-15 million symptomatic infections/ year in the United States. Virus presence in the community wastewater reflects virus infections among the population. Enteric viruses are present in relatively small numbers in water and wastewater. Therefore, environmental samples of 10-1000 L must be concentrated in order to detect these pathogens. An ideal method should fulfil the following criteria: applicability to a wide range of viruses, processing of large sample volumes with small-volume concentrates, high recovery rates, reproducibility, rapidity, and low cost. A number of approaches have been considered for accomplishing this task. The most widely used approach is based on the adsorption of viruses to electronegative and electropositive microporous filters of various compositions (e.g., nitrocellulose, fiber-glass, chargemodified cellulose, epoxy-fiberglass, cellulose + glass fibers, positively charged nylon membranes). This step is followed by elution of the adsorbed viruses from the filter surface. Further concentration of the sample can be obtained by membrane filtration, organic flocculation (using heef extract or casein) or aluminum hydroxide hydroextraction. The concentrate is then assayed using animal tissue cultures, immunological or genetic probes. Other adsorbents considered for virus concentration include glass powder, glass wool, bituminous coal, bentonite, iron oxide, modified diatomaceolls earth or pig erythrocyte membranes. From an epidemiological standpoint, enteric viruses are mainly transmitted via person-to-person contacts. However, they may also be communicated by water transmission either directly (drinking water, swimming, aerosols) or indirectly via contaminated food (e.g., shellfish, vegetables). Some enteric viruses (e.g., hepatitis A virus) persist on environmental surfaces, which may serve as vehicles for the spread of viral infections in day-care centers or hospital wards.

INTRODUCTION

23

Table lA Some human enteric viruses Virus Group A. Enteroviruses Poliovirus Coxsackievirus A

B

Echovirus

Enteroviruses (68-71) Hepatitis A virus (HAy) Hepatitis E virus (HEY) B. Reoviruses C. Rotaviruses D. Adenoviruses

Serotypes 3 23

6

34

4

3 4 41

E. Norwalk agent (calicivirus) E Astroviruses

5

Some diseases caused Paralysis Aseptic meningitis Herpangia Aseptic meningitis Respiratory illness Paralysis Fever Pleurodynia Aseptic meningitis Pericarditis Myocarditis Congenital heart Anomalies Nephritis Fever Respiratory infection Aseptic meningitis Diarrhea Pericarditis Myocarditis Fever, rash Meningitis Respiratory illness Infectious hepatitis Hepatitis Respiratory disease Gastroenteritis Respiratory disease Acute conjunctivitis Gastroenteritis Gastroenteritis Gastroenteritis

The infection process depends on the minimal infectious dose (MID) and on host susceptibility, which involves host factors (e.g., specific immunity, sex, age) and environmental factors (e.g., socioeconomic level, diet, hygienic conditions, temperature, humidity) factors.

24

MICROBIAL PHYSIOLOGY. GENETICS AND ECOLOGY Excreta from humans and animals

•

Wastewater

Figure 1.4 Waterborne transmission of enteric viruses.

Although the MID for viruses is controversial, it is generally relatively low as compared with bacterial pathogens. Experiments with human volunteers have shown an MID of 17 PFU (plaque-forming units) for echovirus 12. Several epidemiological surveys have shown that enteric viruses are responsible for 4.7-11.8 percent of waterborne epidemics. Epidemiological investigations have definitely proved the waterborne and food-borne transmission of viral diseases such as hepatitis and gastroenteritis.

1.2.2.1 Hepatitis Hepatitis is caused mainly by the following viruses: 1. Infectious hepatitis is caused by hepatitis A virus (HAV) , a 27 nm RNA enterovirus (enterovirus type 72 belonging to the family picornaviridae) with a relatively short incubation period (2-6 weeks) and displaying a fecal-oral transmission route. Although it can be replicated on primary and continuous human or animal tissue cultures, it is hard to detect because it does not always display a cytopathic effect. Other means of detection of HAV include genetic probes, use of peR, and immunological methods. 2. Serum hepatitis is caused by hepatitis B virus (HBV), a 42 nm DNA virus displaying a relatively long incubation time (4-12 weeks). This virus is transmitted by contact with infected blood or by sexual contact. The mortality rate (1-4 percent) is higher

INTRODUCTION

25

than for infectious hepatitis «0.5 percent). Hepatitis B virus is responsible for approximately 60 percent of the 434,000 cases of liver cancer worldwide. 3. Non-A, non-B infectious hepatitis is caused by hepatitis E virus (HEy). Hepatitis A virus causes liver damage with necrosis and inflammation. After the onset of infection, the incubation period may last up to 6 weeks. One of the most characteristic symptoms is jaundice. In the United States, approximately 140,000 persons are infected annually with HAY. Hepatitis A is transmitted via the fecal-oral route either by person-to-person direct contact, waterborne, or food-borne transmission. Concentration of HAV in feces can reach 107 to 109/ g. This disease is distributed worldwide and the prevalence of HAV antibodies is higher among lower socioeconomic groups and increases with the age of the infected individuals. Direct contact transmission has been documented mainly in nurseries (especially among infants wearing diapers), mental institutions, prisons, or military camps. Waterborne transmission of infectious hepatitis has been conclusively demonstrated and documented worldwide on several occasions. It has been estimated that 4 percent of hepatitis cases observed during the period 1975-1979 in the United States were transmitted through the waterborne route. The hepatitis cases are due to consumption of improperly treated water or contaminated well water. Hepatitis A outbreaks were also associated with swimming in lakes or public pools. Food-borne transmission of HAV appears to be more important than waterborne transmission. Consumption of shellfish grown in wastewater-contaminated waters accounts for numerous hepatitis and gastroenteritis outbreaks documented worldwide. Passive immunization by means of pooled immunoglobulin is used for the prevention of infectious hepatitis. Vaccines against hepatitis A are available around the world. Hepatitis E virus (HEy) is a single-stranded RNA virus, the classification of which is not known. It is believed that this virus may be a calicivirus. This virus is not well characterized due to the lack of a tissue culture cell line for its assay. Unlike HAY, it is mainly transmitted via fecally contaminated water, while the personto-person transmission is very low. Hepatitis E virus epidemics generally involve thousands of cases. A notorious non-A, non-B hepatitis (now recognized as hepatitis E

26


virus) epidemic broke out in 1956 in New Delhi, India, and resulted in approximately 30,000 cases. A more recent outbreak involved approximately 79,000 people in I

..

~ pyruvate kinase

pyruvIc acid

\ , \;>

NADH + H

'"---

~

NAD lactic acid

Figure 2.8 The Embden-Meyerhoff pathway used by homofermentative lactic aCid bacteria.

76


lack cytochrome or electron transport proteins, and therefore cannot derive energy via respiratory activity. Thus, substrate-level phosphorylation reactions that occur during glycolysis are the primary means by which ATP is obtained. There are, however, other means by which these organisms can conserve energy and save ATP that would ordinarily be used to perform necessary fUllctions, such as nutrient transport. Although there are some important differences between how various genera and species use and metabolize specific carbohydrates, lactic acid bacteria generally lack metabolic diversity and instead rely on two principal pathways for catabolism of carbohydrates. In the homofermentative pathway, hexoses are metabolized via enzymes of the Embden-Meyerhoff pathway, yielding 2 mol of pyruvate and 2 mol of ATP per mole of hexose. Pyruvate is subsequently reduced to lactate by lactate dehydrogenase, so that more than 90% of the starting material (i.e., glucose) is converted to lactic acid. The NADH formed via the glyceraldehyde-3-phosphate dehydrogenase reaction is also reoxidized (forming NAD+) by lactate dehydrogenase, thus maintaining the NADH/NAD+ balance. Among lactic acid bacteria used as dairy starter cultures, most are homofermentative, including Lactococcus iactis, Streptococcus thermophilus, Lactobacillus helveticus, and Lb. delbrueckii subsp. bulgaricus. In heterofermentative metabolism, hexoses are catabolized by the phosphoketolase pathway, wl'ich results in approximate equimolar production of lactate, acetate, ethanol, and COl' Only 1 mol of ATP is made per hexose. In actuality, however, product yields for both homo- and heterofermentative metabolism can vary, depending on the source and amount of available substrate, growth temperature, atmospheric conditions, and other factors. Under certain conditions, fur example, ·some homofermentative organisms can divert pyruvate away from lactate and toward other so-called "heterolactic" endproducts. Importantly, the pathway used by a particular strain or culture may have a profound effect on flavor, texture, and overall quality of fermented dairy products. Although several species of Lactobacillus are heterofermentative, Leuconostoc spp. are the only heterofermentative lactic acid bacteria used as starter cultures 111 dairy products.

2.7.1.1 Metabolism of lactose by lactic acid bacteria As described earlier, lactic acid bacteria generally rely on either the Embden-Meyerhoff or phosphoketolase pathway for metabolism

MICRORIAL METABOLISM AND GROWTH

77

g,uFcoseATP hexokinase ADP glucose-6-phosphate NAD glucose-6-phosphate dehydrogenase [ ; NADH + H 6-phosphogluconate 6-phosphogluconate dehydrogenase

k-:

