Diss. ETH No. 11905

THE PHYSIOLOGY OF A DEFINED FOUR-MEMBERED MIXED BACTERIAL CULTURE DURING CONTINUOUS CULTIVATION WITH MIXTURES OF THREE POLLUTANTS IN SYNTHETIC SEWAGE

A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH for the degree of Doctor of Natural Sciences

Presented by ALBERT JUNG TIEN M.S. (Life Sciences) New Mexico Highlands University, USA born 1 August, 1965 citizen of the United States of America

accepted on the recommendation of Prof. Dr. Th. Leisinger, examiner Prof. Dr. P. Peringer, co-examiner PD. Dr. Th. Egli, co-examiner TABLE OF CONTENTS

Summary ...... , ...... 1

Zusammenfassung •...•....••.....•.•••.•.•..••.•...... ••.• 4

1 . Introduction ...... 8

2 . Regulation of pollutant degradation in the presence of an alternative substrate by the para-toluenesulfonate utilizing Comamonas testosteroni strain T-2 and the dichloromethane utilizing Methylobacterium strain DM4 during growth in batch and continuous culture ..••••. 31

3. Growth of Methylobacterium strain DM4 in continuous culture: Effects of dilution rate and response to substrate shifts during continuous cultivation ...... 5 8

4. Dynamics of substrate consumption and enzyme activity by Comamonas testosteroni strain T-2 in response to carbon substrate shifts in carbon-limited continuous cultures ...... •.....•...... •....•...... ••...•..• 82

5 . Dynamics and regulation of mixed pollutant degradation by a four-membered defined bacterial consortium in continuous culture•.•.•...•..•...... •••.•....•...• 101

6. Conclusions ...... 126

7. Appendix 1) Combined ozonation and biological treatment for the removal of a model refractory compound, toluenesulfonate, from simulated industrial wastewater ...... 139

2) Microbially mediated formation of struvite by Methylobacterium strain DM4: Biotechnological

applications ...... 1' ••••••• 143

3) Microbial mediation as a possible mechanism for natural dolomite formation at low temperature ...... 16 6 I Summary

SUMMARY

The influence of mixed substrate utiHzation on the growth and survival of three pollutant utilizing microorganisms: Comamonas testosteroni strain T-2, a toluenesulfonate (TS) degrader; Methylobacterium strain DM4, a dichloromethane (DCM) degrader; Chelatobacter heintzii strain 29600, a nitrilotriacetate (NT A) degrader; and the non-pollutant degrading Escherichia coli strain ML30; was investigated using pure cultures and a defined mixed culture composed of the four bacterial strains under both batch and continuous cultivation conditions. The purpose of these investigations was to determine the following: 1) Does the presence of easily utilizable substrates inhibit the utilization of a pollutant as a carbon/energy source? 2) Do common trel1lds exist concerning mixed substrate utilization (pollutant and easily utilizable carbon substrates) in evolutionarily distinct microbial species which utilize structurally different chemical pollutants? 3) Does the ability to simultaneously utilize pollutant and easily utilizable substrates give these organisms a competitive advantage over other microorganisms found. in the environment? 4) Can patterns of regulation, especially with respect to the degradation of pollutants in the presence of other easily utilizable substrates be extrapolated to more complex systems; i.e. Is what we observe in pure culture (mixed substrate/pollutant utilization) also seen in nature? In batch culture, C. testosteroni strain T-2 utilized acetate and TS simultaneously. For Methylobacterium strain DM4, substrate utilization patterns were highly dependent on precultivation conditions. Whereas DCM pregrown cultures of Methylobacterium strain DM4 were able to simultaneously utilize acetate and DCM, acetate precultivated cultures of strain DM4 exhibited diauxic growth with acetate utilized first. 2 Summary

Simultaneous utilization of both pollutant and altemati ve substrate was always demonstrated during carbon-limited growth in continuous cultures. In both strains induction of the pollutant degrading systems occurred when the pollutant contributed to approximately 1% of the total carbon supplied in the feed. Maximal induction levels were achieved when the pollutant contributed to approximately 3% in the case of C. testosteroni strain T-2, and 5% in the case of Methylobacterium strain DM4. When steady-state cultures (D=0.05 hr-1) experienced shifts from acetate to DCM in the feed, a two stage induction pattern was observed (as demonstrated by excess substrate specific oxygen uptake measurements) with approximately 30 hrs elapsing before maximal induction was acheived (twenty fold over constitutive level). Levels of DCM dehalogenase protein (quantified by Western blotting) paralleled excess substrate specific oxygen uptake rates. A similar induction pattern as that seen during a transient from acetate to DCM was observed when Methylobacterium strain DM4 cultivated with synthetic sewage, was shifted to a medium consisting of synthetic sewage containing 10% DCM. In a continuous culture of C. testosteroni strain T-2, replacing acetate with TS in the feed, a sigmoidal induction pattern in pollutant degrading activity was seen. Similar induction patterns were observed when acetate was replaced with 10% TS/90% acetate and also 40% TS/60% acetate. As with the case of Methylobacterium strain DM4, simultaneous utilization of alternative substrates by C. testosteroni strain T-2 increased the rate of new enzyme synthesis. During batch growth with the four strains in a synthetic sewage containing the specific pollutants, E. coli strain ML30 outnumbered its nearest competitor, C. testosteroni strain T-2, approximately 2:1. Methylobacterium strain DM4 and C. heintzii strain 29600 were present 3 Summary

at approximately half the levels as C. testosteroni strain T-2. In continuous culture (D=0.05 hr-1) coexistance of the four strains was achieved (55%£. coli strain ML30, 15% C. testosteroni strain T-2, 15% Methylobacterium strain DM4 and 15% C. heintzii strain 29600 ). When NTA, TS or DCM was omitted from the feed, total cell counts were only slightly lower and no significant changes in the ratio of individual str,ains were measured. When NT A, TS or DCM was replaced in the feed, induction of the specific pollutant degrading system, as monitored by increased excess substrate specific oxygen uptake rates, were observed after a lag period. No enrichment in the population of the specific pollutant degrading strain was observed. The stability of our model system can be attributed to the ability of the pollutant degrading strains to utilize not only their specific pollutants but also synthetic sewage (mixed substrate utilization) under the carbon-limited conditions imposed on the four microorganisms. Data indicate that in our case, induction of the necessary enzyme systems and not enrichment of the pollutant degrading strains is the primary mechanism that leads to pollutant removal, especially when the ratio of pollutant to available carbon is low.

-----·------r------r---· 4 Zusammenfassung

ZUSAMMENFASSUNG

Der Einfluss der Verwertung von Gemischen von Kohlenstoffsubstraten auf das Wachtum und Uberleben der drei schadstoffabbauenden Mikroorganismen [Coma.monas testosteroni Stamm T-2, p-Toluolsufonat (TS) abbauendes Bakterium; Methylobacterium Stamm DM4, baut Dichlormethan (DCM) ab; Chelatobacter heintzii Stamm 29600, verwertet Nitrilotriacetat (NTA)] und Escherichia coli Stamm ML30 (vermag keinen der drei Schadstoffe abzubauen) wurde in Batch und kontinuierlicher Kultur sowohl an Reinkulturen, als auch in definierten · Mischkulturen untersucht. Das Ziel dieser Untersuchungen war es, die folgenden Fragen zu beantworten: 1) Hemmt oder verhindert die Anwesenheit leicht verwertbarer Kohlenstoffsubstrate den Abbau der Schadstoffe? 2) Gibt es fiir entfernt verwandte Bakterienstiimrne gememsame (generelle) Verhaltensmuster fiir den Abbau von Mischsubstraten (bier leicht verwertbare Kohlenstoffquellen gemeinsam mit den drei strukturell sehr unterschiedlichen Schadstoffen)? 3) Verleiht die Fahigkeit Gemische von Kohlenstoffquellen gleichzeitig zu verwerten (Mischsubstratwachstum) einem Bakterium in der Umwelt gegeniiber anderen Bakterienstammen einen Vorteil im Wettbewerb um Nahrung? 4) Konnen Verhaltensmuster beziiglich der gleichzeitigen Verwertung von leicht abbaubaren Kohlenstoffquellen und Schadstoffen, welche in Reinkulturen im Labor beobachtet werden, auf die komplexen Umwelt- oder Klaranlagenbedingungen iibertragen werden? 5 Zusammenfassung

Reinkulturen von C. testosteroni Stamm T-2 verwerteten Acetat und TS in Batchkultur immer simultan. Filr Methylobacterium Stamm DM4 war das Verwertungsmuster von den Vorkulturbedingungen abhangig: Wahrend DCM vorgewachsene Zellen Acetat und DCM gleichzeitig verwerteten, zeigten Acetat vorgewachsene Kulturen eine klare Diauxie mit Praferenz fiir Acetat. In kohlenstofflimitierter kontinuierlicher Kultur (D=0.05 hr-1) verwerteten beide Stamme Acetat zusammen mit TS, respektive DCM, unabhangig vom angebotenen Mischungsverhfiltnis der beiden Kohlenstoffquellen. Bei beiden Stammen wurden die am Schadstoffabbau beteiligten Enzyme deutlich induziert, wenn der Anteil des Schadstoffs am gesamten im Medium angebotenen Kohlenstoffs 1 % ilberschritt. Maximale Induktion der Enzyme wurden im Fall von C. testosteroni Stamm T-2 erreicht wenn die angebotenen Mischungen 3% und mehr TS enthielten, im Fall von Methylobacterium Stamm DM4 waren 5% DCM- Kohlenstoff ausreichend fiir eine maximale Induktion. Wenn im zugefiihrten Medium einer C-limitierten Chemostatkultur von Methylobacterium Stamm DM4, welche bei einer Verdilnnungsrate von 0.05 hr-1 mit Acetat als einziger C-Quelle wuchs, Acetat durch DCM ersetzt wurde, so beobachtete man ein zweistufiges Induktionsmuster. Es brauchte etwa 30 Stunden bis die maximale spezifische Aktivitat der DCM- Dehalogenase (zwanzigfach erhoht gegeniiber dem konstitutiven Niveau beim Wachtum mit Acetat) erreicht wurde. Die Menge des vorhandenen DCM-Abbauproteins, quantifiziert durch Western-Blotting, folgte dabei dem Verlauf der spezifischen Aktivitat des Enzyms. Ein vergleichbares 6 Zusammenfassung

Induktionmuster wurde bei emem Wechsel vom Wachtum mit synthetischem Abwasser auf ein Medium mit synthetischem Abwasser plus 10% DCM beobachtet. Beim C-limitierten Wachstum von C. testosteroni Stamm T-2 im Chemostaten (D=0.05 hr-1) wurde bei einem Wechsel von Acetat als einziger C-Quelle zu TS als alleinige C-Quelle ein sigmoides Induktionsmuster der TS-Abbauenzyme beobachtet. In vergleichbruren Experimenten wurde dieses Induktionsmuster auch beobachtet wenn im zufliessenden Medium Acetat

testosteroni Stamm T-2, 15% Methylobacterium Stamm DM4 and 15% C. heintzii Stamm 29600. Wurde NTA, TS oder DCM im zufliessenden Medium weggelassen, so nahm die Gesamtzellzahl leicht ab, die Zusammensetzung der Mischkultur anderte sich jedoch nicht signifikant. Die Schadstoffabbaukapazirat der Kultur reduzierte sich aber auf einen konstitutives niedriges Niveau (gemessen iiber Sauerstoffaufnahmerate in Gegenwart eines Uberschusses der Schadstoffe). Wurden die spezifischen Schadstoffe dem Medium wieder zugefiigt, so beobachtete man nach einer lag-Zeit eine Induktion der Schadstoffabbaukapzitat der Kultur. Wie beim W eglassen des Schadstoffs beobachtete man auch hier keine signifikante Anderung der Zusammensetzung der Mischkultur. Die Stabilirat der Mischkultur unter diesen Bedingungen kann damit erkart werden, dass die einzelnen Bakteriensramme nicht nur auf ihrem spezifischen Schadstoff, sondern gleichzeitig immer auch mit Kohlenstoffverbindungen aus dem synthetischen Abwasser wuchsen. Die Ergebnisse unser Untersuchungen lassen den Schluss zu, dass die Abbauleistung fiir die hier untersuchten Schadstoffe in Okosystemen vielfach iiber die Induktion der Abbauenzyme und weniger Uber die Anreicherung der Abbauorganismen abHiuft.