NAD

~~~NADH+H

t

ribulose-5-phosphate ribulose-5-phosphate-3-epimerase

xyulose-5-phosphate

Ph~Pho~ glyceraldehyde-3-phosphate

acetyl phosphate

I

i i i

i

EM pathway

i i i

i

i

i

~COA

phosphotransacetylase ~ P acetyl-CoA

NAD acetaldehyde dehydrogenase [ ; NADH + H

'-V lactic acid

acetaldehyde ethanol dehydrogenase

~

J

NAD NADH + H

ethanol

Figure 2.9 The phosphoketolase pathway used by heterofermantative lactic acid bacteria.

of sugars. In fact, these catabolic pathways are only a part of the overall metabolic process used l~y these bacteria. The first, and

78


perhaps most important, step in carbohydrate metabolism involves transport of the sugar substrate across the cytoplasmic membrane and its 5ubsequent accumulation in the cytoplasm. This process of transport and accumulation is important for several reasons. First, active transport of sugars requires energy, and much of the energy gained by cells as a result of catabolism must then be used to transport more substrate. Second, the transport system used by a particular strain dictates, in part, the catabolic pathway used by that organism. The transport machinery also plays a regulatory role and can influence expression of alternative transport systems. Finally, the metabolic behavior of a particular strain and how that strain functions in fermented dairy foods may be influenced by the actual operation of the transport system itself.

2.7.1.2 Lactose pltospltotrallsferase system of Lc. lactis There are, in general, two different systems used by lactic acid bacteria to transport carbohydrates, and it is convenient to group lactic acid bacteria according to the system used to transport their primary substrate, lactose. The phosphoenolpyruvate (PEP)dependent phosphotransferase system (PTS) is used by most mesophilic, homofermentative lactic acid bacteria, especially lactococci used as starter cultures for cottage, Cheddar, Gouda, and other common cheese varieties. In contrast, other starter culture bacteria, such as S. ther1110philus and Lactobacillus spp. that are used for yogurt, Swiss, and mozzarella cheese production, transport lactose via a lactose permease. Dairy Leuco11ostoc bacteria also rely on a lactose permease for uptake of lactose. Some lactococci and lactobacilli have the ability to use both systems. Not only do these two systems differ in biochemical characteristics, but energy sources used to drive transport and accllmulated intracellular products differ as well. These differences have practical implications. The LactococCLlS lactose PTS, first described by McKay et al. (1969), consists of a cascade of cytoplasmic and membrane-associated proteins that transfer a high -energy phosphate group from its initial donor, PEp, to the final acceptor molecule, lactose. Phosphorylation of lactose occurs concurrent with the vectorial movement of lactose across the cytoplasmic membrane and results in intracellular accumulation of lactose phosphate. There are two cytoplasmic proteins, enzyme I and histidine-containing protein (HPr), that are nonspecific and function as the initial phosphorylating proteins for all PTS substrates. The substrate-specific PTS components comprise

MICROBIAL METABOLISM AND GROWTH

«-----

Glucose-6-P

PEPI pyruvate

Enzl

Xft~~P)8~

Enz I-P

-_

->

~

EIIB

HPr

AT? AD?

;;jt HPr Kmase ~I

IRPrI

l_~

-

~

--~

HPr

Ser-P

I

eRE

Figure 2.10 Signal transduction and the phosphotransferase system in gram-positive bacteria.

the enzyme II complex, which for the lactose PTS in Le. lactis, represents three protein domains (Enz rIAlae and Enz IIBClac). The phosphoryl group is transferred first from PEP to enzyme r, then to HPr, then to the cytoplasmic protein, Enz lIAlae, which then transfers it to the cytoplasmic domain of Enz IIBCloc. As lactose is translocated across the membrane by the integral membrane domain of Enz lIBClac, it becomes phosphorylated. The product of the lactose PTS, thus, is lactose-phosphate, or more specifically, glucose-[)-1,4-galactosyl-6-phosphate. Hydrolysis phospho-Il-galactosidase

lactose-6-P

> gala,tose-e-p

galactose-6-P isomerase

~

tagatose-GoP tagatose-6-P ki,)ase

~

+ glucose

tEM lactic acid

ATP

ADP

tagatose-1,6-di-P

~

glyceraldehyde-3' phosphate

tEM

\ dihydroxyacetone phosphate

lactic acid Figure 2.11 Tagatose pathway in lactococci.

80


of this compound occurs via phospho-l3-galactosidase, yielding glucose and galactose-6-phosphate. Glucose is phosphorylated by hexokinase (via an ATP) to glucose-6-phosphate, which then feeds directly into the Embden-Meyerhoff pathway, as described earlier. Galactose-6-phosphate, in contrast, takes a different route altogether, as it is first isomerized to tagatose-6-phosphate and then phosphorylated to form tagatose-l,6-diphosphate. The latter is then split by tagatose-l,6-diphosphate aldolase to form the triose phosphates, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, in a reaction analogous to the aldolase of the EmbdenMeyerhoff pathway. It is important to note that in Le. laetis, glucose and galactose moieties of lactose, despite taking parallel pathways, are fermented simultaneously.

2.7.1.3 Regulation oj the pllOspllOtransjerase system In Le. laetis, lactose fermentation is regulated at several levels. First, several glycolytic enzymes are allosteric, and their activities are therefore influenced by the intracellular concentration of specific glycolytic metabolites via feedback inhibition. During active lactose metabolism (i.e., when lactose is plentiful), the high intracellular concentration of fructose-l ,6-diphosphate (FDP) and low level of inorganic phosphate stimulate pyruvate kinase. Thus, much of the PEP made via glycolysis is used to drive ATP synthesis, which is consistent with a period of active cell growth. The activity of the NADH-dependent lactate dehydrogenase is also stimulated, which is important because reduced NAD+, formed via the glyceraldehyde3-phosphate dehydrogenase reaction, must be reoxidized to maintain the NAD+ /NADH balance. In contrast, when lactose is limiting, pyruvate kinase activity decreases causing PEP to accumulate, which forms a "bottleneck" in glycolysis. The concentration of triose phosphates subsequently increases, forming a pool of PEP equivalents. Thus, during a period when lactose is unavailable, a PEP "potential" exists, poising the cell for when lactose is available. A second and more effectual mechanism for controlling or regulating lactose metabolism is exerted at the level of the transport machinery itself. In particular, the phosphorylation state of HPr, the cytoplasmic PTS phosphocarrier protein, plays a major role in sugar metabolism. As noted earlier, HPr is phosphorylated by enzyme 1. This phosphorylation occurs specifically at the histidine-IS (His-IS) residue of HPr. However, HPr can also be phosphorylated at a serine residue (Ser-46) by an ATP-dependent HPr kinase, which


81

is activated by fructose-I ,6-diphosphate (as would occur during active sugar metabolism). When BPr is in this state, that is, HPr (Ser46- P), phosphorylation at His-I5 is inhibited; thus, PTS activity is also inhibited and entry of other potential PTS substrates is prevented. Additional experimental evidence that HPr (Ser-46-P) can directly inhibit transport of sugars was provided by Saier and coworkers, who showed that HPr (Ser-46-P) can bind to or otherwise inactivate sugar permeases, a process known as inducer exclusion. Yet another means by which HPr (Ser-46-P) regulates sugar flux is via inducer expulsion. Presumably, this occurs when sugar phosphates have accumulated intracellularly beyond the rate at which metabolism can occur or when nonmetabolizable sugars have been taken up. Since these sugar phosphates could inhibit metabolism, they must first be dephosphorylated and then effluxed. In inducer expulsion, therefore, HPr (Ser-46-P) activates a sugar-specific phosphatase that dephosphorylates the sugar phosphates so that efflux of the free sugar can then occur. HPr not only exerts biochemical control on transport, but HPr (Ser-46-P) also plays an important role at the gene level through its interaction with the DNA-binding protein, CcpA, or catabolite control protein A. HPr (Ser-46-P) and CcpA (with the participation of fructose-l ,6-diphosphate) affect metabolism by blocking transcription of catabolic genes, including other PTS genes, a process called catabolite repression. CcpA or CcpA-like proteins appear to be widely distributed among gram-positive bacteria, including several species of lactic acid bacteria, and this mechanism of gene regulation, therefore, may be common. According to this model of carbon source-mediated gene regulation, HPr exists in one of two phosphorylation states, HPr (His-15- P) or HPr (Ser-46- P). The former accumulates when lactose (or another PTS sugar, such as glucose) is unavailable, since the enzyme II complex is without its substrate. In contrast, when lactose is available and the energy state of the cell is high, intracellular FOP levels increase and HPr kinase is activated, causing HPr (Ser-46- P) to accumulate. A complex is then formed between HPr (Ser-46-P) and CcpA. This complex, along with a glycolytic activator (fructose-I,6-diphosphate or glucose6-phosphate), binds to 14-base pair DNA regions called catabolite responsive elements (CREs) located near the transcription start sites of catabolic genes. When these CRE regions are occupied by the HPr (Ser-46-P)-CcpA complex, transcription by RNA polymerase is effectively blocked or reduced. In contrast, mutations in ccpA or

82


deletions of cre regions eliminate catabolite repression. Since eRE regions are found in the promoter regions of several catabolic genes, the phosphorylation status of HPr can have a profound effect on whether these genes are expressed. Identified gene clusters preceded by CRE regions in lactococci include genes coding for galactose (and thus lactose) and sucrose metabolism. For example, when Lc. lactis is grown on glucose, a PTS substrate, transcription of genes coding for galactose metabolism is repressed. Even the presence of galactose fails to induce expression of gal genes as long as glucose, the repressing sugar, is present. Not only does HPr have a negative regulatory role, but it was recently shown that HPr (Ser-46-P) and CcpA can also activate gene expression. Specifically, expression of the las operon, coding for lactate dehydrogenase, phosphofructokinase, and pyruvate kinase, is activated at high sugar conditions. The net effect, therefore, is that the phosphorylation state of HPr serves as a signal for activating expression of genes coding for glycolytic enzymes when the cell is actively metabolizing sugars. Recent genetic evidence indicates that HPr is also important in influencing sugar uptake by establishing a hierarchy for different sugars preferentially fermented by Lc. lactis. Finally, lactose metabolism is also genetically regulated via expression and repression of the lactose PTS genes. The lactose metabolism genes in Lc. lactis, like the genes coding for other important metabolic pathways, are often located on plasmids of varying size. Strains cured of the lactose plasmid, which encodes lactose metabolism genes, are unable to ferment lactose. In Lc. lactis MG 1820, the lac genes are organized as an 8-kb operon, consisting of eight genes in the order l£lcABCDFEGX. The first four genes, lacABCD, actually code fol' enzymes of the tagatose pathway and are necessary for galactose utilization. The lactosespecific genes, lacFEG, code for PTS proteins and phospho-13galactosidase. The operon is negatively regulated by LacR, a repressor pl'otein encoded by the lacR gene, which is located upstream of the lac promoter and which is divergently transcribed. In the presence of lactose, lacR expression is repressed, and transcription of the lac operon is induced. During growth on glucose or when lactose is unavailable (and cells are uninduced), LacR is expressed and transcription of the lac genes is repressed. A CRE site is also located nem the transcriptional start site of the lac operon. However, when lacR is inactivated, expression of lac genes becomes constitutive regardless of cmbon source (i.e., under conditions that presumably


83

would activate CcpA-mediated repression). This implies that LacR, along with inducer expulsion-exclusion, have primary responsibility for regulating sugar metabolism, rather than CcpA, and that catabolite repression in lactococci is mediated mainly via the concentration of inducer. The lactose PTS, as described earlier for Lc. lactis, also exists in other dairy lactic acid bacteria, including Lb. casei. However, in Lb. casei, lac genes are chromosomally encoded and the nucleotide sequence and genetic organization are different from those in Lc. lactis. The Lb. casei lac cluster (lacTEGF) encodes, respectively, for a regulatory protein, two PTS proteins, and phospho-~ galactosidase. Genes coding for galactose metabolism (lacABCD in Le. lactis) are absent in the Lb. casei lac cluster. Although expression of lac genes is repressed by a CcpA-mediated process, as in Le. lactis, an additional regulatory mechanism dependent on an antiterminator also exists in Lb. casei.

2.7.1.4 Lactose transport alld hydrolysis by S. thermophilus Although the PTS is widely distributed among lactic acid bacteria, several important dairy species rely on a lactose permease for transport and a ~-galactosidase for hydrolysis. Some species have both pathways for lactose, and some have a PTS for one sugar and a permease_ for another. The best example of the lactose permease/ ~-galactosidase system is that which occurs in S. therl11ophilus, Lb. helveticlls, and Lb. delbruecki subsp. bulgaricus. In these bacteria, lactose accumulates in an unmodified form via the LacS permease. A similar system also exists in some strains of Lc. lactis, but clearly it is not the primary system. The lactose permease in S. ther1710philus is dramatically different from other, well-studied lactose permeases, such as the LacY system in Escherichia coli. In E. coli, lactose transport is fueled by a proton motive force (PMF), and the permease binds and transports its substrate lactose in symport with a proton. In S. ther171ophilus, lactose transport can also be fueled by a PMF, but that is not the main way the permease can function. Instead, the transporter has exchange or antiporter activity, so that lactose uptake can be driven by efflux of galactose. That is, "uphill" lactose transport (uptake against a concentration gradient) occurs as a result of "downhill" galactose efflux. Since generation of a PMF requires ATP (or its equivalent), not having to llse the PMF for lactose uptake conserves energy. The lactose: galactose exchange

84


reaction is actually quite remarkable, in that, as discussed below, galactose efflux, rather than galactose utilization, appears to be the preferred pathway for most strains of S. thermophilus. Why this phenomenon occurs and the important practical implications for this will be discussed later. Detailed analysis of the S. thermophilus LacS system has revealed that the permease protein itself is a hybrid consisting of two distinct regions or domains. The deduced amino acid sequence of the amino- terminal region is very similar to the melibiose permease of E. coli. However, the carboxy-terminal region is structurally similar to an E. coli PTS enzyme IIA domain. In fact, this enzyme IIA-like domain can be phosphorylated by HPr, reducing transport activity of LacS. It now appears that the permease region functions as the lactose carrier and the enzyme IIA-like domain functions as a regulatory unit. Hydrolysis of lactose in S. thermophilus occurs via a ~ galactosidase that has modest amino acid homology to other LacZlike enzymes (30-50%). After hydrolysis, S. thermophilus rapidly ferments glucose to lactic acid by the Embden-Meyerhoff pathway, yet most strains, es[!ecially those used as dairy starter cultures, do not ferment the galactose moiety of lactose. Rather, galactose is effluxed and accumulates in the extracellular medium. In the manufacture of dairy products made with an S. thermophilllScontaining culture, such as yogurt or mozzarella cheese, galactose may appear in the finished product. With yogurt, accumulated galactose is of little consequence, but for mozzarella cheese, even a small amount of galactose can present problems. This is because of the nonenzymatic browning reaction that occurs when galactose, a reducing sugar, is heated in the presence of free amino acids. Since most mozzarella cheese is used for pizzas, high-temperature baking accelerates nonenzymatic browning reactions. Cheese containing galactose can brown excessively, a phenomenon considered as a defect by many pizza manufacturers. Therefore, mozzarella producers may be asked by their customers to satisfy specifications for "lowbrowning" or low-galactose cheese. Although some cheese manufacturers can rely on their cheese-making know-how and simply modify the production procedures to remove unfermented galactose, other manufacturers have chosen to use cultures that have lowbrowning potential, as described below. Why are most strains of S. thermophilus phenotypically galactose negative (Gal-) and unable to ferment either free or lactose-derived galactose? Evidence from several laboratories indicates that S.

85


thermophilus does contain genes necessary for galactose metabolism, but that these genes are not ordinarily expressed even under inducing conditions. Mutants have been isolated, however, that ferment free galactose, but when these strains are grown on lactose, galactose utilization is still repressed. Thus, it has been suggested that of the two routes that galactose can take, the efflux reaction is favored over the catabolic pathway.

2.7.1.5 Lactose metabolism by Lactobacillus and other lactic acid bacteria Most other lactic acid bacteria rely on one or the other of the two pathways described earlier. With the exception of Le. lactis and Lb. casei, however, most dairy lactic acid bacteria do not have a lactose PTS, and instead use a lactose permease/~-galactosidase system for metabolism of lactose. Some strains have more than one system; for example, Lc. lac/is and Lb. casei have both a lactose PTS and a lactose permease/~-galactosidase. It is important to note that not all strains or species that use non- PTS pathways for lactose metabolism excrete galactose into the medium, as described for S. thermophilus. Many of the lactobacilli and Leuconostoc spp. that transport and hydrolyze lactose by a permease and a ~-galactosidase, respectively, also ferment glucose and galactose simultaneously. This is important, because in almost all fermented dairy products made with a culture containing S. thermophilus, a galactose-fermenting Lactobacillus sp. is also present. For some products, such as Swissstyle cheeses, the galactose that is effluxed into the curd by S. thermophilus is subsequently fermented by Lb. helveticus. Otherwise, the free galactose could be fermented by other members of the microflora, resulting in heterofermentative end products that could contribute to off-flavors and other product defects. Table 2.1 Lactose transport and metabolic systems in dairy lactic acid bacteria Organism

Lactose transport system

Galactose pathway

Streptococcus thermophilus Lactococcus lactis Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus helveticus Lactobacillus casei Leuconostoc lactis

Lac permease PTS Lac permeas'e

Leloir Leloir, tagatose Leloir

Lac permease PTS, Lac permease Lac permease

Leloir Leloir, tagatose Leloir

86


2.7.1.6 Galactose metabolism During growth in milk, lactic acid bacteria ordinarily encounter free galactose only after intracellular hydrolysis of lactose. For lactococci and those lactobacilli that transport lactose via the PTS, galactose-6-phosphate is the actual hydrolysis product (resulting from hydrolysis of lactose-phosphate by phospho-I)-galactosidase). Galactose-6-phosphate feeds directly into the tagatose pathway. However, as noted earlier, free galactose will appear and accumulate in fermented dairy products made with thermophilic starter cultures containing S. thermophilus, Lb. bulgaricus, or other galactosenonfermenting strains. Yogurt and mozzarella cheese, for example, can contain up to 2.5 and 0.8% galactose, respectively. Therefore, metabolism of free galactose may be of practical importance. For the lactococci and some lactobacilli, free galactose appears to be transported by either a galactose-specific PTS or by a galactose permease. The intracellular product of the galactose PTS (galactose6-phosphate) simply feeds into the tagatose pathway. When galactose accumulates via galactose permease. the intracellular product is free galactose. Subsequent metabolism occurs via the Leloir pathway, which phosphorylates galactose, and then converts galactose-lphosphate into glucose-6-phosphate. The latter then feeds into the glycolytic pathway. Interestingly, in Le. lactis, galactose permease may be the primary means for transporting galactose, since it has a much higher apparent affinity for galactose than the PTS transporter. galactose galactokinase

F

t

ATP ADP

galactose-l-P galactose-1-P

uridyl transferase

UDP glu

~UDP

galactose , ) eoimcrase

UDP gal

gJucose-1-P phosphoglucomutase

J

glucose-6-P EM

t.:

/'

"

PK ~

Figure 2.12 Leloir pathway in lactic acid bacteria.


87

The Leloir pathway is used not only by lactococci, but it is also the pathway used by Lb. helveticus, Leuconostoc spp., and galactosefermenting strains of S. thermophilus. During growth on lactose, these bacteria rely on a lactose permease/~-galactosidase system and therefore generate free intracellular galactose. In some instances, they will also encounter free extracellular galactose, especially if they are grown in the presence of galactose-nonfermenting strains, as described earlier. Subsequent galactose fermentation by Lb. helveticus and Leuconostoc lactis occurs via the Leloir pathway. Transport is mediated by a permease, apparently driven by a PMF. A mutarotase (the product of the galM gene) may also be necessary to convert ~-D-galactose (the product of lactose hydrolysis) to its anomeric isomer, a-D-galactose, before it can be efficiently phosphorylated by galactokinase. Despite the inability of most strains of S. thermophilus to ferment galactose, genes coding for enzymes of the Leloir pathway appear to be present and functional. The S. thermophilus gal operon consists of four structural genes (gaIKTEM) and one divergently transcribed regulatory gene (gaLR). Transcription of these genes, however, does not occur in most wild-type strains, accounting for the galactose nonfermenting phenotype. Mutations in the gal promoter/regulatory region led to isolation of galactose-fermenting mutants that expressed gal genes and fermented galactose. Such efforts suggest that genetic modification of S. thermophilus may provide the basis for obtaining stable galactose-fermenting derivatives that would be of considerable value to the dairy industry. Although the gal genes in S. thermophilus, Leuc. lactis, Lc. lactis, Lb. casei, and Lb. helveticus share significant amino acid sequence homology and are chromosomally encoded, they are organized in a somewhat different order. All contain galK (galactokinase), gaIT(galactose-l-phosphate uridyl transferase), and galE (UDP-galactose-4-epimerase), and some clusters also contain the galM gene coding for mutarotase. In S. thermophilus, the gal genes are located immediately upstream of the lacS-IacZ cluster. There is also rather significant variation with respect to genetic structure even between strains of the same species. For example, a gaLA gene, thought to encode for a permease, is the first gene in the Lc. lactis MG 1363 gal cluster, but this gene does not appear in gal clusters from other organisms. The ability of these strains, especially lactobacilli, to ferment galactose can be quite variable, and strain selection is important.


88

Galactose fermentation by lactobacilli has also been used as a basis for distinguishing between Lb. helveticus (Gal+) and Lb. delbrueckii subsp. bulgaricus (Gal-). As noted earlier, some culture suppliers promote "non browning" cultures for use in mozzarella cheese production; invariably, these cultures contain galactose-fermenting lactobacilli.

2.7.1.7 Altemate routes for pyruvate As described earlier, lactic acid bacteria are ordinarily considered as being either homofermentative or heterofermentative, with some species being able to metabolize sugars by both pathways. However, sugar metabolism, even by obligate homofermentative strains, can result in formation of endproducts other than lactic acid by a variety of pathways. In general, these alternative fermentation products are formed as a consequence of accumulation of excess pyruvate and the requirement of cells to maintain a balanced NADH/NAD+ ratio. That is, when the intracellular pyruvate concentration exceeds the rate at which lactate can be formed via lactate dehydrogenase, other pathways must be recruited not only to remove pyruvate but also to provide a means for oxidizing NADH. These alternative pathways may also provide cells with the means to make additional ATP. Under what conditions or environments would pyruvate accumulate? As noted earlier, when fermentation substrates are limiting, and the glycolytic activator, fructose-! ,6-diphosphate, is in short supply, activity of the allosteric enzyme, lactate dehydrogenase, is reduced

acetate

a-aceto/actate

CO 2

~tOI"t.t. a-as~enthase

/

pyruvate

pvruvate-::rmay IY'~

ethanol acetate formate

~eh~~~tate

~ogen.se lactate

Figure 2.13 Alternative routes of pyruvate metabolism


89

and pyruvate accumulates. Low carbon flux may also occur during growth on galactose or other less preferred carbon sources, resulting in excess pyruvate. When the environment is highly aerobic, NADH that would normally reduce pyruvate is instead oxidized directly by molecular oxygen and is unavailable for the lactate dehydrogenase reaction. Several enzymes and pathways have been identified in lactococci and other lactic acid bacteria that are responsible for diverting pyruvate away from lactic acid and toward other products. In anaerobic conditions, and when carbohydrates are limiting and growth rates are low, a mixed-acid fermentation occurs, and ethanol, acetate, and formate are formed. Under these conditions, pyruvate-formate lyase is activated, and pyruvate is split to form formate and acetylCoA. Acetyl-CoA can be converted to ethanol and/or acetate. The latter also results in formation of an ATP via acetate kinase. If the environment is aerobic, pyruvate-formate lyase is inactive, and instead pyruvate is decarboxylated by pyruvate dehydrogenase to form acetate and COr Finally, excess pyruvate can be diverted to a-acetolactate via a-acetolactate synthase. This reaction has other important implications, since a-acetolactate is the precursor for diacetyl formation. Although these alternative pathways for pyruvate metabolism are influenced largely by environmental conditions, mutants unable to produce lactate dehydrogenase also must deal with excess pyruvate and, therefore, produce other endproducts. Under certain conditions, cells may divert excess pyruvate to highly desirable products, specifically the aroma compound diacetyl. Ordinarily diacetyl is made from citrate, but even citrate-nonfermenting cells will make diacetyl from lactose if appropriate conditions are established or if cells are genetically modified. For example, overexpression of NADH oxidase in Le. lactis decreases lactate formation from pyruvate, and instead a-acetolactate, the precursor for diacetyl, is formed. Enhancing diacetyl production by metabolic engineering will be discussed later.

2.7.2 Protein Metabolism Just as dairy lactic acid bacteria are well suited to utilize lactose as a source of energy and carbon, they are also well adapted to use casein as a source of nitrogen. Lactic acid bacteria cannot assimilate inorganic nitrogen and, therefore, they must be able to degrade proteins and peptides to satisfy their amino acid requirements. The absolute requirement for a system to degrade milk casein was first