------Chapter 1 8 Introduction

1. INTRODUCTION

1.1 The carbon cycle, microbes, and pollutants

It is estimated that the earth's atmosphere contains 6.4x1017 g of carbon as C02 (16). Per annum, approximately 78% (5xI017 g) of this carbon is assimilated into biomass by autotrophic organisms (27). In the absence of human interference, homeostasis is assumed to operate regarding the transfer of carbon among the carbon reserv01rs representing living and dead organic matter and the atmosphere (15, 30). Heterotrophic microorganisms, by mineralizing detritus derived from primary biomass, serve as a crucial link in global carbon cycling (17). Human activities have recently introduced changes in the cycling and accumulation of carbon as C02 that are large enough to be measured (30). The flux of synthetically produced. organic materials m industrialized countries has increased over the last two centuries to approximately 40 g of carbon per m2 per annum (12). In these ecosystems pollutant carbon contributes to a significant proportion of the available carbon pool making the ability of heterotrophic microorganisms to mineralize these compounds all that the more important.

1.2. Natural environments and carbon-limitation

Natural environments are oligotrophic

Although ecosystems have differing levels of orgamc matter, carbon availability is the limiting factor for the growth of heterotrophic bacteria in most natural environments (12). For example, average Chapter I 9 Introduction

concentrations of organic carbon in the environment range from 25 mg organic carbon per gram loamy soil to 10 mg organic carbon per liter for aquatic systems (41). However, most of this organic carbon cannot be utilized directly by heterotrophic microorganisms and despite often high concentrations of organic matter in aerobic aquatic ecosystems, sediments and soils, dissolution and decomposition of rather recalcitrant particulate and polymeric organic materials limits the level of available carbon (17, 32). Therefore, most ecosystems are low in microbiologically utilizable carbon compounds, i.e. they are oligotrophic, due to a balance between uptake and production of utilizable carbon. Addition of readily utilizable forms of organic matter to such environments, as demonstrated ll>y nutrient pulse experiments, results in the rapid decomposition of this material, given favorable nutritional conditions.

Carbon I energy limitation in the laboratory

Elemental assays of different microorganisms demonstrate that varying growth environment and the state of growth with respect to the available nutrients have profound effects on microbial biochemical composition (4, 29, 42). Nutrient limitation such as carbon-limitation can be experimentally studied in the laboratory using two differing approaches. The first method is based upon stoichiometric nutrient limitations where exponentially grown cells grown from batch culture are transferred to a starvation medium where a certain nutrient, i.e. carbon, has been omitted (41 ). The second approach, based upon kinetic limitation, is that of continuous cultivation where growth rates below µmax can be controlled and maintained by adjusting the dilution rate of inflowing fresh medium where a particular substrate is present in limiting concentrations (28, 47, 48). Chapter 1 JO Introduction

1.3. Starvation and mixed substrates

As indicated above, two methods exist for studying carbon- limitation in the laboratory, however, none are able to exactly reproduce the conditions found in the environment. The typical lifestyle for heterotrophic microorganisms in the environment 1s that of starvation/survival (41). Starvation, as applied in this context, refers to a state of slow or arrested growth as observed in nature, where cells are physiologically active and in turn have been adapted to the low nutrient fluxes present. In the laboratory, slow growth in carbon-limited continuous culture best mimics the growth conditions bacteria encounter in natural environments (40).

The hydrolysis of particulate orgamc matter results in tllle production of a complex mixture of organic compounds (carbon pool), all of which are found at extremely low concentrations. Under these conditions it seems logical that microorganisms do not rely on a single carbon compound but rather utilize as much of the available carbon compounds for energy as possible. Indeed, all the available data indicate that the simultaneous utilization of multiple carbonaceous substrates is the norm (14, 26). This is in contrast to the generally accepted patterns of diauxic/sequential carbon substrate utilization found in many textbooks [e.g. Stanier et al., (46); Brock and Madigan, (10); Schlegel, (45)]. Therefore, it is believed that diauxic growth does not occur in nature (12). For a more detailed compilation of mixed substrate utilization in the laboratory by batch and continuous culture~, please refer to the review by Egli (12). Chapter 1 11 Introduction

1.4. Harnessing heterotrophic microorganisms: wastewater treatment

The unconscionable discharge of pollutants into aquatic systems results in two extreme forms of ecological impact; (a) eutrophication due to increased phosphates, nitrates or other nutrients; or (b) dead or dying ecosystems-characterized by a lack of species diversity, due to the presence of toxic compounds. Cumulative effects of the introduction of chemicals, chronic and synergistic - especially refractory compounds - into the environment in large quantities has unknown ecological ramifications. In order to reduce such environmental impacts, treatment of wastewaters are recommended (25). One of the oldest yet most robust methods is that of using activated sludges (3). Quite simply, wastewaters are fed to an undefined, sometimes immobilized, partially recirculated consortium of mainly heterotrophic microorganisms which remove much of the organic matter by mineralization and incorporation as biomass. Wastewater treatment is not "rocket science" but rather the intensification, enhancement, and concentration of the natural process of heterotrophic growth; first carbon removal was adopted, then nitrogen removal (nitrification/denitrification) and later phosphorous removal were also introduced in wastewater treatment processes. When compared to nature, activated sludge treatment systems can handle approximately 100 to 1000 times the amount of organic matter normally degraded in terrestrial or aquatic ecosystems (35).

Pathogens to pollutants

Initially wastewater treatment by activated sludges were intended to stem the transmission of pathogenic microorganisms (e.g. cholera) and Chapter 1 12 Introduction parasites by reducing the organic content and other nutrients (N, P) found in domestic wastewater which contributed to the growth and transmission of disease producing microorganisms (8). Presently, in addition to the removal of bulk carbon, wastewater treatment facilities serve as the frontline defense in the battle to removal/reduce toxic or carcinogenic compounds before discharge into the environment. Moreover, these facilities are under attack from increasing spectrum and levels of toxic compounds now found in domestic wastes, industrial wastes and also from combined sewer systems {34, 35, 50).

1.5. Understanding the "black box": community structure and function

Wastewater engmeers still treat activated sludge systems, the backbone of wastewater treatment facilities, as "black boxes 11 where their only concern is process engineering {e.g. chemical oxygen demand (COD), biological oxygen demand, nitrification/denitrification, phosphate removal). Little attention has been given to the individual groups of organisms which contribute to the collective removal of pollutants such as detergents and other organic compounds which increasingly make up a larger proportion of COD. Consequently, our current microbiological knowledge of community structure/function relationships of activated sludge systems are very limited. Much of our understanding of the degradative abilities of pollutant utilizing heterotrophic microorganisms isolated from activated sludges has been obtained in the laboratory with pure cultures under batch conditions. However, m nature, microorganisms are rarely exposed to pollutants as sole carbon sources or at the high concentrations used in most laboratory studies {49) and as a Chapter 1 13 Introduction result of this, it is not certain that the most frequently isolated strains are the most prevalent (52) or if isolated pollutant degrading strains are responsible for the bulk pollutant removal in a particular environment (6).

Autecological Methods

In 1901, Beijerinck (7) wrote in a paper concerning urea utilizing bacteria that, "Everything is everywhere, the environment only selects." This statement is the basis of microbial ecology and has evolutionary implications. If one holds this statement to be true, then tho$e microorganisms which are found in nature are highly adapted to their particular niche and the reasons for the adaptation and subsequent survival in such environments can be found in the genetic make-up and the physiological functions of these microorganisms (9). Although Beijerinck's statement was made nearly a century ago, its implication still ring true today and are the foundations of the present approaches in the study of individual species or guilds of microorganisms in the environment. This type of study is often referred to as autecological studies. For much of the last century, the approach for autecological studies has been an emphasis towards enrichment culture, selective plating, and morphological differences. However, the present emphasis is that of using probes based upon unique nucleic acid sequences to determine community structure and function without the biases seen with enrichment culture [e.g., DeLong et al. (11); Ward et al. (52), Amman et al. (1, 2). This emphasis on purely molecular methods for the understanding of community structure/function lacks one important aspect for autecological studies; that is, the physiology of the microorganisms in question. Hobbie (31) stated that "In order to understand what microbes Chapter 1 14 Introduction

are actually doing in nature, as opposed to what microbes are capable of doing in the laboratory, ecologists must make measurements of rates of microbial processes in the real world. Mills and Bell (39), in their classic review of autecological approaches, advocated the use of combining measurements of physiological activity with methods of direct microbial determination such as fluorescent antibodies for a better understanding of community structure/function. Unfortunately, the paucity of knowledge of the exact physicochemical conditions, the precise identity and number of available growth substrates and the influence of the numerous other microorganisms present in the same habitat make the study of bacteria in their natural habitats quite difficult. The use of the chemostat with two-, three- (or even more) membered mixed cultures under precisely defined growth conditions may be used to elucidate the basic principles governing natural ecosystems (22).

Competition and coexistence

As discussed previously, use of continuous cultures for ecological studies has many advantages. Since activated sludges are composed of numerous species of microorganisms, understanding how competition for natural resources affects the coexistence of important pollutant degrading strains is a crucial part of understanding how to optimize pollutant . removal in wastewater treatment facilities. In a review by Gottschal (21) aspects of interspecies microbial competition using continuous culture are discussed. The results of competition experiments found in the literature provide support for the conclusion that complete competitors cannot coexist. However, under conditions of multiple substrate limitation and discontinuous or alternating supply of nutrients, coexistence of species has been frequently observed (19, 20, 23; 24, 52, 53). Very little is known of Chapter 1 15 Introduction the enzymological and the general physiological state of the individual populations or cells within competing mixed cultures. Furthermore, it is not clear how the presence of one competing population affects the regulation of enzymes and nutrient uptake systems in the other population. Application of autecological methods along with defined mixed cultures studies may help answer some of these questions.

1. 6 Aim of thesis

In order to study how the utilization of mixed substrates influence the survival of pollutant degrading microorganisms in nature, it was decided to first develop a less complex model system where autoecological methods could be developed, tested and later applied to more complex systems. Fortunately, over the last ten years the degradative pathways and in some cases, the genetic regulations of the three pollutant degrading isolates (Methylobacterium strain DM4, Comamonas testosteroni strain T- 2, and Chelatobacter heintzii strain ATCC 29600), have been studied in the laboratories of Th. Leisinger (36), A. M. Cook (38, 44) and Th. Egli (13). Since the genes and enzymes for many of these organisms were already known, construction of gene probes and antibodies against key enzymes could be developed. Furthermore, species specific 16s rRN A probes and, as a backup, specific surface antibodies could be produced to track individual populations. Therefore a nearly ideal model system could constructed were individual microbial populations, presence of degradative pathways, key enzyme activity and quantity could be monitored. Chapter 1 16 Introduction

Microorganisms and degradative pathways

The mam uses for dichloromethane (DCM) are as solvents or extractants. However, this compound is often found as a pollutant in aquifers, surface waters and soils (43). Methylobacterium strain DM4 (18) is a pink pigmented, aerobic, Gram-negative, facultative serine pathway methylotroph from the a-subgroup of the Proteobacteria, which is able to utilize DCM as a sole carbon and energy source. The first step of DCM utilization is catalyzed by a glutathione-dependent DCM dehalogenase with the formation of formaldehyde, a central metabolite of methylotrophic organisms (Figure 1). For more information on the microorganisms, enzymes and genetic regulation of DCM degradation, please refer to Leisinger et al. (36). Toluenesulfonate (TS) is typically used as a cloudpoint depressant in many liquid detergents and is the least substituted member of a group of compounds collectively known as linear alkylbenzenesulfonates which are used as surfactants. Comamonas testosteroni strain T-2 (DSM 6577) is a tan colored, aerobic, Gram-negative microorganism from the ~-subgroup of the Proteobacteria, which is able to utilize TS as a sole source of carbon and energy. Degradation of TS proceeds by the NADH dependent TS monooxygenase where it is converted into the alcohol. After many steps, the compound is finally converted to protocatechuate where it enters the central metabolic pathways (Figure 2). For more information on metabolic pathways and genetic regulation of TS degradation refer to Locher et al. (38) and Schlafli Oppenberg et al. (44 ). Nitrilotriacetate (NTA) is a chelating agent which has been used for a wide range of applications such as to replace pentasodiumtriphosphate in household detergents or for the decontamination of nuclear materials [For more information please refer to Egli, (13)]. Chelatobacter heintzii strain Chapter 1 17 Introduction

ICH2Cl2I GSH -i ©I Dicbloromethane dehydrogenase [GS-CHz Cl] + HCl H2 0~

(C~) S [GS-Cf1i OH] + HCI CH30H CH3Nf1i 2 ~ii/ GSH r;:;;:;;;:i ~ >-Assimilation !®I Formaldehyde dehydrogenase I HCOOH +@ j Fonnate dehydrogenase I co2

Figure 1. Degradative pathway for dichloromethane metabolism by Methylobacterium strain DM4. Enzymes involved: © dichloromethane dehydrogenase; ®

fonnaldehyde dehydrogenase; @ fonnate dehydrogenase. Dichloromethane dehalogenase is strictly dependent on glutathione (GSH). (Adapted from

Leisinger et al., 1994)

ATCC 29600 was first described as a new genus by Auling et al. (5) and is a member of the a-subgroup of the Proteobacteria. It was found to utilize NT A as a sole source of carbon and nitrogen. The degradation of NT A by strain 29600 is first catalyzed by a two-component NT A monooxygenase in which iminodiacetate (IDA) and glyoxylate are formed. IDA is further degraded by IDA dehydrogenase to glycine and glyoxylate (Figure 3). For more information concerning the so 3-

~ NADH + H+ NAD+ NAD+ NADH + H+ #>~ 0 2 H20 Hpr lmonooxygenase I aco o ...------.aldehyde CH 3 CH20H dehydrogenase CHO dehydrogenase COOH p-Toluenesul fonate p-Sulfobenzyl p-Sulfo p-Sulfobenzoate alcohol benzaldehyde

1-03s_ OH I ~ OH NADH + H+ NAD+ I ~ Hso; -00 .../ ... _ _.,... Central metabolic pathways ~r: spontaneous 02 I 13,4 dioxygenase COff I...... : COff coo- Protocatechuate p-Sul fobenzoate unstable intermediate

Figure 2. Metabolic pathway for p-toluenesulfonate (TS) by Comamonas testosteroni strain T-2. Enzymes of the TS pathway are TS monooxygenase; 4-sulfobenzylalcohol dehydrogenase; 4-sulfobenzaldehyde dehydrogenase; protocatechuate 4,5 dioxygenase. (Adapted from Locher, 1991) o ff o~ ~o '\c/ + ;c "' I H ff H-C-H Glyoxylate 0 H OH 0 0 H H 0 ~ I I I /} spontaneous ~ 1 I /f 'c--C-N--C--C/ -+----...... "C-C-N--C--C/" ·o/ ~ 14 "a- -o/ ~ A 'b- Nitri lotriacetate NTA Momoxygenase u-hydroxyl-NTA Iminodiacetate (Component A and B) 0 0 + ~-~ 1/2 02 H/ 'b- Glyoxylate 0 0 . H H 0 H H 0 0 H H ~ -t-N-~-C,f' _ _;:;,,.___...,::;._....i +B:zO ~ I I~ ~-~-N=~-C,:7 ,... C--C-N-H-... __,...Central "ff - / I I -0 / if J fl -0/ HI ""'OH 0 H H metabolic Iminodiacetate Glycine pathways

IDA Dehydrogenase (membrane bound)

Figure 3. Reactions catalyzed by the key enzymes in the nitrilotriacetate (NTA) degradative pathway by Chelatobacter heintzii strain ATCC 29600. The key enzymes are the two-component NTA monooxygenase and the membrane bound iminodiactetate dehydrogenase. (Adapted from Egli et al., 1994) Chapter 1 20 Introduction microorganisms, metabolic pathways, and physiological regulation, please refer to Egli (13). The "non-pollutant" utilizing Escherichia coli strain ML30 (DSM 1329) is a gram-negative Enterobacteriaceae belonging to they-subgroup of the Proteobacteria. Strain ML30 was derived from the original isolate strain ML found by J. Monod which was permease negative strain which he called ML3. The revertant strain designated ML 30 contains an inducible galactoside permease and was very important in the early phases of molecular biology (33). Lendenmann et al. (37) demonstrated that this strain in a carbon-limited chemostat, independent of mixture and dilution rate, could simultaneously utilize six different sugars. Screening studies proved that ML 30 could utilize synthetic sewage in the presence of various pollutants (each 100 ppm as carbon of NT A, TS or DCM) without an inhibitory effect on the growth rate. This organism is not presently known to possess pollutant degradative pathways.

Selection of autecological methods

Many of the autecological techniques used in this study were originally designed for use with batch grown pure cultures and had not been tested in heterogeneous systems under continuous cultivation. Since RNA content of cells are highly dependent upon the growth rate and growth conditions of the cells (42) and due to the low growth rates (µ = o.os-1) used in our system, the use of in situ 16s rRNA probes for enumeration was deemed not fully reliable. Cells growing at slower rates have fewer 16s rRNA target sites and reduced fluorescent signal. Moreover, the relative concentration of rRNA and probe availability of rRNA target regions can vary for different species (2). In contrast, direct counts using specific surface antibodies for monitoring individual Chapter 1 21 Introduction microbial populations were found to be highly accurate, independent of growth rate. Levels of expressed enzymes in pure culture experiments were measured by Western blotting and immunoquantification. Unfortunately, during mixed culture experiments, cross-reactivity between certain enzyme components occurred. As a result, in all experiments, pure and mixed culture, specific pollutant degrading activity was also assessed by excess substrate specific oxygen uptake rates.

Model system and experimental questions

After confirming that the specific pollutant degrading microorganisms could not utilize the other pollutants and could grow in synthetic sewage, a model system consisted of the nitrilotriacetate (NT A) degrader, Chelatobacter heintzii, the p-toluene sulfonate (TS) degrader, Comamonas testosteroni strain T-2, the dichloromethane (DCM) degrader, Methylobacterium strain DM4, and the non-pollutant degrading Escherichia coli strain ML30 was assembled and cultivated in a carbon- limited continuous culture on a synthetic sewage containing mixtures of the specific pollutants (Figure 4). In this system we examined whether patterns of regulation, especially with respect to the degradation of pollutants in the presence of other easily utilizable substrates can be extrapolated to more complex systems; i.e. what we observe in pure culture (mixed substrate/pollutant utilization) is also seen in nature. Additionally, the question was asked if the simultaneous utilization of pollutant and alternative substrates guarantee the survival of pollutant degrading strains in nature? Furthermore, what are the effects of transient addition or omission of pollutants on levels of expressed pollutant catabolizing enzymes/activities and on population dynamics, and also, do the pollutant degrading strains remain in the synthetic sewage when no Environment