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demonstrated by McKay and Baldwin (1974), who showed that Le. lactis C2, cured of a plasmid containing the proteinase gene, was unable to grow to high cell density in milk. However, if milk was supplemented with hydrolyzed milk protein, the plasmid-cured strain grew like the parental strain. We now know that dairy lactic acid bacteria have evolved highly efficient systems for reducing large casein subunits to smaller pieces and for supplying cells with all of the amino acids necessary for growth in milk. The proteolytic system consists of three main components. The first involves the proteolysis of casein to form a large collection of peptides. In the second step, peptides are transported into cells by one of several transport systems. Once inside the cell, peptides are further hydrolyzed by a diverse group of peptidases to form free amino acids which are ultimately either metabolized or assimilated into protein.

2.7.2.1 Proteinase system Although lactic acid bacteria vary considerably in their ability to degrade 'milk protein, most organisms possess similar systems, as typified by the extensively studied proteolytic system of Lactococcus. For Le. lactis and other dairy lactic acid bacteria, casein is the primary source of amino acid nitrogen, since the non-protein nitrogen and fr,ee amino acids available in milk «300 mg/L) are quickly depleted. Because Le. lactis is a multiple amino acid auxotroph and requires as many as eight amino acids, casein hydrolysis is essential. Casein utilization by Le. lactis begins with elaboration of a cell envelope-associated serine 'proteinase. This proteinase, Prtp, is expressed as a large (>200 kD), inactive preproproteinase. The leader sequence, which is responsible for directing the protein across the cytoplasmic membrane, is removed, leaving the remaining protein anchored to the cell envelope. However, the propr6teinase is not active until it is further processed by the maturation protein, PrtM. The latter presumably acts by inducing an autocatalytic cleavage event that results in hydrolysis of the pro region of the enzyme, leaving a mature PrtP with a molecular mass of 180-190 kD. Although the proteinases among different strains of Lc. lactis are all genetically related and show only minor differences with respect to their amino acid sequence, the specific casein substrates and hydrolysis products of PrtP enzymes from lactococci can vary considerably. For example, proteinases belong to group A (formerly Pili-type) hydrolyze U SI -' 13-, and K-caseins, whereas group E proteinases (formerly PI-type) have a preference for l3-casein and


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relatively little activity for u s ,- and K-caseins. Still, the functional organization of the PrtP and PrtM system varies little among lactococci. Both are required for rapid growth in milk, and genes for both (prtP and prtM, respectively) are induced when cells are grown in low-peptide media (e.g., milk) and repressed in peptiderich media. Over 100 caseinolytic products result from action of PrtP on ~-casein. Most are large oligopeptides (4-30 amino acid residues) with a major fraction between 4 and 10 residues. Free amino acids, dipeptides, and tripeptides are not formed. The first and most abundant oligopeptides formed by PrtP are generated from the Cterminal end of ~-casein, and it now appears that initial hydrolysis events cause casein to unfold so that other cleavage sites are exposed.

2.7.2.2 Amino acid and peptide transport systems Although it was once believed that extracellular peptidases must be present to degrade further these peptides before transport, it is now well established that extracellular hydrolysis of peptides formed by PrtP does not occur, at least not by peptidases. Instead, lactococci and other lactic acid bacteria possess an array of amino acid and peptide transport systems able to transport substrates of varying size, polarity, and structure. Some of these are highly specific, whereas others have rather broad substrate specificity. They also vary as to energy Sources used to fuel active transport. As described earlier, the concentration of free amino acids in milk is too low to support growth of lactic acid bacteria. Still, lactococci possess at least 10 amino acid transporters, most of which are specific for structurally similar substrates. If the medium contains an adequate concentration of free amino acids, these transport systems can deliver enough amino acids to the cytoplasm to support growth. However, it has been suggested that the primary function of these transporters may be simply to excrete or efflux excess amino acids from the cytoplasm to maintain appropriate intracellular pool ratios. That is, if peptides are indeed the primary source of amino acids, then some amino acids, generated from intracellular peptidases, may accumulate faster than they can be assimilated. These free amino acids could then diffuse out of cells down their concentration gradient via the amino acid transporter operating in the reverse or efflux direction. If efflux of an amino acid is accompanied by a coupling ion (e.g., proton extrusion), then a net increase in the PMF is obtained. It may even be possible for amino acid efflux to provide

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enough energy to drive uptake of peptides. In contrast to the amino acid transporters, peptide transport is clearly necessary for lactic acid bacteria to grow in milk. Three groups of peptide transport systems have been identified. Two of these, DtpT and DtpP, transport dipeptides and tripeptides. DtpT is a large (463 amino acid residues) monomeric, PMF-dependent transporter that has affinity for hydrophilic peptides. Mutants with a deletion in the dtpT gene have been obtained and are unable to express DtpT and transport some peptides. In a defined medium, dtpT mutants grew poorly; however, growth of these mutants in milk was unaffected, indicating that DtpT is not essential in milk. DtpP, the other transport system in lactic acid bacteria that transports dipeptides and tripeptides, is an ATPdependent transporter that has high affinity for hydrophobic peptides. It also appears to be unnecessary for growth of lactococci in milk. The third and most important peptide transport system in lactic acid bacteria is the oligopeptid.e transport system (Opp). Since dipeptides and tripeptides are not released from casein, neither DtpT nor DtpP is required for growth in milk; lactococci instead rely on oligopeptides and Oppto satisfy all amino acid requirements. Indeed, mutants unable to express genes coding for the Opp system are unable to transport oligopeptides and ase unable to grow in milk. Although it was initially not known which oligopeptides were actually transported by Opp, many of the structural and genetic features of the Opp system in Le. lactis are .10W well defined. The Opp complex belongs to the ABC (ATP binding casette) family of transporters and consists of five subunits: two transmembrane proteins (OppB and OppC), two ATP binding proteins (OppD and OppF), and a membrane-linked substrate-binding protein (OppA). The five genes coding for Opp are organized as an operon in the order oppDFBCA. A gene coding for an oligopeptidase (pepO) is also located immediately downstream of oppA and is cotranscribed with the opp operon. The Opp system transports a diverse population of oligopeptides. Although PrtP releases over 100 peptides from p-casein, only 1014 pep tides apparently serve as substrates for Opp. All of these oligopeptides contain more than 4 and fewer than 11 amino acid residues. Detailed analysis revealed that they contain proportionally higher levels of valine, proline, and glutamate and moderate levels of alanine, leucine, isoleucine, lysine, and serine. Importantly, these oligopeptides provide all essential amino acids, with the exception of histidine, needed by lactococci for growth in milk.


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2.7.2.3 Peptidases The third and final step of protein catabolism involves peptidolytic cleavage of Opp accumulated peptides. Over 20 different peptidases have been identified and characterized, either biochemically and/or genetically, in lactococci and lactobacilli. Both endopeptidases (those that cleave internal peptide bonds) and exopeptidases (those that cleave at terminal peptide bonds) are widely distributed. Of the latter, only aminopeptidases have been reported; carboxypeptidases apparently are not produced. In general, concerted efforts of endopeptidases, aminopeptidases, dipeptidases, and tripeptidases are required fully to utilize peptides accumulated by the Opp system. Although there was once considerable debate on the location of these peptidases, it is now well accepted, based on genetic as well as physical evidence (e.g., lack of signal peptides and anchor sequences, cell fractionation, and immunogold labeling experiments), that they are intracellular enzymes. Substrate size and specificity and other properties of peptidases from lactic acid bacteria have been of considerable interest, not only because of their physiological importance but also because of the significant role peptidases play in cheese manufacture and ripening. 2.7.2.3.1 Endopeptidases Several endopeptidases have been described, including PepF and PepO in Lc. lactis and PepE, PepG, and PepO in Lb. helveticus. Most of these endopeptidases are metalloenzymes that contain sequences typical of zinc-binding domains and hydrolyze oligopeptides of varying lengths as substrates. It is interesting to note that some endopeptidases (e.g., PepF) have pH optima in an alkaline range (7.5-9.0) and that peptidase activity at pH levels typical of ripened cheese (e.g., R), and a negative value where the community is net heterotrophic (P < R). The balance between P and R is determined by the relative metabolic contributions of autotrophic and heterotrophic organisms, which in turn relates to environmental parameters such as light availability (promoting photo trophy) and availability of externallyderived (allochthonous) carbon-promoting heterotrophy.

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Benthic communities vary from net heterotrophy (e.g., profundal sediments, dominated by anaerobic heterotrophic bacteria) to net autotrophy (e.g., shallow lake sediments, dominated by benthic algae). Pelagic communities typically have greater light exposure than benthic systems, and (in lentic water bodies) often have a greater autotrophic contribution. The balance of available nutrients is important, and low nutrient (oligotrophic) lakes are typically net heterotrophic while high nutrient (eutrophic) lakes show net autotrophy. This differ.ence arises because oligotrophic conditions support only limited carbon uptake by phototrophy (low inorganic nutrient availability) but significant heterotrophy can still occur due to input of allochthonous carbon. In eutrophic lakes, both modes of nutrition are increased, but the increased availability of soluble inorganic nutrients promotes autotrophy to a greater extent. The pelagic communities of lotic systems may also show great variation in net ecosystem production. This is seen particularly well in some estuaries, where the value for NEP shows marked seasonal fluctuation with river inflow. 5.2.4 Planktonic and Benthic Microorganisms Freshwater organisms can be divided into two main groups, according to where they spend the major part of their growth phase - pelagic organisms (present in the main body of water) and benthic organisms (associated with the sediments). Pelagic biota can be further subdivided into nekton (strongly swimming organisms such as fish) and plankton (free-floating). The latter tend to drift within the water body, though they may have limited motility and achieve vertical movement. Pelagic microorganisms are essentially plankton, so the distinction within this assemblage is between planktonic and benthic states. Although freshwater organisms can be categorized as planktonic or benthic, most species have both planktonic and benthic phases within their life cycle. Biofilm bacteria, for example, have a dynamic equilibrium between attached and planktonic states, and freshwater algae also show clear planktonic/benthic interactions in lake and stream environments. Planktonic and benthic phases of the same species typically show considerable differences in metabolic activity. This is particularly the case for freshwater bacteria, where quorumsensing mechanisms lead to the induction of stationary-phase physiology i~ biofilm communities.

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5.2.4.1 Planktonic microorganisms These biota frequently have specialized mechanisms to migrate or maintain their position vertically within the water column, and are particularly well represented by micro-algae and bacteria. Planktonic organisms can be further divided into holoplanktonic forms (where the organism is present in the water column for a major part of the annual cycle) and meroplanktonic organisms (where the planktonic phase is restricted over time) biota. The distinction between holoplanktonic and meroplanktonic species is particularly well shown by the algae, where the meroplanktonic state can beviewed as an adaptation for short-term competition. 5.2.4.2 Benthic microorganisms Present at the surface and within sediments, these are dominated by biota such as fungi, protozoa, and bacteria that are able to break down sedimented organic debris. The diversity of benthic life forms is particularly well represented by the protozoa, which include both attached and freely-motile organisms, with a variety of feeding mechanisms. A number of key terms have been used to define particular groups of benthic organisms, including one major group - the periphyton. This term is used to describe all the 'plant-like' microorganisms (microflora) present on substrata, including microscopic algae, bacteria and fungi. This term excludes 'animal-like' organisms, such as micro-invertebrates and protozoa, but includes filamentous algae (e.g., Cladophora, Spirogyra, Chara, and Vaucheria). Differences occur within the periphyton in terms of the nature of the substrate, which may be living (e.g., plant surfaces) or nonliving (organic or inorganic, with different particle sizes). 1. Epiphytic organisms are associated with the surfaces of higher plants and macroalgae. The substratum in this case is often metabolically active, and the epiphytic association may be important in terms of competition for light, metabolic exchange and nutrient availability. 2. Epilithic, epipsammic and epipelic microorganisms grow on non-living substrates which differ in particle size. Epilithic biota occur on hard, relatively inert substrata such as gravel, pebbles and large rocks. Decrease in particle size leads to epipsammic organisms (present on sand) and epipelic organisms (present on fine sediments such as mud). Relatively few of the larger microorganisms, such as algae, live in sand, since particles are