Comamonas testosteroni © __ strainT2 -}~ ~

~~~ --~. nitriJo.. p -toluene triacetate suJfonate (NTA) (TS) Eseheriehia coli strainML.30

TS + ac:etate NTA +acetate ? • ~; dichloro- acetate methane (DCM)

DCM+ acetate acetate I 0 © ©©...-1-----' defined mixed cuJture defined mixed substrates

Figure 4. Overview of the model system composed of three pollutant degrading strains and one non-pollutant degrading strain: 1) Chelatobacter heintzii strain ATCC 29600, a nitrilotriacetate utilizer; 2) Escherichia coli strain ML30, non-pollutant degrading strain; 3) Methylobacterium strain DM4, a dichloromethane utilizer; 4) Comamonas testosteroni strain T-2, a p-toluenesulfonate utilizer. Scheme of experimental design starting from batch and continuous culture experiments with pure cultures to continuous culture experiments with mixed cultures. Model system was set up to determine whether regulatory patterns for pollutant degradation in the presence of easily degradable carbon sources (mixed substrate utilization) with pure cultures can be extrapolated to more complex systems.

-----·----··········------··········------··-·------··------·····----- Chapter 1 23 Introduction pollutant is present? A final question addressed was can the autecological methods used in this study be applied to heterogeneous natural or engineered environments?

1. 7 Contents of this thesis

The major aspects studied are: 1) the ability of evolutionarily distinct microorganisms (pure cultures) to utilize pollutants in the presence of easily utilizable alternative carbon substrates (simultaneous utilization of mixed substrates) and what proportion of pollutant carbon in the total available carbon pool is necessary to induce pollutant degrading systems/enzymes above constitutive levels (Chapter 2); 2) the temporal relationship of induction of pollutant degrading systems/enzymes in pure cultures of pollutant degrading organisms due to shifts in carbon substrates (Chapters 3 and 4); 3) the influences of mixed substrate utilization (pollutant/alternative substrates) on the survival of specific pollutant degrading organisms cultivated with synthetic sewage with competing populations of other microorganisms and the establishment of a stable defined mixed culture consisting of four members (Chapter 5); 4) the effect of carbon substrate shifts on the community structure and physiological response of a defined mixed culture (Chapter 5).

Additionally, the Appendix section contains the abstract of a manuscript in preparation, one submitted manuscript and one published article which were not directly part of the thesis but whose contents are a direct result of questions which either arose during the thesis or were Chapter 1 24 Introduction further extensions of phenomena observed during the course of the thesis. All of these studies were the result of other collaborative research during my doctoral studies. The contents are as follows: Appendix 1) assessment of the degradation of a model refractory waste by combined ozonation and biological treatment by methods stemming from the thesis; Appendix 2) the discovery that Methylobacterium strain DM4, one of the primary organisms used during the thesis, was able to produce struvite, an ammonium magnesium phosphate mineral, under both batch and continuous cultivation; Appendix 3) the discovery that the carbonate mineral, dolomite, could be formed at earth surface temperatures by a sulfate reducing bacterium.

References

1. Amman, R. I., L. Krumholtz, and D. A. Stahl. 1990. Fluorescent-oligonucliotide probing of whole cells for the determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol. 172: 762-770. 2. Amman, R. I., B. J. Binder, R. 0. Olson, S. W. Chisolm, R. Devereux, and D. A. Stahl. 1990. Combination of 16s rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56: 1919-1925. 3. Ardern, E., and W. T. Lockett. 1914. Experiments on the oxidation of sewage without the aid of filters. J. Soc. Chem. Ind. 33: 523-539. 4. Atkinson, B., and F. Mavituna. 1991. Biochemical Engineering and Biotechnology Handbook. Stockton Press. New York. 1271 p. Chapter 1 25 Introduction

5. Auling, G., H.-J. Busse, T. Egli, T. EI-Danna, and E. Stackbrandt. 1993. Description of the gram-negative, obligately aerobic, nitrilotriacetate (NT A)-utilizing bacteria as Chelatobacter heintzi, gen. nov., and Chelatococcus asaccharovorans, gen. nov., sp. nov. System. Appl. Microbiol. 16: 104-112. 6. Bally, M. 1994. Physiology and ecology of nitrilotriacetate degrading bacteria in pure culture, activated sludge and surface waters. Diss. ETH Nr. 10821. Ph.D. Swiss Federal Institute of Technology Zurich. 7. Beijerinck, M. W. 1901. Anhaufungsversuche mit Ureumbakterien. Zentralblatt fiir Bacteriologie, II. 7: 33-61. 8. Bitton, G. 1994. Wastewater Microbiology. Wiley-Liss. New York. 478 p. 9. Brock, T. D. 1961. Milestones in Microbiology. Prentice-Han. Englewood Cliffs, New Jersey. 273 p. 10. Brock, T. D., and M. T. Madigan. 1988. Biology of Microorganisms. Prentice-Hall. Englewood Cliffs, New Jersey. 835 p 11. Delong, E. F., G. S. Wickham, and N. M. Pace. 1989. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 243: 1360-1363. 12. Egli, T. 1995. The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates. p.305-386. In J. Gwynfryn Jones (ed.) Advances in Microbial Ecology, Plenum Press, New York. 13. Egli, T. 1994. Biochemistry and physiology of the degradation of nitrilotriacetic acid and other metal complexing agents, pp. 179-195. In C. Ratledge (ed.). Biochemistry of Microbial Degradation. Kluwer Academic Publishers, Dordrecht, Netherlands. Cha.pter 1 26 Introduction

14. Egli, T., U. Lendenmann, and M. Snozzi. 1993. Kinetics of microbial growth with mixtures of carbon sources. Antonie van Leeuwenhoek. 63: 289-298. 15. Ehrlich, H. L. 1996. Geomicrobiology. Marcel Dekker, Inc. New York. 719 p. 16. Fenchel, T., and T. H. Blackburn. 1979. Bacteria and Mineral Cycling. Academic Press. London. 17. Fenchel, T. M. and B. B. Jorgensen. 1977. Detritus food chains of aquatic ecosystems: The role of bacteria. Adv. Microb. Ecol. 1: 1-58. 18. Galli, R., and T. Leisinger. 1988. Plasmid analysis and cloning of the dichloromethane-utilization genes of Methylobacterium sp. DM4. J. Gen. Microbiol. 134: 943-952. 19. Gerritse, J., F. Schut, and J. C. Gottschal. 1992. Modelling of mixed chemostat cultures of an aerobic bacterium, Comamonas testosteroni, and an anaerobic bacterium, Veillonella alcalescens - Comparison with experimental results. Appl. Environ. Microbiol. 58: 1466-1476. 20. Gerritse, J., F. Schut, and J. C. Gottschal. 1990. Mixed chemostat cultures of obligately aerobic and fermentative or methanogenic bacteria grown under oxygen limiting conditions. FEMS Microbiol. Lett. 66: 87-93. 21. Gottschal, J. C. 1993. Growth kinetics and competition - some contemporary comments. Antonie van Leewenhoek. 63: 299-314. 22. Gottschal, J. C. 1990. Different types of continuous culture in ecological studies, In R. Grigorova and J. R. Norris (ed.). Methods in Microbiology. Academic Press, London. Chapter 1 27 Introduction

23. Gottschal, J. C., and J. G. Koenen. 1980. Mixotrophic growth of Thiobacillus A2 on acetate and 'thiosulfate as growth limiting substrates in the chemostat. Arch. Microbiol. 126: 33-42. 24. Gottschal, J. C., H. J. Nanninga, and J. G. Koenen. 1981. Growth of Thiobacillus A2 under alternating growth conditions in the chemostat. J. Gen. Microbiol. 126: 85-96. 25. Gutherie, F. E. 1980. Nonagricutural pollutants, pp. 1-23. In F. E. Gutherie and J. J. Perry (ed.). Introduction to Environmental Toxicology. Elsevier, New York. 26. Harder, W., and L. Dijkhuizen. 1976. · Mixed substrate utilization, 297-314. In A. C. R. Dean, D. C. Ellwood, C. G. T. Evans and I. Melling (ed.). Continuous Culture 6. Applications and New Fields. Ellis Horwood, Chichester, England. 27. Hedges, J. I. 1992. Global biogeochemical carbon cycles: Progress and problems. Marine Chem. 39: 67-93. 28. Herbert, D. 1961. A theoretical analysis of continuous culture systems. Soc. Chem. Ind. Monogr. 12: 21-53. 29. Herbert, D. 1976. Stoichiometric aspects of microbial growth, pp. 1-30. In A. C. R. Dean, D. C. Ellwood, C. G. T. Evans and J. Melling (ed.). Continuous Cultures 6: Applications and New Fields. Ellis Horwood, Chichester. 30. Hobbie, J. E. 1990. Measuring heterotrophic activity in plankton. pp. 235-250. in R. Grigorova and J.R. Norris (eds). Methods in Microbiology, vol. 22. Academic Press, London. 31. Hobbie, J. E., and J. M. Melillo. 1984. Role of microbes in global carbon cycling. ASM Press. Washington, D.C. 32. Jannasch, H. W., and R. I. Mateles. 1974. Experimental bacterial ecology studied in continuous culture. Advances Microbial Physiol. 11: 165-212. Chapter 1 28 Introduction

33. Koch, A. L. 1985. The macroeconomics of bacterial growth, In M. Fletcher and G. D. Floodgate (ed.). Bacteria in Their Natural Environments. Academic Press, London. 34. Kumaran, P., and N. Shivaraman. 1988. Biological treatment of toxic industrial wastes, pp. 227-283. In D. L. Wise (ed.). Biotreatment Systems. CRC Press, Boca Raton, Florida. 35. La Rivier, J. W. M., and D. H. Eikelboom. 1986. Role of specific microbial groups in wastewater treatment; Filamentous organisms as an example. pp. 59-65. in F. Megusar, M. Gantar (eds) Perspectives in Microbial Ecology. Mladinska Knjiga. Ljubljana, Yugoslavia. 36. Leisinger, T., R. Bader, R. Hermann, M. Schmid-Appert, and S. Vuilleumier. 1994. Microbes, enzymes and genes involved in dichloromethane utilization. Biodegradation. 5: 237-248. 37. Lendenmann, U., M. Snozzi, and T. Egli. 1996. Kinetics of simultaneous utilization of sugar mixtures by Escherichia coli in continuous culture. Appl. Environ. Microbiol. 62: 1493-1499. 38. Locher, H. H., C. Malli, S. W. Hooper, T. Vorherr, T. Leisinger, and A. M. Cook. 1991. Degradation of p-toluic acid (p-toluenecarboxylic acid) and p-sulphonic acid via oxygenation of the methyl sidechain is initiated by the same set of enzymes 1n Comamonas testosteroni T-2. J. Gen. Microbiol. 137: 2201-2208. 39. Mills, A. L., and P. E. Bell. 1986. Determination of individual organisms and their activities in situ, 27-60. In R. L. I. Tate (ed.). Microbial Autecology- A Method For Environmental Studies. John Wiley & Sons, New York. 40. Morita, R. Y. 1993. Bioavailability of energy and the starvation state, 1-23. In S. Kjelleberg (ed.). Starvation in Bacteria. Plenum Press, New York. Chapter 1 29 Introduction

41. Morita, R. Y. 1992. Low-nutrient environments. pp. 617-624. In J. Lederberg (ed.). Encyclopedia of Microbiology. Academic Pres's, London. 42. Neidhardt, F. C., J. L. lng~aham, and M. Schaechter. 1990. Physiology of the Bacterial Cell: A Molecular Approach. Sinauer Associates, Inc. Sunderland, Massachusetts. 506 p. 43. OECD. 1994. Risk reduction monograph no. 2: Methylene chloride. Environment Directorate. OECD Environment Monograph Series. 96. 44. Schliifli Oppenberg, H. R., G. Chen, T. Leisinger, and A. M. Cook. 1995. Regulation of the degradative pathways ofrom 4- toluenesulphonate and 4-toluenecarboxylate to protocatechuate in Comamonas testosteroni T-2. Microbiology. 141:1891-1899. 45. Schlegel, H. G. 1993. General Microbiology. Cambridge Press, Cambridge, England. 46. Stanier, R. Y., J. L. Ingraham, M. L. Wheelis, and P. R. Painter. 1987. General Microbiology. Prentice-Hall Inc. Englewood Cliffs, New Jersey. 689 p. 47. Tempest, D. W. 1970. The continuous cultivation of microorganisms. 1. Theory of the chemostat. Meth. Microbiol. 2: 259-276. 48. Tempest, D. W ., 0. M. Neijssel, and W. Zevenboom. 1983. Properties and performance of microorganisms in laboratory culture: Their relevance to growth in natural ecosystems, pp. 119- 152. In J. H. Slater, R. Whittenbury and J. W. T. Wimpenny (ed.). Microbes in Their Natural Environment. Cambridge University Press, Cambridge, England. 49. Veldkamp, H., and H. W. Jannasch. 1972. Mixed culture studies with the chemostat. J. Appl. Chem. Biotechnol. 22: 105-123.

------Chapter 1 30 Introduction

50. Vestal, J. R. 1980. Pollution effects of storm-related runoff, pp. 450-456. In F. E. Guthrie and J. J. Perry (ed.). Introduction to Enviromental Toxicology. Elsevier, New York. 51. Ward, D. M., R. Weller, and M. M. Bateson. 1990. 16s rRNA sequences reveal numerous uncultivated microorganisms in a natural environment. Nature. 345: 63-65. 52. Wimpenny, J. W. T., and H. Abdollahi. 1991. Growth of mixed cultures of Paracoccus denitrificans and Desulfovibrio desulfuricans in homogeneous and in heterogeneous culture systems. Microb. Ecol. 22: 1-1352. 53. Wimpenny, J. W. T., R. W. Lovitt, and J. P. Coombs. 1983. Laboratory model systems for the investigation of spatially and temporally organized microbial ecosystems, pp. 67-117. In J. H. Slater, R. Whittenbury and J. W. T. Wimpenny (ed.). Microbes in Their Natural Environments. Cambridge University Press, Cambridge, England. Chapter2 31

Regulation of Pollutant Degradation in the Presence of an Alternative Substrate by the para-Toluenesulphonate Utilizing Comamonas testosteroni Strain T-2 and the Dichloromethane Utilizing Methylobacterium Strain DM4 During Growth in Batch and Continuous Culture

Albert J. Tien and Thomas Egli

Key words: Comamonas testosteroni, dichloromethane, Methylobacterium, mixed substrates, toluenesulfonate, regulation

ABSTRACT

The regulation of pollutant degradation in the presence of an alternative substrate (acetate) was investigated in both batch and continuous cultures of Comamonas testosteroni strain T-2, a para-toluenesulphonate (TS) degrader, and of Methylobacterium strain DM4, a dichloromethane (DCM) degrader. In batch culture, strain T-2 utilized acetate and always TS simultaneously. With the case of strain DM4, substrate utilization patterns were highly dependent on precultivation c9nditions. DCM pregrown cultures of strain DM4 were able to simultaneous utilize acetate and DCM, while acetate precultivated cultures of strain DM4 exihibited diauxic growth with acetate utilized first. During simultaneous utilization, the maximum specific growth rates achieved exceeded those observed during growth with the individual substrates. During cultivation with different mixtures of the pollutant and acetate under carbon-limited conditions in the chemostat (dilution rate of 0.05 hrl ), both the pollutant

------Chapter2 32 and acetate were simultaneously utilized, independent of the ratio of pollutant to acetate supplied in the medium feed. In both strains induction of the pollutant degrading systems above a constitutive level occurred when the pollutant contributed to approximately 1% of the total carbon supplied in the feed. Maximal induction levels were achieved when the pollutant contributed to approximately 3% in the case of C. testosteroni, and 5% in the case of Methylobacterium. Monitoring inflowing medium and residual steady-state substrate concentrations demonstrated that at pollutant to acetate ratios of less than 1%, pollutant concentrations in the culture remained below the detection limit (approximately 0.1 mg 1-1 as carbon for TS and DCM) which infers that these strains have a low constitutive capacity for pollutant degradation, even when it contributed only to a minor fraction of the total carbon utilized by the cell.

INTRODUCTION The field of microbiology entered a "renaissance" when microbes from the environment were found to degrade pollutants, previously thought of as recalcitrant. Great hope existed for the application of such organisms for environmental remediation or hazardous waste treatment. However, most of the research on biodegradation of pollutants was carried out under laboratory conditions with pure cultures grown under batch conditions and typically with the pollutant supplied as the sole source of carbon and energy at high concentrations. In nature, microbial growth and turnover of organic matter proceeds at much slower rates as compared to those obtained in artificial culture media commonly prepared to produce high bacterial or product yields. Decomposition of organic material occurs under the influence of a myriad of differing organisms culminating with the complex flow of energy through the "food chain". Dissolution and decomposition of Chapter2 33 particulate and polymeric organic materials is the rate-limiting step over great periods of time in aerobic aquatic ecosystems, sediments, and soils despite the presence of high concentrations of organic matter (14, 22, 36). It is believed that most natural environments are virtually oligotrophic and that heterotrophic organisms adapted to such environments survive and thrive due to their ability to utilize various substrates from the available carbon pool (20, 34). Limitation by carbon and energy is the crucial factor for the degradation of carbonaceous pollutants in the environment. Morita (35) remarked that slow growth in continuous culture under carbon-limitation represents a method that closely mimics the growth conditions bacteria encounter in the natural environment and that this system allows for better control of growth conditions than any other cultivation system available. Under such conditions simultaneous utilization of mixtures of carbon sources has been reported to be the rule rather than the exception (9, 16). Little is known concerning the effects of concentration and spectrum of other naturally ocurring potential carbon substrates on the regulation of the degradative abilities of pollutant utilizing organisms in the environment. The emphasis of this paper is to determine whether or not the degradation of pollutants occurs in the presence of alternative substrates, the factors which govern degradation, and how these findings are applicable to removal of pollutants from the environment. In this model system the regulation of pollutant degrading activities of Methylobacterium strain DM4 (dichloromethane utilizer) and Comamonas testosteroni strain T-2 (toluenesulfonate utilizer) were examined during growth in batch and continuous culture with their specific pollutant, an alternative substrate (acetate) or mixtures thereof. Chapter2 34

MATERIALS AND METHODS

Microbial strains and cultivation conditions. In experiments with mixtures of dichloromethane (DCM) and acetate, Methylobacterium strain DM4 (15) was used. Conditions for culture maintenance and batch growth have been previously described (26). For continuous culture, the medium contained per liter 0.3 g MgS04·7H20, 0.02 g CaCl2·2H20, 0.14 g KCl, 1.07 g NH4Cl, 0.02 g (NH4)2S04, 2.46 g Na2HP04, 5 ml of trace elements (13), 1.8 ml H3P04 (85%) and 10 mg silicon antifoam (Fluka, Buchs, Switzerland) with the substrate concentration adjusted accordingly. Total carbon in the medium was 200 mg-C 1-1 either from DCM, acetate, or varying proportions of both. The bioreactor (MBR, Zurich, Switzerland, 2 liter working volume) was aerated at 0.1 1 min-1 while stirred at 1,500 rpm. The pH was controlled at 6.8 by automatic addition of H3P04 (1 M) and KOH/NaOH (0.5 M, each). The temperature was maintained at 30°C. C. testosteroni strain T-2 was used in experiments with para- toluensulphonate (TS), acetate and mixtures of both. Strain T-2 was precultured and maintained as previously described (30). For continuous cultures, the medium composition was as before with the exception that the total carbon in the feed was 200 mg-C 1-1 either from acetate, TS or varying proportions of both. The bioreactor (MBR, Zurich, Switzerland, 2 liter working volume) was aerated at 0.5 1 min-1 while stirred at 1,500 rpm with pH controlled at 7.5 with H3P04 (1 M) and KOH/NaOH (0.5 M, each). The temperature was maintained at 30°C. Determination of specific oxygen uptake rates. Culture liquid (80 ml) was collected directly from the chemostat, cells were collected by centrifugation, washed twice with and resuspended in 20 ml carbon-free medium. Substrate (DCM, TS, or acetate) stimulated specific

. ···---~-~·------·~·--··~------Chapter2 35 oxygen uptake rates were recorded at 30°C in a glass Clark-type oxygen electrode (Rank Brothers, Great Britain). The total volume of the assay was 3.0 ml, consisting of 2.8 ml of cell suspension plus 0.2 ml of a 0.1 M substrate solution. Sampling and assay times were standardized. All assays were preformed in triplicate. Quantification of biomass. Biomass was quantified by measuring dry weight or optical density. Dry weight (Dwt) was obtained by filtering cells through 0.2 µm pore size polycarbonate filters (Nuclepore, Pleasanton, CA, U.S.A.) which together were washed with distilled water to remove salts. Filters were dried at 105°C overnight to constant weight. Optical density was determined at 480 nm in 1 cm cuvettes using a Uvikon 860 spectrophotometer (Kontron, Zurich, Switzerland). Analysis of substrates. Residual concentrations of acetate were measured by high pressure ion exclusion chromatography as described by Schneider et al. (41). The detection limit was I mg I-1 acetate carbon. DCM measurements were performed by gas chromatography (Hewlett Packard 5892 series II, U.S.A.) using a 60 m long, 0.32 mm diameter glass capillary column (J&W DB624), fitted with an ECD detector, by direct analysis. The injector and detector temperatures were maintained at 250°C. The oven temperature was maintained at 30°C with helium as the carrier gas. Total flow rate was 32 ml min-1. The detection limit was 0.1mg1-l DCM carbon. Determination of TS concentrations were performed by high pressure liquid chromatography after Locher et al. (30). The detection limit was 0.1 mg 1-1 TS carbon. Cha.pter2 36

RESULTS

Growth and regulation of DCM degradation by Methylobacterium strain DM4 in batch culture. The growth of strain DM4 on a one to one mixture of DCM and acetate, based on carbon, varied depending on the history of the inoculum. When the inoculum was cultured on DCM and then transferred into batch culture containing the two substrates at an initial concentration of 100 mg of carbon each, a µmax of 0.191 hr-1 was observed. This value was significantly higher than that found during growth with either DCM (µmax = 0.079 hr-1) or acetate alone (µmax = 0.078 hr-1 ). Measurement of the substrate concentration during the course of the growth curve with the mixture confirmed that both substrates were simultaneously utilized (Fig. la). However, when specific substrate consumption rates were analyzed, a preference towards DCM was observed in the initial phase of growth. In the later phase of growth, the cells consumed approximately equal amounts of both substrates, i.e. 1/3 of the carbon originated from DCM whereas 2/3 came from acetate. (Fig. lb). Further analysis ofµ= f(t) indicates a rapid decrease in µ from 5 hrs until 10 hrs then a the specific growth rate declined gradually until stationary phase was reached (Fig. le). When the inoculum was derived from an acetate pregrown inoculum, a diauxic growth pattern was observed (Fig. 2a). Residual substrate concentrations of the culture during the growth time course generally agree with classical substrate utilization patterns observed during diauxic growth with the exception that, as acetate concentrations decreased, a small percentage of the DCM also was utilized indicating a low constitutive activity of DCM pathway enzymes in acetate grown cells. By 15 hrs the acetate concentration was below Chapter2 37

a. 1.0 100 ,-... 8 IJ,. !. = IJ,. u =QQ 80 I .., 0.8 • ••• Ill) ._.8 ...QI • •• .0 • • .a 0.6 • •• 60 QI ·~ • _, Q.. ll= C"l:I= -; ti. 40 .:! 0.4 •• -; c. • .a :g= 0 • :I 0.2 • A 20 ~ • 0.0 • 0 b. 1.2 I\ ...~ 1.0 ~ II* I "':' I I .. Z' 0.8 I I ~ -~= I I ~"' .....Ill) 0.6 I \ C"l:I= -.. r::r I .s 0.4 I ~"' '".. I C"l:I= ~ 0.2 ~~::iria::~, ·~ 0.0 c. 0.2 ,._ ,-... "':' ._... -= I: I .!! QI I .I ~ I I -= I 4p ~ 0.1 -Q • tt tJ.. I • t.I I I ·oi= I ..Cl. C"l:I , .....I ...... 0.0 49 ·------.,_ 0 10 20 30 40 50 Time (hrs)

Figure 1. Growth of Methylobacterium strain DM4 in batch culture at pH 6.8 and 30C)C

with a one to one mixture of acetate and dichloromethane ( 100 mg-C 1-1 eacm).

(a.) Simultaneous utilization of both substrates by a culture inoculated from

dichloromethane grown preculture: optical density [e], acetate concentration [.1], and dichloromethane concentration [•]. (b.) Specific substrate