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too unstable and may crush them. Quite large algae are present on fine sediments, however, including large motile diatoms, motile filamentous algae and large motile flagellates such as Euglena. A further group of plant-like benthic organisms includes the metaphyton, which are loosely associated (but not attached) to the substratum. These are characterized by clouds of filamentous green algae such as Spirogyra, Mougeotia or Zygnema, which become loosely aggregated and accumulate in regions of substratum that are free from currents and waves. Metaphyton usually arises from other substrata, such as higher plant surfaces (epiphyton). 5.2.5 Metabolically Active and Inactive States Although freshwater microorganisms are often thought of as dynamic, metabolically active biota, this is not always the case. All species have inactive or inert periods for at least part of their life cycle; these may occur as temporary dormant phases (surviving adverse conditions) or terminal phases of senescence, leading to death.

5.2.5.1 Dormant phases In temperate climates, particularly, many microorganisms overwinter on the sediments in a dormant state. The formation of resistant spores typically occurs as environmental conditions deteriorate (overcrowding, reduced nutrient and light availability, accumulation of toxic metabolites, reduced temperature), and may be preceded by sexual reproduction. In some cases, dormancy appears to relate to a specific environmental change, such as oxygen concentration. In the water column of lakes, obligate anaerobes such as photosynthetic purple bacteria are metabolically active in the anaerobic hypolimnion during stratification, but overwinter in flocs of organic material once the lake becomes oxygenated at autumn overturn. In bacteria and viruses, metabolic inactivity is also nutrientrelated. Comparisons of total and viable bacterial counts suggest that only a small fraction of aquatic populations are metabolicallyactive. Under certain environmental conditions, bacteria remain inert until they encounter appropriate growth conditions - particularly in relation to nutrient supply. Viruses are also metabolically inactive within the water medium, and are only activated when they encounter a healthy host cell. In the inert state, they occur as free particles (virions) within the water medium, and are vulnerable to inactivation and loss processes.

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5.2.5.2 Senescence Senescence and cell death are just as much a part of population development as growth processes, and play an important role in the dynamics of microbial communities. In some situations, death is induced by specific environmental factors such as nutrient limitation, solar irradiation and parasitism. In other situations, senescence may occur due to internal changes, particularly after a long sequence of divisions and probably due to programmed cell death. The occurrence of growth and senescence within a single-species population is taken from a stationary phase in a laboratory monoculture of the green alga Micrasterias. Mature cells (M) of this placoderm desmid have two halves (semi-cells) of equal size, each with a single chloroplast. Cells that are undergoing senescence (S) show condensation of the chloroplasts to the centre of the cell, leading to degeneration and the formation of colourless dead cells that just have the remains of the cell wall. Actively growing cells that have recently divided (inset) have one normal sized semicell (b) derived from the mother cell, plus a new semicell (a) that will attain full size on completion of the growth cycle. Although cell differences in growth cycle and the onset of senescence are particularly clear in populations of this organism, they are also characteristic of other populations where they are not so easily observed. Witl1in phytoplankton populations at the top of the water column, for example, senescence and cell death occurring in a wide range of species results in a continuous rain of mixed organisms to the lower part of the water column and the sediments. These can be collected in sediment traps suspended in the water column and subsequently analy~ed.

5.2.6 Evolutionary Strategies: r-selected and K-selected Organisms In the population of cells, regeneration (formation of new organisms) and death continuously occur. The growth of populations that include continuous cycles of regeneration and death is best described by 'models of continuous growth', and leads to a consideration of the importance of competition in population increase and the designation of two opposing evolutionary strategies - adopted by r-selected and K-selected organisms.

5.2.6.1 Exponential and sigmoidal models of continuous growth The speed with which a single-species population (N) increases with time will be denoted by dN Ielt. The increase in size of the

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whole population is the sum of the growth contribution of all individuals, so the rate of increase per individual (r) will be:

r= dN[~] dt N or

rN= dN dt The parameter r is also referred to as the 'per capita rate of increase' and describes the fundamental ability of individual organisms to grow and reproduce. The above equation describes population growth in conditions of unlimited resources and generates an exponential increase in population with time. In practice, intra-specific competition for resources (particularly nutrients and space) must also be taken into account, and the population growth curve then becomes sigmoidal. In this situation, the population (N) rises to a maximum value (K), which is the maximum value (the carrying capacity) that the environment can support. The growth equation which describes the sigmoidal curve is referred to as the 'logistic equation' and has the form:

dN =rN[K-N] dt K This sigmoidal curve is well-known to microbiologists, where the growth of microorganisms in batch culture follows a sequence of lag, logarithmic and stationary phases. In the lower part of the curve (lag phase), when population is sparse, the population is beginning to colonize the new environment and relatively little competition occurs between cells. As the population approaches the carrying capacity (K), resources (nutrients, space) become limited and high levels of competition occur between cells.

5.2.6.2 r-selected and K-selected organisms The conditions seen in the single-species growth curves of organisms cultured under laboratory conditions can also be applied to freshwater environments, where mixed populations occur. Some environments have low population densities, and are dominated by organisms that are adapted for high rates of growth and rapid colonization, while others have high population densities and are dominated by organisms that are adapted to survive in these highly competitive conditions.

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This fundamental distinction between two major types of adaptation was first described by MacArthur and Wilson (1967) in relation to differences in selection pressure at different phases in the colonization of oceanic islands. These authors separated organisms into r-strategists, which were the initial colonizers of the island environment, and K-strategists - adapted to the more crowded, later conditions. In uncrowded environments, organisms able to grow and reproduce rapidly, with high productivity, will be most suited to dominate the environment. They are subsequently replaced by organisms which are more suited to a crowded community that is approaching its population limit and are referred to as K-strategists. The terms rand K are derived from the logistic equation for population growth, which specifies that the per capita rate of increase (r) is maximized under sparse conditions. With increasing crowding, a decline in the per capita rate of increase occurs until the population density equilibrates to its upper level or carrying capacity (K). The adaptation of r- and K-strategists to environments of differing population density has a range of biological implications, including differences in biological diversity, competition, accumulation of metabolites, resource availability, and liability to parasitic attack. The incidence of ecological stress can be high or low in either situation, depending on the particular circumstances. The adaptation of r-strategists to uncrowded conditions also makes them suitable to unstable environments, where growth is limited to short periods of time and high population levels cannot become established. Under such conditions the ability of these organisms to grow rapidly and exploit growth opportunities as they become available gives them a competitive advantage. In line with this, r-strategists are typically small, with a short life cycle and high growth rate. In contrast, Kstrategists are adapted to stable environmental conditions, where dense populations develop and rapid growth is not an advantage. All groups of freshwater microorganisms contain r- and K-selected species, and all aquatic environments have situations (during colonization, and at different times in the mature community) when r- and K-strategists are respectively adapted to the prevailing situation. Planktonic bacteria are particularly good examples of r-strategists, existing in a metabolically inert form for much of their life cycle, but able to grow and multiply within a short space of time when nutrients become available. Phytoplankton contain well-adapted examples of both types of organism, with changes in dominance of r- and Kselected algae during the seasonal progression. One particular group

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of algae, the dinoflagellates, are perhaps the best example of all microbial K-strategists. 5.3 BIODIVERSITY IN ECOSYSTEMS, COMMUNITIES, AND SPECIES POPULATIONS Individual microorganisms are part of larger groups or communities, and any consideration of biodiversity must take into account the wider community and the environment in which it occurs. A community is a naturally occurring group of organisms living in a particular location, such as a lake or stream. The occurrence and interactions of organisms living within a discrete environment constitutes a functional unit, referred to by Tansley (1935) as an ecological system or 'ecosystem'. This was considered by Tansley to consist of two major components - the biome or ecosystem community (the entire group of organisms) and the habitat (physical environment). Each particular ecosystem can be regarded to some extent as self-contained and as a basic unit of ecology. Ecosystems are themselves part of a larger geographic or global unit, the biosphere, which is the sum of all ecosystems within a particular zone. ECOLOGICAL UNIT

l\lAIN ECOSYSTEM le g. J.. kC. nver. v.·ell.and)

SUBSIDIARY COl\lMUNITIES 'c.g .• hh,lill11. pdaglc communlly)

SINGLE SPECIES POPULATION te g.• I,lIlglc !tlgal 'p~t'I(!'" waler column)

III

HlOLOGICAL DIVERSITY MIxture uflubsidiary ecosystems, each with il.l' ()WII c01llmunity

Single cOll/IlI/mity, with: • Different types of organism • VlIrietyof species

iJil't'rsity within 6pecies - TIIaleeular, chemical, and pizysiait)gil"al variation

Figure 5.1 Diversity wIthin species, communities, and groups of communities at different levels of organization in freshwater systems.

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The nature and evaluation of community diversity partly depends on environmental scale and can be considered as a hierachy of three levels - main ecosystems, subsidiary communities, and species populations. 5.3.1 Main Ecosystems Major aquatic environments such as lakes, rivers and wetlands form a discrete ecological unit, with their own characteristic community of organisms; they can be referred to as main ecosystems. Each of these main ecosystems contains a diverse array of distinctive groups of organisms (subsidiary communities), each in their own particular environment, forming subsidiary ecosystems. As an example of this, a typical temperate lake (main ecosystem) can be divided into two major regions - the peripheral shoreline (littoral) zone and the central zone, each with its own subsidiary communities and ecosystems. The central zone can be separated into pelagic and benthic groups of organisms, with further resolution within the pelagic zone of neuston, phycosphere and photosynthetic bacterial communities. These occur in discrete small-scale environments, and are entirely composed of microorganisms. The littoral zone of the lake is dominated by attached organisms, which in eutrophic lakes can be divided into three major groups macrophyte, upper periphyton and lower periphyton communities. Within these, various distinctive micro-communities occur including epiphytic communities on macrophyte leaves, and algal and bacterial biofilms on exposed rock surfaces. Although these various groups of organisms have a degree of autonomy, they all interrelate within the main ecosystem. The pelagic and benthic communities, for example, may appear very distinct during the summer growth phase of eutrophic lakes but the benthic community depends on continuous biomass input from the pelagic zone by sedimentation, and populations of planktonic organisms arise by recruitment from the benthic zone. 5.3.2 Diversity within Subsidiary Communities Different communities have their own distinctive pattern of organisms with their own level of biodiversity. Within these communities, this diversity can be measured in various ways including variation in size of organisms, presence of mucilaginous and non-mucilaginous types, attached and free-floating biota and proportions of autotrophic and heterotrophic species. In practice,