consumption rates: qAcetate [.1] and qDCM [•]. (c.) µ [e]. Chapter2 38 detection and it was at this point when DCM concentrations decreased the most rapidly. In both cases (Fig. 1 and Fig 2.) no exponential growth was seen in the late growth phase. The specific growth rate on DCM during the transition from acetate utilization to DCM utilization slowed down at relatively high concentrations indicating either poor affinity for DCM or excretion/formation of toxic metabolites such as formaldehyde. These phenomena could be better illustrated when qacetate and qDCM are plotted against the experimental time course (Fig. 2b). Specific substrate consumption rates concur with the previous trends. Initially, a preference towards acetate was seen with a maximal qacetate around 4 hrs. During this time a low qDCM is exhibited which increased to a maximal value of 18 hrs. Examination ofµ= f(t) demonstrate a two phase increase in µ; one corresponding to acetate utilization and the second with DCM utilization (Fig. 2c ). Growth and regulation of TS degradation in batch cultures of Comamonas strain T-2. In the case of strain T-2, no differences in growth patterns were seen during batch cultures derived from precultures grown with either acetate or TS. A µmax of 0.210 hr 1 was recorded during growth of strain T-2 with a one to one mixture of acetate and TS (carbon equivalents). This value is within the same range as that observed with the individual substrates (acetate µmax= 0.231 hrl and TS µmax = 0.180 hr-1 ). No clear exponential growth phase was observed. The substrate utilization patterns for TS and acetate indicate that both substrates were simultaneously utilized (Fig 3a). Suprisingly, examination of specific substrate consumption rates indicate a preference towards acetate. qTS remained at a low level in the initial phase of growth and increased only towards the end of the batch culture (Fig. 3b). Chapter2 39

a. 1.0 00

s 6 \ '[ 0.8 80 u <:::>= .4 • • • • • 00 ell' ~ • e 6 • '-' =;.., 0.6 6 • • 60 "' ;::- - r:l A j Q"' 0.4 ...·' 40 ~ 'ii ':cla "Cl= c. ·;;i 0 0.2 • 20 6 ~"' 6 • 0.0 • 0 b. 1.0

:::;-< 0.8 4 I.. :~~ ;:;"'-= I .I!! .:.. 0.6 I A'I =J.. ! I I .c-"' ~ ~ ~ tl.l= "' 0.4 I I.. \ C' liJ.. I 4 I .... I \ 111111 lS I 0.2 tl.l= I •' ' ~ • ... a : ... ':t I ...... o.o c. 0.2

,..... 1. 6 flP •II .... ,~," "' 9 I c2 I I .::: 0.1 I i I e t I Cl I e. I ••, 9>:.; I ' "'c. , 00 ...... o.o . .. - -·--- .. 40 0 10 20 30 50 Time(hrs)

Figure 2. Growth of Methylobacterium strain DM4 in batch culture at pH 6.8 and 30°C

with a one to one mixture of acetate and dichloromethane (100 mg-C 1-l each).

(a.) Diauxic substrate utilization pattern by culture originating from inoculum

originating from acetate-grown preculture: optical density [e], acetate concentration [A], and dichloromethane concentration [•]. (b.) Specific

substrate consumption rates: qAcetate [A] and qDCM [•]. (c.) µ [e]. Chapter2 40

a. 1.2.,....------120 s = 1.0 ~ c ~ 0.8 A D •••••• ~ •• ••• J 0.6 ••A.·• ~ 0.4 • 8 • 0.2 •• • •• b.

~I \ I \

II \\ I\" I \ I \ I \ I \ I \ I \ o.s I \...... or ...i·-~ ', I ~ I \ A I ,,JI...... / \. \ I ,,...... • \i)( \ .... Ci .. o.om-....-...... --...... --.----,...... !::=:.:::t~._---1 c. 0.2.,....-.,.------. r \ ; " ...\IJ-.• \

~ 'I i •,•, J5 0.1. ....,...... o.o+-~-...... -"""""-....- ...... -..--..--,....---.-,---1 2 4 6 8 10 12 14 Time (hrs)

Figure 3. Growth of Comamonas testosteroni strain T-2 in batch culture at pH 7.5 and

30°C with one to one mixtures of acetate and toluenesulphonate ( 100 mg-C L -

1 each). (a.) Simultaneous utilization of both substrates occurred, irregardless

of inoculum origin, i.e. preculture with acetate or toluenesulphonate: optical

density [e], acetate concentration [A], and toluenesulphonate concentration

[•]. (b.) Specific substrate consumption rates: qAcetate [A] and qTS [•]. (c.)

µ [•]. Chapter2 41

Analysis of µ = f(t) shows that µ is gradually decreasing until the stationary phase was reached (Fig. 3c). Carbon-limited growth with mixtures of acetate and DCM in continuous cultures of Methylobacterium strain DM4. In order to investigate the regulation of DCM degradation in the presence of other substrates under carbon-limited conditions, strain DM4 was cultivated in a chemostat at a constant dilution rate of O.OS hr- I and the culture was supplied with different mixtures of acetate and DCM. The concentration of carbon in the feed from acetate, DCM or mixtures of both was maintained at 0.2 g I-1. Independent of the mixture composition, strain DM4 was able to simultaneously utilize both substrates to levels below the detection limit. The specific consumption rates for both acetate (qAc) and DCM (qDCM) exhibited a dependence on the ratio of DCM to acetate in the feed. The geometry of the curves are a result of similar yields on the individual substrates (Fig. 4a). The ability to utilize DCM was assessed via oxygen uptake measurements when washed cultures were exposed to excess DCM. Excess substrate specific oxygen consumption rates indicated that more than O.S% DCM carbon in the feed would elicit DCM degradation above the constitutive levels (Fig. Sa). Nevertheless, at DCM feed concentrations less than 0.5% DCM (So of DCM in feed between 0.1 and 1 mg 1-1 ), no residual DCM was detected in the culture medium indicating that DCM was still degraded (loss of DCM by aeration was found to be negligible as determined by abiotic controls). At DCM carbon concentrations contributing to 5 % of the feed, the DCM degrading enzyme system was fully induced to levels approximately 40 times that of the constitutive level of 0.6 nmol 02 (mg Dwt)-1 min-1 (Fig. Sa). Growth of C. testosteroni strain T-2 with mixtures of acetate and TS in carbon-limited continuous culture. The Chapter2 42

a. 0.2~------.

0.1

0.0 0 20 40 60 80 100 Fraction DCM Carbon (%) in Feed b. 0.2

_4' ...... c.'11 ... =:: .c i::";' ;ic ,.-.. .§~ 0.1 =~ ;;J r:: u c i: .c... -8,Uu = rl!I~

0.0 0 20 40 60 80 100 Fraction TS Carbon ( % ) in Feed

Figure 4. (a.) Specific substrate utilization rates of Methylobacterium strain DM4 during

growth with mixtures of dichloromethane and acetate in carbon- limited

chemostat culture at a dilution rate of D= 0.05 hr-1. Specific dichloromethane consumption rate qDCM £•] and specific acetate consumption rate qAcetate

[.&.] as a function of dichloromethane carbon in the feed. (b.) Specific substrate utilization rates of Comamonas testosteroni strain T-2 during growth

with mixtures of toluenesulphonate and acetate in carbon-limited chemostat

culture at a dilution rate of D= 0.05 hr 1. Specific toluenesulphonate uptake rate qTS [•] and specific acetate consumption rate qAcetate [.&.] as a function

of toluenesulphonate carbon in the feed. [---] represents the theoretical values

calculated after Bally (4). Chapter2 43

a. /.... ----7._LG------. \.& / \ ,. ,, I \ I ".t...., I• ' " ----??-"'----->" /,{ 0 -- 0 s 10 40 60 80 100 Fraction DCM Carbon (%) in Feed b.

0 0 s 10 40 60 80 100 Fraction TS Carbon (%)in Feed

Figure 5 (a.) Excess dichloromethane stimulated maximal oxygen consumption rate as a function of dichloromethane carbon exhibited by cells of Methylobacterium strain DM4 in carbon-limited chemostat cultures grown at constant dilution rate of D=0.05 hr 1 with mixtures of acetate and dichloromethane at different compositions. The concentration of total carbon in the feed was 200 mg i-1. Regulation of specific dichloromethane stimulated excess oxygen uptake rate [•] and acetate stimulated excess oxygen uptake rate [AJ. (b.) The toluenesulphonate stimulated excess oxygen uptake rate as a function of toluenesulphonate carbon expressed by cells of Comamonas testosteroni strain T-2 grown in carbon-limited chemostat cultures at constant dilution rate of D=0.05 hr-1 with mixtures of acetate and toluenesulphonate at different compositions. Concentration of total carbon in the feed was 200 mg i-1. Regulation of specific stimulated toluenesulphonate excess oxygen uptake rate [O] and acetate stimulated excess oxygen uptake rate [.AJ. Chapter2 44 regulation of TS degradation was examined under carbon-limited conditions with different mixtures of acetate and TS. As in the previous experiment a constant dilution rate of 0.05 hr-1 and a combined carbon concentration of 0.2 g 1-1 was maintained. Residual substrate concentrations in the culture were below the experimental detection limit indicating that during growth with all mixtures tested acetate and TS were simultaneously utilized. The yield on TS (Y xis= 0.36) is slightly higher than on acetate (Y xis= 0.32) and this is reflected in the shape of the substrate consumption curves (Fig. 4b ). Similar induction patterns measured via the excess substrate specific oxygen consumption rate, as with strain DM4 were seen. Induction of the TS degrading pathway above constitutive level of 1 nmol 02 (mg Dwt)-1 min-1 was observed when TS contributed to as little as 0.5% of the total carbon in the feed. Full induction of TS degradative systems was achieved when TS carbon in the feed contributed to approximately 3% of the total and a further increase in the proportion of TS did not result in an additional increase in the excess substrate consumption rate (Fig. 5b ).

DISCUSSION

Degradation of pollutants occurs in the presence of alternative substrates. Simultaneous utilization of acetate and pollutant was seen in our batch growth experiments (carbon-excess) with strain T -2 and strain DM4 with the exception of strain DM4 derived from an acetate grown preculture. Similar substrate utilization patterns have been seen for Pseudomonas pudita Pl, with glucose and phenol, and· another pseudomonad, with phenol and p-cresol (21, 40). The specific growth rates of both strain DM4 (slightly) and strain T-2 (significantly) were Chapter2 45

higher on the mixtures, when simultaneously utilized, than on the individual substrates. The observation that µmax was enhanced during growth with the mixtures compared to growth with single substrates is not unusual. This has been reported frequently in the literature during batch growth with mixtures of carbon sources and has been compiled in a recent review by Egli (9). It has also been frequently observed that the ability for microorganisms to utilize certain substrates may depend on the history of exposure of the microorganism by the particular substrate. When cultured on a certain substrate the microorganism retains the ability to utilize this substrate for a period of time on the condition that transport of the substrate is not inhibited. Substrates that were often utilized secondarily could be utilized simultaneously if the organism had been previously grown on this compound (3, 23, 33, 44, 49). Similarly in our study. with respect to strain DM4, the history of the preculture influenced the utilization patterns of DCM and acetate. When strain DM4 was pregrown on acetate and inoculated into a mixture of acetate and DCM (100 mg carbon each), a diauxic growth pattern was seen. The phenomenon that certain organic acids can inhibit the utilization of glucose or other carbon substrates is frequently observed m pseudomonads and this effect has been termed "reverse catabolite repression" (16, 17). In the case of DCM pregrown Methylobacterium, DCM enters the cell by diffusion and cannot be inhibited by acetate or its metabolites. Once DCM has entered the cell, expression of the glutathione dependent DCM dehalogenase is necessary to convert the compound to formaldehyde which is further metabolized via the serine pathway (27, 28). Our results indicate that acetate is not able to interact with and inhibit the expressed/existing DCM dehalogenase enzyme. Yet other chlorinated aliphatics (non growth substrates) could serve as reversible Chapter2 46 competitive inhibitors for the DCM dehalogenase enzyme (26). When strain DM4 was pregrown on acetate, DCM degrading enzymes were not fully expressed as demonstrated by the low DCM utilization rates.