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diversity is often determined simply in terms of species content. The variety of algae within phytoplankton is considered in terms of indices of species diversity. Significant changes occur in these indices during seasonal development of phytoplankton, indicating fundamental changes in community diversity throughout the growth season.

5.3.3 Biodiversity within Single-species Populations Phenotypic and genetic diversity within the ecosystem populations of individual species represents an important but relatively unexplored area of biological diversity in freshwater systems. One of the problems in studying variability at this level is that classical biochemical (testtube) techniques are difficult to apply to single species within mixed populations. The use of analytical microscopical techniques, however, has gone some way to overcoming this problem since these have sufficient spatial resolution to determine the chemical and molecular composition of single microorganisms within mixed populations. Some of these techniques are discussed in relation to algal populations and include the use of light microscope infrared spectroscopy to study the vibrational states of different molecular groups and the use of scanning electron microscope X-ray micro-analaysis to determine variations in elemental composition. Both of these approaches have demonstrated considerable variability within the single-species populations that make up the mixed phytoplankton assemblage, indicating that intra-specific variation in planktonic algae is an important feature of the pelagic environment. Molecular techniques also have considerable potential for looking at biodiversity within single species populations, and have been used to study various aspects of genetical variation within natural populations. These include sub-species (strain) variation in blue-green algae and variations in plasmid-borne resistance in aquatic bacteria. Differences between planktonic and attached (biofilm) bacteria in terms of the expression of stationary phase genes also constitute an important an important intraspecific variable within benthic systems. Ecosystems vary in size and composition, and contain a wide range of organisms which interact with each other and with the environment. Individual ecosystems have a number of important properties: (i) a distinct pattern of interactions between organisms, (ii) defined routes of biomass formation and transfer, (iii) maintenance of the internal environment, and (iv) interactions with the external environment.

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The range of organisms present in aquatic communities define and characterize the system concerned, and are involved in the generation and transfer of biomass. They have distinct roles and interactions within the ecosystem, occupying particular trophic levels and forming an interconnected system of feeding relationships (the food web). The balance of individual species within the food web is determined primarily by resource (light, nutrient availability) and competition. This in turn affects variety in the range and proportions of the different organisms (biodiversity), with important implications for the overall functioning of the system. Community structure is closely related to ecosystem stability and physical stress levels in the environment. Interactions between ecosystems and their surrounding environment occur in various ways. One example of this is the net exchange of carbon between the aquatic ecosystem and the adjacent atmosphere, which can be quantified in relation to net ecosystem production (NEP). 5.4 BIOFILM COMMUNITY: A SMALL-SCALE FRESHWATER ECOSYSTEM This section considers the dynamic properties of freshwater communities within two ecosystems where microorganisms have a key role - the microbial biofilm and the pelagic ecosystem of lakes. The microbial biofilm is a small-scale ecosystem composed entirely of microorganisms, while the pelagic ecosystem is a large-scale functional unit containing a wide range of biota. Contrasts between the diagrams to some extent reflect diversity in research approach rather than fundamental ecosystem differences. In the case of biofilms, for example, a lot of information has been obtained on genetic interactions, but relatively little is known about food webs. The reverse is true for pelagic ecosystems. Microbial biofilms provide a useful model system for considering fundamental aspects of community interactions and ecosystem function. Their small scale makes them amenable to laboratory as well as environmental experimentation, and the close proximity of organisms within the biofilm leads to high levels of biological interaction. Within the context of this book. the exclusively microbial composition of biofilms also makes them particularly appropriate for consideration. Microbial biofilms occur as discrete communities within a gelatinous matrix, and are present as a surface layer on rocks and stones in lakes and rivers. The biological composition of biofilms

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varies with environmental conditions, including factors such as ambient light intensity, water flow rate and prior colonization history. In some cases they are entirely bacterial, while in others they initiate with the settlement of diatoms and develop into larger scale periphyton communities. The biofilm is a mature mixed biofilm, such as might occur on a river substratum under conditions of limited light. and consists of a balanced community composed mainly of bacteria, with algae, protozoa, and fungi also present. The organisms are largely present within a gelatinous matrix, which defines the extent of the ecosystem. The matrix typically has a columnar appearance, with channels or pores between the columns through which water percolates. The microbial biofilm shares with other small-scale microbial (e.g., neuston and phycosphere) ecosystems and with larger ecosystems the four key aspects of ecosystem function listed above - interactions between organisms, biomass transfer, homoestasis and interactions with the external environment.

5.4.1 Interactions between Microorganisms Microbial interactions occur both within the main population of bacteria and in relation to other microorganisms. Bacterial interactions determine the structure and diversity of the biofilm and include gene transfer, quorum sensing and specific adhesion processes. Trophic (feeding) interactions occur between different groups of biota and can be considered in terms of food webs.

5.4.1.1 Gene transfer Microbial biofilms are particularly important in relation to gene transfer between bacterial cells, since they are a part of the aquatic environment where the transfer process is optimized due to the close proximity of the organisms concerned. Gene transfer between bacterial cells has been studied under both laboratory and environmental conditions. 5.4.1.2 Quorum sensing The physiological activities of bacteria vary considerably in relation to population density (number of cells per unit volume of medium). The pattern of gene activity, in particular, differs markedly within a single bacterial species between the sparse planktonic populations that occur in the general water medium and the dense community of cells present in the microbial biofilm. The mechanism underlying this difference in gene expression is referred to as 'quorum sensing'

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and depends (in Gram-negative bacteria) on the release of the signal molecule acyl homoserine lactone (AHL) into the water medium. At low population density, release of AHL results in the formation of low signal concentrations in the water medium, and no quorum response. At high population density, the concentration of AHL in the water reaches a critical level, activating DNA transcription factors (by binding to them) and triggering the activation of AHL-responsive genes. These AHL-responsive genes have been shown to be present in a wide range of biofilm bacteria (including Pseudomonas aeruginosa) and to operate in environmental (stream) biofilms. They encode a variety of cell functions, including population density regulation, pseudomonad virulence factors and stationaryphase characteristics. The induction of stationary-phase physiology is an important feature of biofilms, distinguishing these organisms from their planktonic counterparts. In Pseudomonas aeruginosa, the stationary phase state is caused by induction of a stationary phase sigma (transcription) factor known as RpoS, which activates a wide spectrum of stationary phase characteristics. These include greater antibiotic resistance, decrease in protease secretion, reduced motility and higher levels of extracellular polysaccharide (EPS) synthesis. These changes promote the formation of the biofilm (EPS) matrix, and the retention of cells within the surface film (reduced motility), and are thus crucial in forming and maintaining the biofilm ecosystem.

5.4.1.3 Specific adhesion processes Specific adhesion mechanisms are important in the secondary colonization and maturation of bacterial biofilms. During biofilm development, different species of bacteria enter the community at different times, and there is a need for specific recognition systems to maintain the necessary sequence and hierarchy of association. This is achieved by specific co-aggregation, which involves receptormediated recognition and specific binding between different species of bacteria. 5.4.1.4 Trophic interactions Ingestion of biofilm bacteria, diatoms and blue-green algae is carried out by protozoa that move over exposed submerged surfaces. These protozoa include hypotrich ciliates, hypos tome ciliates (with ventral mouths) plus bodonid and euglenoid flagellates. Ingestion of biofilm microorganisms is the first stage in the sequence of biomass

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transfers that forms the food web of the biofilm ecosystem, but also connects with the food web of the wider lake or river ecosystem. In addition to protozoa, surface biofilms are also grazed by larger invertebrates such as snails and larvae of Ephemeroptera and Trichoptera. These larger organisms are extraneous to the biofilm community. 5.4.2 Biomass Formation and Transfer Relatively little is known about biomass formation and transfer in aquatic biofilms. The presence of autotrophic algae such as diaroms and blue-green algae generates fixed carbon by /' photosynthesis, and some release of dissolved organic carbon (DOC) would be expected. Biomass transfer occurs via ingestion of cellular and other particulate material by protozoa and invertebrates (see above), and also by assimilation of secreted organic material such as DOC and matrix by bacteria and fungi. Extraneous organic material may also enter biofilms and become part of the biomass transfer. 5.4.3 Maintenance of the Internal Environment The microbial biofilm is a mature film that has arisen from a sequence of colonization processes. This has resulted in a stable and balanced internal environment which has two major components: 1. The gelatinous matrix, secreted by the bacteria, and forming a complex architecture that includes internal spaces (pores and channels) with a water circulation connecting to the outside medium. 2. Populations of different microorganisms that are in a state of balanced equilibrium. Once the biofilm has become established, the internal environment will be maintained and controlled by the resident microorganisms. This involves the following processes. (a) Continued production of gelatinous matrix. Continued secretion of the extracellular polysaccharide (EPS) matrix by bacterial cells tends to balance the loss caused by detachment of pieces of biofilm at the water surface and entrainment in the current. (b) Controlled population growth. Growth rates of bacteria within the biofilm are controlled by the quorum sensing system that acts as a negative feedback process. High population densities trigger the induction of stationary phase characteristics, including reduced rates of cell division. If the population of bacteria

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becomes depleted (e.g., due to ingestion) the quorum control will cease to operate, and division will increase. (c) Balance of mixed populations. The balance of different organisms will be determined by differential growth rates, stable food webs and specific adhesion mechanisms. The latter is particularly important during biofilm development and maturation, when the changing pattern of species composition that accompanies the colonization process is determined by differential recognition and adhesion characteristics. These features may also control the entry of bacteria into the mature biofilm and their retention within the community. 5.4.4 Interactions with the Exter.nal Environment Although microbial biofilms operate as a functional (and to some extent self-contained) unit, they are not separate from either their physicochemical or biological surrounding environment. Important physicochemical characteristics include light (required for photosynthesis), inorganic nutrients, soluble organic nutrients and dissolved oxygen. The entry and exit of soluble components into and out of the biofilm is promoted by internal water circulation, and leads to concentration gradients within the gelatinous matrix. These concentration gradients may be important determinants of local biofilm physiology and microstructure. The potential importance of nutrients is indicated by laboratory studies on monospecies biofilms, which have suggested that biofilm structure is greatly influenced by the concentration and quality of nutrients. The external biotic environment also has direct influences on biofilms. These are exerted through invertebrate grazing activities, and also entry and loss of resident microbes at the water interface. The entry of particulate (by sedimentation) or soluble (water flow) organic materials into the biofilm is a further biotic effect that provides important substrates for heterotrophic nutrition. 5.5 PELAGIC ECOSYSTEM Pelagic ecosystems occupy the main water body of the lake and contain the largest subsidiary community within the main lake ecosystem, encompassing all the free floating (planktonic) and strongly swimming (nektonic) organisms. Pelagic ecosystems of lakes occupy the largest volume of all freshwater environments (excluding snow and ice systems), and show close similarities to the marine pelagic ecosystems of seas and oceans.

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5.5.1 Interactions between Organisms A range of defined interactions occur between microorganisms in pelagic lake ecosystems, including competition for resources, antagonism, trophic interactions and epiphytic associations. Competition for resources such as light and inorganic nutrients (silicate, phosphate, nitrate) are an important contributory factor in determining the relative ability of different species populations to grow and dominate the pelagic environment. In cases where competition for more than one nutrient is involved, a change in algal dominance may occur with shift in nutrient balance. Antagonistic interactions may also occur as part of the competition for resources, allowing the population of the successful antagonist to access the resources and also generating nutrients from the target organism. Freshwater bacteria in particular, which are known to produce a range of anti-algal metabolites, may be important as antagonists in the termination of algal blooms, and are also potentially useful as biological control agents. Dominance of bluegreen algae in bloom conditions may also be regarded as antagonistic activity, since the low CO/ high pH micro-environment created by these organisms inhibits the growth of eukaryote algae in the top part of the water column. Trophic interactions in the pelagic zone are often very specific, and may result in close coupling of particular microbial populations. This is the case for heterotrophic bacteria and phytoplankton, which are linked by algal DOC production. A trophic link also occurs between bacteria and predatory heterotrophic nanoflagellate (HNF) protozoa, with a buildup of HNF populations in some lakes at times of high bacterial count. Parasitic viral and fungal infections of bke bacteria and phytoplankton provide examples of highly spec ific microbial interactions and are ecologically important in limiting host population growth and productivity. Epiphytic associations are common in the pelagic environment, with many unicellular organisms (bacteria, protozoa and algae) occurring on (or within) the surface of larger biota such as colonial algae and zooplankton. With the exception of blue-green algal heterocyst bacteria, little is known about the possible exchange of nutrients between epiphyte and host. Whether this occurs or not, epiphytic sites such as colonial blue-green algae are trophically significant in providing microcosms of microbial activity, each with its own localized food ·'\·eb. Epiphytic sites also provide a point of

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attachment in a part of the lake that is otherwise devoid of substratum, and allow planktonic organisms the opportunity to exist in equilibrium with a sedentary (non-planktonic) phase.

5.5.1.1 Seasonal changes in a temperate lake The importance of microbial interactions in the pelagic ecosystem of temperate lakes is seen in the seasonal progression of biomass and populations that occurs during the growth season. This sequence is considered in the section on algae, since it is driven primarily by the response of phytoplankton populations to environmental alteration, and all other lake biota show correlated changes. The role of other lake biota within the seasonal transition is important and includes: 1. Control of phytoplankton spring and autumn blooms by fungal and viral epidemics; 2. Control of phytoplankton populations by protozoon and zooplankton grazing, with the occurrence of a clear-water pha50 per cent) of the more labile low molecular weight DOC compounds, particularly those of human origin. Studies on the elemental composition of DOC in relation to bioavailability have suggested that the atomic ratios of Hie and ole in DOC can be used to predict the ability of bacteria to degrade these compounds. The Hie ratio in particular is directly proportional to readily degradable aliphatic compounds and inversely proportional to less degradable aromatic material. Because of the variation in DOC bioavailability, the amount of DOC entering the system is not a direct measure of DOC supporting the microbial food web.

5. B.1.2 Particulate organic carbon Particulate organic matter (diameter >0.2 JLm) ranges from finely dispersed material (including bacteria) to large particulate matter such as leaf litter and other plant debris. Much of this material is directly deposited into the flowing water from surrounding vegetation. Large particulate matter such as leaf litter is an important carbon source for the microbial food web, serving as a substrate for fungal and bacterial growth and as a source of DOC. On entering a stream or river, leaf litter releases an initial pulse of rapidly leachable, water soluble material, followed by a slow release of DOC due to microbial degradation. The breakdown and release of organic material from leaf litter is accelerated by the feeding activities of invertebrates.

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5.8.1.3 Changes in the concentrations of dissolved and particulate carbon during the seasonal cycle Concentrations of dissolved and particulate carbon show wide fluctuations in many streams and rivers during the annual cycle, with recorded DOC values generally in the range 1-10 mg 1-1, but reaching much higher levels in some rivers. In most cases, this annual variation reflects the allochthonous derivation of these compounds and the seasonal fluctuations of entry into the river system. The large rivers of Southern Asia, for example, have large changes in DOC concentration, with close correlations between seasonal flow patterns and the timing of DOC maxima and minima. The dissolved organic carbon levels in the Indus and GangesBrahmaputra Rivers reach a maximum near the end of rising water levels, due to overflow and entrainment from highly productive flood plains. The pulse of DOC then rapidly declines as water levels recede due to mixing, metabolic removal, and dilution. In the upper Mississippi River (USA), the very high autumn allochthonous DOC levels are derived not from soil, but from leaching of leaf litter. Although many rivers show elevated allochthonous DOC concentrations at times of flood and terrestrial runoff, wide differences in seasonal patterns can occur. The Shetucket River (USA), for example, is unusual in showing minimal DOC levels during high inflow winter months, but maximum levels during the low-flow summer period. The summer maximum was attributed to the generation of autochthonous carbon by secretion and senescence of benthic algae. In winter, the allochthonous DOC input was overridden and diluted by high discharge due to ice melt and heavy rain. Seasonal patterns in some rivers are at least in part driven by in situ planktonic primary production. In the Gambia River (West Africa), allochthonous DOC concentrations reach a maximum at the time of maximum discharge into the system, but accumulation of autochthonous DOC occurs during the low-flow period. This was linked to phytoplankton production and was associated with . elevated river water pH levels. 5.8.2 Pelagic and Benthic Communities Lotic systems differ considerably in the extent to which pelagic and benthic communities are able to develop. This depends particularly on size and flow, with a major distinction between large, slow-flowing rivers and small, turbulent streams.

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5.8.2.1 Large rivers: the development of a phytoplankton community In'most lotic systems, phytoplankton are simply displaced by the current, and are not able to form standing populations. Because of this continuous displacement, net production within a defined section can only occur when local growth rates exceed downstream losses. Phytoplankton growth in lotic systems tends to be limited by ambient light intensity (overhanging foliage), turbidity, and circulation within the water column (no stratification), while downstream loss is largely a function of current velocity. Phytoplankton production in riverine systems, and the development of a pelagic community, is thus largely regulated by light availability in combination with hydrological processes. Recent studies by Sellers and Bukaveckas (2003) have demonstrated that phytoplankton production may be significant in large rivers. Local biomass accumulation occurs particularly in shallow reaches during peaks of low discharge and turbidity, when phytoplankton experiences prolonged exposure to favourable light conditions. Observations on a large navigational pool in the Ohio River (USA), for example, showed that at times of high discharge, phytoplankton productivity within the pool was < 10 per cent of phytoplankton input from upstream and tributary sources. At times of low discharge, phytoplankton production in the pool exceeded external algal sources.

5.8.2.2 Small rivers and streams: development of a benthic community Most lotic systems are dominated by benthic communities, with little development of pelagic food webs. Solid surfaces are rapidly colonized by microbial organisms, leading to the development of bacterial and algal biofilms. These permanent microbial communities are an important aspect of the lotic environment, with algal biofilms making a significant contribution to primary productivity and both algal and bacterial biofilms forming part of the ecosystem food web. Bacterial biofilms typically produce copious amounts of extracellular matrix, creating a distinctive micro-environment that limits the extent of the community.

5.8.2.2.1 Benthic bacteria Bacteria present on the surface, and in subsurface regions, of stream-bed sediments are involved in a number of key ecosystem

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processes - including the breakdown (mineralization) of organic matter, assimilation of inorganic nutrients, and acting as a food source for consumer organisms. While quantitative aspects of the supply of organic matter clearly influence the abundance and productivity of sediment bacteria, qualitative aspects are also important. These include delivery, particle size distribution, and chemical composition - all of which affect the spatial and temporal composition of bacterial communities. Chemical composition is particularly important, and recent studies by Findlay et al. (2003) have emphasized differences between labile (easily assimilable) and recalcitrant (poorly assimilable) carbon sources in promoting bacterial community responses such as oxygen consumption, productivity, extracellular enzyme activity, and community composition. The growth of heterotrophic bacterial populations in benthic environments relates to both productivity (availability of organic carbon, inorganic nutrients, terminal acceptors) and loss processes such as grazing and viral infection.

5.8.2.2.2 Ecological pyramids and food webs The comparatively low level of phytoplankton and zooplankton in lotic systems means that ecological pyramids from rivers and streams look very different from those of standing waters. Food webs of lotic systems are also very different. The grazing (phytoplankton, zooplankton, fish) food web that dominates lakes is of much reduced importance, and the low level of internal (autochthonous) DOC production by phytoplankton and other photosynthetic organisms means that the pelagic microbial loop recycling carbon released from phytoplankton back into the macrobiota - has little application. The metabolic coupling and correlation in populations between planktonic algae and bacteria seen in lakes is not a feature of lotic systems. 5.8.2.3 Microbial loop of lotic systems Although the pelagic microbial loop has little relevance in most lotic systems, the benthic microbial community is important in cycling externally derived DOC into multicellular organisms within the stream environment. This benthic microbial loop is initiated by the arrival of externally-derived carbon into the river system as dissolved organic carbon (DOC) and particulate organic matter. The latter is broken down by invertebrates (conversion of coarse to fine particulate matter) then digested by fungal and bacterial exoenzymes to form DOC, with subsequent uptake by these two groups of organisms. Protozoa

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are involved in the direct uptake of particulate material and the ingestion of bacteria. Comparison between pelagic and benthic microbial loops emphasizes a number of key differences in addition to the internal or external origins of the carbon supply. Non-microbial biota have a greater impact on the benthic microbial loop, carrying out the initial shredding or breakdown of the coarse particulate organic (CPOM) matter and generating fine particulate material ahd DOC which are ingested by microorganisms. Connections with the overall food web also differ, with relatively good linkage between benthic and planktonic biota in lotic systems, but fewer biomass transfers between microorganisms and top carnivores within the river ecosystem.