Induction of DCM dehalogenase and subsequent enzymes in the C1 pathway was required before DCM was able to be fully utilized by the Methylobacterium and significant induction of the DCM dehalogenase occurred obviously only after acetate had been consumed to a low concentration. The observation that strain DM4 did not grow exponentially in batch culture may be a result of excretion of formaldehyde during growth. Successful management of the formaldehyde flux by Methylobacterium depends on a delicate balance between production and utilization of formaldehyde. Excretion of formaldehyde by facultative methylotrophs such as Methylobacterium and other methylotrophs during unbalanced growth has often been observed (2, 8). The toxic effect of formaldehyde and its influence on µ have also been reported for methylotrophic bacteria and yeasts (2, 46). Factors which govern degradation. Little is available in the literature concerning factors which govern the level of induction of pollutant degrading enzyme systems in the presence of alternative substrates. Bally et al. (4) reported that in Chelatobacter heintzii, grown in continuous culture at a dilution rate of 0.06 hr-1 with mixtures of glucose plus NT A, induction of the NT A-degrading system occurred when NTA contributed to more than 1-3% of the total carbon in the substrate mixture supplied. In addition, NTA was also degraded when the proportion of NTA in the mixture was lower than 1%. A similar trend was seen in our experiments with strains DM4 and T-2. It has been demonstrated for mixtures of NT A and glucose with Chelatobacter heintzii (4, 5, 6) and for methylotrophic yeasts with mixtures of methanol Chapter2 47 and glucose (7, 10, 11, 12), that during mixed substrate growth at constant dilution rate, the ratio of substrates rather than the actual concentrations in the feed are controlling factors in enzyme regulation. Although not experimentally tested we expect this also to be the case with C. testosteroni and Methylobacterium. Environmental and ecological implications. It is thought that in the environment, growth of heterotrophic bacteria is limited by the availability of carbon substrates (1, 25, 34). Previously, investigations of microbial growth with carbon substrate mixtures focused upon identifying what factors contributed to diauxic patterns and whether this phenomenon would occur in continuous culture. (3, 18 , 19, 45). From these and other studies with sugar mixtures, it was shown that at low carbon concentrations and low dilution rates, simultaneous utilization of "diauxic substrates" is the rule rather than the exception (9). The results of our continuous culture experiments demonstrate that even at the lowest pollutant to acetate ratios, both substrates were simultaneously utilized. In a group report on life under low nutrient conditions, Hirsch et al. (20) discuss the fact that most natural environments are oligotrophic and that in spite of this bacteria not only survive but prosper. They predicted that a "model oligotroph" or rather a heterotrophic bacterium m an oligotrophic environment must possess certain characteristics which facilitate the simultaneous utilization of substrates. Most provocative are the predictions that such an organism would possess large proportions of catabolic enzymes which are inducible and that the carriers would be constitutive, constantly capable of uptake, and that the uptake systems are of high affinity; possibly for the simultaneous uptake of mixed substrates. Several investigations demonstrate that in addition, not only the carriers, but many catabolic pathways are derepressed or show a low constitutive level. This enables the organism to immediately utilize substrates that Cha.pter 2 48 become transiently available [please refer to the review by Egli, (9) for a comprehensive overview of this topic]. Our results generally agree with these predictions and indicate that under "enviromental conditions 11 both pollutant degrading strains, C. testosteroni strain T-2 and Methylobacterium strain DM4, were able to degrade their specific pollutant (TS and DCM, respectively) with acetate at low pollutant to acetate ratios. In our case two regulation strategies appear to play a role in the degradation of pollutants in the presence of other substrates; on one hand a low constitutive enzyme expression which operates when the pollutant contributes only to a small fraction of the total carbon utilized by the cell. On the other hand additional induction is triggered when pollutant carbon flux is > 1% of the available carbon. Typical dissolved organic carbon concentrations in freshwaters are reported to be in the range of a few milligrams per liter (20, 37). However, only a small fraction of this DOC is microbially available (20, 38). Acetate and other low molecular weight organic acids often contribute to a significant portion of utilizable carbon pool in freshwaters and wastewaters (37, 42). Much of this acetate is produced under anaerobic conditions in sediments (24, 43, 48) and consumed rapidly when released into aerobic waters. Turnover times of acetate in freshwaters range from 7 .8 to 290 hours (37). Data in the literature indicate that in surface waters, typical background concentrations for DCM are reported to be 0.05 mg 1-1 in fresh waters and 0.5 mg 1-I in urban areas (39). Data on TS concentrations in the environment are often lumped into the linear alkylbenzene sulphonate (LAS) category. The background concentrations· for LAS in urban freshwaters range from 2- 130µg1-l (47). For these reported concentrations, our data indicate that in carbon-limited environments such as lakes or streams, pollutants such Chapter2 49 as DCM and TS should be readily degradable by organisms posessing the respective degradative pathways in the presence of other substrates by low constitutive expression of the pollutant-degrading enzymes. In highly polluted natural environments or wastewater treatment facilities, inducible catabolic enzyme pathways appear to play a major role. One example of this was reported by Bally (4) where he compared levels of NT A enzymes in various wastewater treatment plants across Switzerland. Only in one case, a specialized wastewater treatment plant on Mt. Santis, designed to handle combined restaurant and tourist wastes, where the restaurants used a specially formulated high NT A containing detergent (NT A contributed to between 5 and 10% of the total DOC), were measurable levels of NT A monooxygenase detected indicating induction of the NT A degradation system. In other wastewater treatment plants in this same study, NTA degrading bacteria were present but appeared not to be induced for NT A monooxygenase, although NT A elimination of these treatment facilities were quite good. For the pollutants in our study, typical concentrations for LAS in sewage range from around 4-15 mg 1-l (47). In contaminated groundwaters DCM concentrations ranging from 4-25,000,000 mg 1-1 have been measured (39). In addition, the median concentration of DCM in many industrial effluents is reported to be 10 mg 1-1 DCM. The total DOC measured from the supernatant of a primary sewage (Berlin, Germany) was approximately 110 mg I-1 as C with acetate contributing to approximately 21 % or 23 mg 1-1 C (4 2). Assuming median DCM concentrations in industrial effluents of 10 mg 1-1 and DOC values of 110 mg 1-1 as carbon, the induction of DCM degrading enzymes can be expected to play a significant role in removal of this pollutant. However, further experimentation is necessary to investigate the effects of transient Cha.pter2 50 substrate conditions on the time course for induction of the pollutant- degrading system and the efficiency of degradation.

CONCLUSIONS

The presented data gives insight into some of the parameters which govern pollutant degradation in the environment or in wastewater treatment facilities and highlight the use of continuous culture techniques for such studies. It was demonstrated that pollutant degrading organisms retain their ability to utilize pollutants in the presence of other substrates and that substrate utilization rates were controlled by the ratio of the substrates rather than the actual feed concentrations. The ability to utilize substrate mixtures are another factor which give such organisms an advantage for survival, ecologically speaking. Additionally, and perhaps even more importantly, the fact that utlilization of mixtures facilitates the reduction in steady-state concentrations of all substrates utilized (29) will allow such cells to more efficiently compete for substrate at the low environmental concnetrations. These findings may have significant implications in designing treatment strategies for environmental remediation and wastewater or wastestream processing. For example, especially in industrial waste treatment plants or municipal wastewater treatment plants receiving a high proportion of industrial wastewater, preinduced organisms or defined mixtures of organisms capable of utilizing a specific pollutant or mixtures of pollutants in the presence of alternative substrates could be used, which in tum would eliminate the time needed for induction to occur and may lead to the reduction of residual pollutant concentrations due to mixed substrate utilization. Chapter2 51

ACKNOWLEDGEMENTS

The authors are indepted to the Swiss National Science Foundation who financed the work of A. J. Tien. (Project No. SPP 5001.35285). We would like to thank Prof. A. M. Cook and Prof. T. Leisinger for their support and interest in this project.

References

1. Alexander, M. 1994. Biodegradation and Bioremediation. Academic Press, San Diego. 2. Attwood, M. M., and J. R. Quayle. 1984. Formaldehyde as a central intermediary metabolite of methylotrophic metabolism. pp.315-323. In R. L. Crawford and R. S. Hanson (ed) Proceedings

of the 4th International Symposium on Microbial Growth of C1 Compounds. ASM Press, Washington, D .C. 3. Baidya, T. K. N., F. C. Webb, and M. D. Lilly. 1967. The utilization of mixed sugars in continuous fermentation I. Biotechnol. Bioeng. 9: 195-204. 4. Bally, M. 1994. Physiology and Ecology of Nitrilotriacetate Degrading Bacteria in Pure Culture, Activated Sludge and Surface Waters. Ph.D. Thesis no.10821. Swiss Federal Institute of Technology Ziirich. 5. Bally, M., and T. Egli. 1996. Dynamics of substrate consumption and enzyme synthesis in Chelatobacter heintzii during growth in carbon-limited continuous culture with different mixtures of glucose and nitrilotriacetate. Appl. Environ. Microbiol. 62: 133-140. Chapter2 52

6. Bally, M., E. Wilberg, M. Kiihni, and T. Egli. 1994. Growth and regulation of enzyme synthesis in the nitrilotriacetic acid (NT A) degrading Chelatobacter heintzii ATCC 29600. Microbial. 140: 1927-1936. 7. Brinkmann, U., and W. Babel. 1992. Simulataneous utilization of heterotrophic substances by Hansenula polymorpha results m enhanced growth. Appl. Microbial. Biotechnol. 37: 98-103. 8. Brooke, A. G., and M. M. Attwood. 1983. Regulation of enzyme synthesis during growth of Hyphomicrobium X on mixtures of methylamine and ethanol. J. Gen. Microbial. 129: 2399-2404. 9. Egli, T. 1995. The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates. p.305-386. In J. Gwynfryn Jones (ed.) Advances in Microbial Ecology, Plenum Press, New York. 10. Egli, T., C. Bosshard, and G. Hamer. 1986. Simultaneous utilization of methanol-glucose mixtures by Hansenula polymorpha in chemostat: Influence of dilution rate and mixture composition on utilization pattern. Biotechnol. Bioeng. 28: 1735-1741. 11. Egli, T., O. Kappeli, and A. Fiechter. 1982. Mixed substrate growth of methylotrophic yeasts in chemostat culture: Influence of dilution rate on the utilization of a mixture of glucose and methanol. Arch. Microbial. 131: 8-13. 12. Egli, T., N. D. Lindley, and J. R. Quayle. 1983. Regulation of enzyme synthesis and variation of residual methanol concentration during carbon-limited growth of Kloeckera sp. 2201 on mixtures of methanol and glucose. J. Gen. Microbial. 129: 1269-1281. Chapter2 53

13. Egli, T., H.-U. Weilenmann, T. El-Banna, and G. Auling. 1988. Gram-negative, aerobic, nitrilotriacetate-utilizing bacteria from wastewater and soil. System. Appl. Microbiol. 10: 297-305. 14. Fenchel, T. M., and B. B. Jorgensen. 1977. Detritus food chains of aquatic ecosystems: The role of bacteria. Adv. Microb. Ecol. 1: 1-58. 15. Galli, R., and T. Leisinger. 1988. Plasmid analysis and cloning of the dichloromethane-utilization genes of Methylobacterium sp. DM4. J. Gen. Microbiol. 134: 943-952. 16. Harder, W., and L. Dijkhuizen. 1976. Mixed substrate utilization, p. 297-314. In A. C. R. Dean, D. C. Ellwood, C. G. T. Evans and I. Melling (ed.). Continuous Culture 6. Applications and New Fields. Ellis Horwood, Chichester, England. 17. Harder, W., and L. Dijkhuizen. 1982. Strategies of mixed substrate utilization in microorganisms. Phil. Trans. R. Soc. Lond. B. 297: 459-480. 18. Harder, W., J. G. Koenen, and A. Matin. 1977. A review microbial selection in continuous culture. J. Appl. Bacteriol. 43: 1- 24. 19. Harte, M. J., and F. C. Webb. 1967. Utilization of mixed sugars in continuous fermentation. II. Biotechnol. Bioeng. 9: 205- 221. 20. Hirsch, P., M. Bernhard, S. S. Cohen, J. C. Ensign, H. W. Jannasch, A. L. Koch, K. C. Marshall, A. Matin, J. S. Pointdexter, S. C. Rittenberg, D. C. Smith, and H. Veldcamp. 1979. Life under conditions of low nutrient concentrations: Group report, p. 357-372. In M. Shilo (ed.). Strategies of Microbial Life in Extreme Environments. Dahlem Konferenzen, Berlin. Chapter2 54