5.8.3 Microbial Food Web The microbial loop of flowing water is completed by the assimilation of bacterial carbon, which then passes into multicellular biota. Bacteria form the main microbial biomass and are present mainly within biofilms and associated with organic debris such as leaf litter. They are ingested by three main groups of benthic organisms protozoa, insect larvae, and meiofauna. These differ in the efficiency with which they ingest bacteria, as determined by bacterial carbon uptake per unit weight of the organism.

5.8.3.1 Protozoa (flagellates and ciliates) This group of unicellular organisms are important grazers of biofilms. They have significant impact on bacterial populations, with assimilation rates of the order of 10- 1 to 10-2 p.g bacterial carbon 111g-1 protozoon biomass d- I •

5.8.3.2 Insect larvae Aquatic insect larvae ingest bacteria that are present mainly on organic debris such as leaf litter. They are both predators and competitors of microbial organisms, directly consuming bacteria and fungi and also digesting the organic detritus that is an important basis for the bacterial food chain. Some insect larvae (e.g., stoneflies and craneflies) ingest bacteria simply as part of the litter that they collect, resulting in relatively low bacterial assimilation rates (about 10-4 p.g mg- I d- I ). Other larvae have specialized bacterial collection strategies, including filtration (black fly) and surface scraping (mayfly), resulting in much higher bacterial assimilation rates.

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5.8.3.3 Meiofauna These are animals inhabiting the bottom of a lake or river that are just visible to the namost important ked eye and include copepods, nematodes and rotifers. These are generally regarded as the most important bacterial predators in lotic systems, and include both filter feeders and biofilm grazers. Their bacterial carbon assimilation rates are typically 1-4 orders of magnitude greater than that of other bacterial consumers. The microbial loop in running waters is part of a more complex food web that also involves photosynthetic carbon production by algae and higher plants, saprophytic and parasitic activities of fungi and viruses and important roles for the large invertebrate and vertebrate predators. Primary production by benthic algae (periphyton) varies in importance in different systems, depending partly on the depth of the water column and light penetration to the substratum. Light is also important in the water column in relation to degradation of non-assimilable (refractory) DOC. UV-irradiation, in particular, has been shown to have a major effect in converting humic acids to more labile forms of DOC. The ecological importance of bacteria, protozoa, and fungi in lotic food webs has been mentioned in relation to the microbial loop. Further information is subsequently given in relation to the breakdown of organic matter by benthic bacteria, saprophytic activities of benthic fungi, and the role of protozoa in the ingestion of both living and non-living particulate matter. The transfer of biomass from microbes to top carnivores does not have such a defined route as pelagic food-webs, and the domination of herbivorous activities in the water column by crustaceans (zooplankton) does not occur in the lotic food web. Herbivory in running waters occurs mainly at the sediment surface and involves the ingestion of either unattached or attached microorganisms. The consumption of microorganisms in lotic communities involves ingestion of: 1. Free-moving biota such as bacteria and protozoa, present as localized populations around organic debris, by meiofauna and protozoa; 2. Microorganisms, particularly fungi and bacteria, that are present within organic debris such as leaf litter - this is carried out by

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invertebrate shredders, gougers, and collector gatherers, prominent amongst which are insect larvae; 3. Biofilm and other microorganisms that are attached to solid substratum - this is carried out by shredding, scraping and rasping. The fragmentation and ingestion of leaf litter by invertebrates requires partial breakdown of this material by fungal activity. The substrate colonization, invasion, and macerating activity of these organisms thus promotes their own ingestion during the final stages of leaf processing.

6 Light as Abiotic Factor Sunlight (solar radiation) is important to aquatic microorganisms in four major and interrelated ways. 1. Determination of their physico-chemical environment. Light is a major determinant of the dynamics and structure of aquatic environments. Energy from the sun provides the heat that generates the Earth's wind patterns, resulting in mixing of the surface layers of lakes and oceans. Transformation of light to heat energy within surface waters results in a localized temperature increase which combines with wind energy to produce thermal stratification. This in turn limits the distribution of nutrients within the water column and thus has a secondary effect on aquatic chemistry. Light also has direct effects on chemical characteristics and is important, for example, in the degradation of humic acids in peaty lakes to more biologically available compounds. 2. Production of biomass. Light penetration of surface waters drives the photosynthetic activity of the primary producers. These include both planktonic and benthic organisms, and comprise three major groups - higher plants, algae, and photosynthetic bacteria. The photosynthetic reducing power which is generated by light is important for both carbon (C0 2 ) and nitrogen (NO" N0 2 , NH 4 ) assimilation. Nitrogen fixation (in colonial blue-green algae) and phosphorus metabolism are also closely linked to photosynthesis. but the cellular energy required for uptake and deposition of silicon in diatoms is derived solely from aerobic respiration without any involvement of photosynthetiC energy. 222

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3. Damage to cell processes. In extreme conditions, where general light intensity is high, or where there is a high level of ultraviolet radiation, damage to cell metabolic and genetic processes (photoinhibition) can seriously impair biological activities. 4. Induction of periodic seasonal and diurnal activities. Temporal changes in the occurrence and activities of freshwater biota are considerably influenced by the periodicity of light. This affects both seasonal activities (where day length is important) and diurnal activities (where alternation of light and dark entrains circadian rhythms). This chapter initially considers aspects of light intensity and wavelength within aquatic systems. The major part of the chapter then deals with light as a resource for the production of biomass, followed by a consideration of the damaging effects of light and the importance of light periodicity.

6.1 LIGHT ENVIRONMENT 6.1.1 Physical Properties of Li&.ht: Terms and Ur-its of Measurement Solar radiation can be considered either as a continuous wave of energy or as discrete packets (photons) of excitation. These two concepts give rise to two fundamental measurements of light: 1. Wavelength, which describes the quality of light and the type of effect it will have on living organisms, and 2. Photon intensity, which describes the quantity of light to which freshwater microorganisms are exposed. Solar energy has a wavelength range of about 100-3000 nm, with most of the energy being present between 300-2000 nm. The spectral range can be separated into three main regions - ultraviolet radiation (100--400 nm), visible light (approximately 400-700 nm), and infrared radiation (700-300 nm). These three groups comprise 3 per cent, 46 per cent and 51 per cent respectively of solar radiation arriving at the Earth's outer atmosphere. The visible radiation also coincidentally corresponds to the 'photosynthetically available radiation' (PAR) which drives the photosynthetic activities of most phototrophic organisms (algae and higher plants). Infrared radiation appears to have little specific metabolic effect on living organisms, although photosynthetic bacteria do absorb some of it for photosynthesis. Ultraviolet light is of major importance in causing damage to microorganisms.

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The measurement of light is based on the physical properties of electromagnetic radiation and is carried out using a 'quantum sensor'. Numbers of photons are usually expressed directly in moles, where 6:02 x 1023 photons equal 1 mol. Commonly used terms and units for different light parameters and include the number of incident photons (photon flux, photon flux density), energy content (irradiance), and wavelength (range of wavelength). Light intensity in both environmental and laboratory systems is typically expressed as photon flux density (PFD) and is an important parameter is assessing the light-response of living organisms in relation to photosynthesis, growth, and photoinhibition. The terms 'photosynthetic PFD' and 'photosynthetic irradiance' are also sometimes used to denote the amount of incident light within the restricted range of photosynthetically active radiation. 6.1.2 Light Thresholds for Biological Activities In the freshwater environment, photon flux density varies with time (seasonal, diurnal), ~ter quality (content of dissolved substances, presence of biota), and depth in the water column. Light intensity is quantitatively important for the photosynthesis and growth of autotrophic microorganisms, and is also qualitatively important in determining the presence or absence of particular biological activities. Threshold levels of light are particularly relevant to the activities of photosynthetic organisms. At the top end of the PFD scale, these include light saturation of photosynthesis in periphyton and phytoplankton. With decreasing light intensity levels, a sequence of thresholds operate for the growth and survival of macrophytes, algae, and photosynthetic bacteria.

6.1.2.1 Macrophytes The threshold PFD level for macrophyte survival (45-90 Ilmol III 1 S-I) is ecologically important and is directly linked to microbial activity, since in eutrophic waters shading by algal blooms reduces PFD below the critical level and causes eradication of the macrophyte flora. 6.1.2.2 Algae In temperate and polar lakes, critical levels of light intensity are required at the beginning of the growing season to trigger the onset of the spring diatom bloom. Studies on laboratory cultures indicate the onset of net photosynthesis and growth at PFD values of 0.1--1 Ilmol mIs-I.

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6.1.2.3 Photosynthetic bacteria These organisms are restricted to low-light anaerobic regions of the water column, and the onset of growth is triggered by very low-light levels « 1 /-Lmol m- I S-I). In addition to the direct effects of light on photosynthetic microorganisms, indirect effects on freshwater microbes also occur via the influence of light on zooplankton and fish. The onset of maximal vertical migration of Daphnia hyalinarequires threshold light levels of 10-3 /-Lmol m- I sI, and has implications for the grazing of microorganisms in surface waters. This light level is equivalent to the irradiance from a full moon, which also allows fish such as perch to feed visually on zooplankton in surface waters at night - indirectly influencing populations of algae and other microorganisms via the food web.

6.1.3 Light Under Water: Refraction, Absorption, and Scattering Both the overall intensity of light and the sp~ctral composition are modified by its passage through water, largely due the effects of refraction, absorption, and scattering. Refraction of light, with separation into different wavelengths, occurs as the radiation passes through regions of different refractive index - including the air/ water interface and strata of different density in the water column. Absorption (conversion of light to heat energy) and scattering (reflection) are mediated by water molecules, dissolved substances, and particulate matter.

6.1.3.1 Decrease in light intensity The intensity of light at the surface of a body of water does not normally exceed 2000 -mol photons m-2 S-I, equivalent to about 400 Wm-2 in energy units. Loss of light due to absorption and scattering results in an exponential decrease in intensity with depth. The fraction of light lost per metre of water is expressed mathematically as the extinction coefficient (eJ. For parallel beams of monochromatic (single wavelength) light, the intensity of light at a particular depth in the water column is given by the LambertBeer law, which can be expressed as: '" (1) I Z -- I oe -E).Z where: I z = light intensity at depth z (mol photon m- 2 S-I), 10 = light intensity penetrating the water surface (mol photon m- 2 S-I), e; = extinction coefficient for the particular wavelength (m- I ), and z = depth (m).

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The extinction coefficient (c) is very wavelength-dependent and can be separated into separate components which relate to light absorption by water molecules (cJ, dissolved substances (cd) and particulate matter (E/ For pure water, where Ed and c p are zero, EA = cw .

6.1.3.1.1 Light zonation of water column The top part of the water column has a distinct zonation in relation to light availability. The depth at which light intensity reaches the point where 02 evolution by photosynthesis equals 02 uptake by respiration is referred to as the compensation point. Light intensity at the compensation point, le' varies with environmental (e.g., temperature) and physiological characteristics, but is normally about 0.1-1 per cent of the value at the lake surface. The vertical distance between the water surface and compensation point is the photic zone, and is the region within which net anabolic (synthetic) processes occur in phototrophic organisms. Below this, in the aphotic zone, respiration exceeds photosynthesis and metabolism is predominantly katabolic. The depth of the photic zone varies widely between water bodies. In eutrophic lakes, light attenuation with depth is pronounced due to high phytoplankton biomass and the photic zone does not extend Incident light 10 (per cent)

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