21. Hutchinson, V., and C. W. Robinson. 1988. Kinetics of the simultaneous batch degradation of p-cresol and phenol by Pseudomonas putida. Appl. Microbial. Biotechnol. 29: 599-604. 22. Jannasch, H. W., and R. I. Mateles. 1974. Experimental bacterial ecology studied in continuous culture. Advances Microbial Physiol. 11: 165-212. 23. Kastner, J. R., and R. S. Roberts. 1990. Simulataneous fermentation of D-xylose and glucose by Candida shahatae. Biotechnol. Lett. 12: 57-60. 24. King, G. M. 1991. Measurement of acetate concentrations m marine pore waters by using an enzymatic approach. Appl. Environ. Microbiol. 57:3476-3481. 25. Kjelleberg, S., N. Albertson, K. FUirdh, L. Holmquist, A. Jouper-Jaan, R. Marouga, J. Ostling, B. Svenblad, and D. W eichart. 1993. How do non-differentiating bacteria adapt to starvation? Antonie van Leeuwenhoek. 63: 333-341. 26. Kohler-Staub, D., S. Hartmans, R. Galli, F. Suter, and T. Leisinger. 1986. Evidence for identical dichloromethane dehalogenases in different methylotrophic bacteria. J. Gen. Microbiol. 132: 2837-2843. 27. La Roche, S., and T. Leisinger. 1990. Sequence analysis and expression of the bacterial dichloromenthane dehalogenase structural gene, a member of the glutathione S-transferase supergene family. J. Bacteriol. 172: 164-171. 28. Leisinger, T., R. Bader, R. Hermann, M. Schmid-Appert, and S. Vuilleumier. 1994. Microbes, enzymes and genes involved in dichloromethane utilization. Biodegradation. 5: 237- 248. Chapter2 55

29. Lendenmann, U., M. Snozzi, and T. Egli. 1996. Kinetics of the simultaneous utilization of sugar mixtures by Escherichia coli in continuous culture. Appl. Environ. Microbiol. 62:1493-1499. 30. Locher, H. H., T. Leisinger, and A. M. Cook. 1989. Degradation of p-toluenesulphonic acid via sidechain oxidation, desulphonation and meta-ring cleavage in Pseudomonas (Comamonas) testosteroni T-2. J. Gen Microbiol. 135: 1969-1978. 31. Locher, H. H., T. Leisinger, and A. M. Cook. 1991. 4- sulphobenzoate 3,4-dioxygenase. Biochem. J. 274: 833-842. 32. Locher, H. H., C. Malli, S. W. Hooper, T. Vorherr, T. Leisinger, and A. M. Cook. 1991. Degradation of p-toluic acid (p-toluenecarboxylic acid) and p-sulphonic acid via oxygenation of the methyl sidechain is initiated by the same set of enzymes in Comamonas testosteroni T-2. J. Gen. Microbiol. 137: 2201-2208. 33. Loubiere, P., E. Gros, V. Paquet, and N. D. Lindley. 1992. Kinetics and physiological implications of the growth and behavior of Eubacterium limosum on glucose/methanol mixtures. J. Gen. Microbiol. 138: 979-985. 34. Morita, R. Y. 1988. Bioavailability of energy and its relationship to growth and starvation in nature. Can. J. Micro biol. 43: 436-441. 35. Morita, R. Y. 1993. Bioavailability of energy and the starvation state, p. 1-23. In S. Kjelleberg (ed.). Starvation in Bacteria. Plenum Press, New York. 36. Morita, R. Y., and C. L. Moyer. 1989. Bioavailability of energy and the starvation state, p. 7 5-79. In T. Hattori, Y. Ishida, Y. Maruyama, R. Y. Morita and A. Uchida (ed.). Recent Advances in Microbial Ecology. Japan Scientific Societies Press, Tokyo. Chapter2 56

37. Munster, U. 1993. Concentrations and fluxes of organic carbon substrates in the aquatic environment. Antonie van Leeuwenhoek. 63: 243-264. 38. Munster, U., and R. J. Chrost. 1990. Origin, composition, and microbial utilization of dissolved organic matter, 8-46. In J. Overbeck and R. J. Chrost (ed.). Aquatic Microbial Ecology, Biochemical and Molecular Approaches. Springer, New York. 39. OECD. 1994. Risk reduction monograph no. 2: Methylene chloride. Environment Directorate. OECD Environment Monograph Series. 96. 40. Reber, H. H., and R. Kaiser. 1981. Regulation of the utilization of glucose and aromatic substrates in four strains of Pseudomonas pudita. Arch. Microbiol. 130: 243-247. 41. Schneider, R. P., F. Zurcher, T. Egli, and G. Hamer. 1988. Determination of nitrilotriacetate in biological matricies using ion exclusion chromatography. Anal. Chem. 173: 278-284. 42. Schneider, C., V. Kircher, and T. Wiegmann. 1993. Mechanismen der erhohten biologischen Phosphateliminierung im Klarwerk Berlin-Ruhleben. Fachgebiet Hygiene des Fachbereichs Umwelttechnik der Technishe Universitat Berlin. 43. Schultz, S., and R. Conrad. 1995. Effect of algal deposition on acetate and methane concentrations in the profundal sediment of a deep lake (Lake Constance). FEMS Microbiol. Ecol. 16:251-260. 44. Silver, R. S., and R. I. Mateles. 1969. Control of mixed substrate utilization in continuous cultures of Escherichia coli. J. Bacteriol. 97: 535-543. 45. Tyree, R. W., E. C. Clausen, and J. L. Gaddy. 1990. The fermentative characteristics of Lactobacillus xylosus on glucose and xylose. Biotechnol. Lett. 12: 51-56. Chapter2 57

46. Veenhuis, M., K. Zwart, and W. Harder. 1978. Degradation of peroxisomes after transfer of methanol-grown Hansenula polymorpha into glucose-containing media. FEMS Microbiol. Lett. 3:21-28 47. Waters, J., and T. C. J. Feijtel. 1995. AIS/CESIO Environmental surfactant monitoring programme: Outcome of five national pilot studies on linear alkylbenzene sulphonate (LAS). Chemosphere. 30: 1939-1956. 48. Wellsbury, P., and R. J. Parkes. 1995. Acetate bioavailability and turnover in an estuarine sediment. FEMS Microbiol. Ecol. 17: 85-94. 49. Wong, T. Y., H. Pei, K. Bancroft, and G. W. Childers. 1995. Diauxic growth of Azobacter vinelandii on galactose and glucose. Appl. Environ. Microbiol. 61: 430-433. Cha.pter 3 58

Growth of Methylobacterium strain DM4 in Continuous Culture: Effects of Dilution Rate and Response to Substrate Shifts during Continuous Cultivation

Albert J. Tien and Thomas Egli

Key words : Acetate, carbon substrate shifts, continuous cultivation, dichloromethane, dilution rate, Methylobacterium sp., synthetic sewage

ABSTRACT

The pink pigmented, aerobic, Gram-negative, facultative methylotroph, Methylobacterium strain DM4, is able to utilize dichloromethane (DCM) as a sole carbon and energy source. Dynamics of DCM dehalogenase induction and DCM utilization were investigated during growth of strain DM4 under DCM limited conditions in continuous culture. Excess DCM specific oxygen uptake and residual DCM concentration in the culture medium was found to increase with dilution rate (0.03 hr 1 to 0.08 hr-1 ). In order to assertain the effects of carbon shifts on degradation of DCM, the feed of steady-state cultures (D=0.05 hr-1) was switched from a medium containing acetate to one containing only DCM and vice versa (carbon concentrations held constant at 200 mg 1-1 ). During steady-state growth on acetate the cells exhibited a low but distinct DCM utilizing capacity (measured via excess DCM induced oxygen uptake) of approximately 0.5 nM 02 (mg dwt)-1 min-1. When shifted from acetate to DCM, a two stage induction pattern was observed with approximately 29 hrs elapsing before maximal induction Chapter3 59

(twenty-fold over constitutive) of DCM dehalogenase activity occurred. Levels of DCM dehalogenase protein also increased proportionally with activity. Coincidently, the start of the second level of induction occurred with the end of a transient increase in the residual dichloromethane concentration within the chemostat. A transient shift from DCM to acetate in the feed resulted in a loss in DCM dehalogenase activity and enzyme concentration, consistant with "washout", to constitutive levels. Loss of the characteristic pink pigmentation to that of a yellow-tan color also occurred. A similar induction pattern as that seen during transient switch from acetate to DCM were seen when steady-state cultures of Methylobacterium strain DM4, cultivated with synthetic sewage, were fed a medium containing 10% DCM and synthetic sewage. As with the previous experiment, DCM was simultaneously utilized along with alternative substrates, in this case, synthetic sewage.

INTRODUCTION

Up until the eighties, dichloromethane (DCM) or more commonly, methylene chloride was considered to be non-degradable (15) or not "readily biodegradable" as defined in the OECD Guidelines for Testing of Chemicals (22). World production of DCM in 1991 was estimated to be 437,000 tonnes. The main uses of DCM are components of paint and varnish strippers, adhesive formulations; solvents in aerosol formulations; extractant solvent and process solvent in the food and pharmaceutical industries; or as a vapor degreasing solvent in the metal-working industries. Release into the enviroment by these industries is mainly into the air and, to a lesser extent, in water and soil (22). Over the past twenty years, DCM has been found to be utilized by a few strains of bacteria as a Chapter3 60 sole source of carbon and energy. The first step of DCM utilization is catalyzed by a glutathione-dependent DCM dehalogenase with the formation of formaldehyde which is a central metabolite of methylotrophic organisms (19). In natural and technical systems microorganisms are confronted with many transitory conditions such as spectrum and quality of carbon sources. Much of the knowledge on growth or degradative potential and the genetic regulation of DCM degrading organisms was elucidated in the laboratory with batch culture where DCM was supplied at the highest possible concentrations (19). Many researchers have pointed out that laboratory media containing high concentrations of single carbon substrates do not reflect true environmental growth conditions (21, 28) and growth on mixtures of substrates has been found to be the rule rather than the exception for heterotrophic organisms in the environment (9). Also DCM utilizing organisms have been found to simultaneously utilize mixtures of acetate and DCM (18, 25). In the latter study with continuous cultures, a few percent DCM carbon was necessary to fully induce the DCM degradative system. It is known that changing environmental conditions can strongly influence the physiological behavior of microorganisms. Continuous culture systems represent excellent tools for studying microorganisms under accurately controlled growth conditions (12, 23). The ability to maintain cells for long periods of time under rigorously controlled environmental conditions ensures that the physiological state of these cells is accurately and reproducibly tuned to the chosen conditions, permitting detailed studies of the manner which microorganisms respond to particular environmental· constraints. Moreover, exploiting the possibilities of using continuous cultures in a discontinuous mode greatly adds to its usefulness in studying microbial adaptability, flexibility and Chapter3 61 ressembles the hetrogeneity which is characteristic of natural systems (12). In our study using continuous cultures, we looked at the effects of changing DCM concentrations, e.g. shock loading or accidental DCM spills, and the possibility of (catabolite) repression of DCM utilization by multiple alternative substrates, e.g. synthetic sewage on DCM degradation in Methylobacterium strain DM4. Given that releases of DCM into the aquatic environment are primarily due to "accidental" wastewater discharges (22) understanding how DCM utilizing organisms respond to fluxes in DCM concentration is essential for designing treatment or cleanup strategies.

MATERIALS AND METHODS

Microbial strains and cultivation conditions. In experiments with mixtures of dichloromethane (DCM) and acetate, Methylobacterium strain DM4 (11) was used. Conditions for culture maintenance and batch growth have been previously described (16). For continuous culture, the medium contained per liter 0.3 g MgS04·7H20, 0.02 g CaCl2·2H20, 0.14 g KCl, 1.07 g NH4Cl, 0.02 g (NH4)2S04, 2.46 g Na2HP04, 5 ml of trace elements (Egli et al., 1988), 1.8 ml H3P04 (85%) and 10 mg silicon antifoarn (Fluka, Buchs, Switzerland) with the substrate concentration adjusted accordingly. Total carbon in the medium was 200 mg-C 1-1 either from DCM, acetate or a synthetic sewage. The synthetic sewage contained per liter 0.14 g peptone, 0.1 g meat extract, 0.009 g urea, 0.006 g NaCl, 0.18 g NaAc, 0.3 g MgS04·7H20, 0.02 g CaCl2·2H20, 0.14 g KCl, 1.07 g NH4Cl, 0.02 g (NH4)2S04, 2.46 g Na2HP04, 5 ml of trace elements (10), 1.8 ml H3P04 (85%) and 10 mg Chapter3 62 silicon antifoam (Fluk:a, Buchs, Switzerland). The bioreactor (MBR, Switzerland, 2 liter working volume) was aerated at 0.1 1 min-1 while stirred at 1,500 rpm. Experiments with synthetic sewage were performed at pH 7 .5 while all others were performed at pH 6.5. The pH was controlled by automatic addition of H3P04 (1 M) and KOH/NaOH (0.5 M, each). The temperature was maintained at 30°C. Determination of excess specific oxygen uptake rates. Culture liquid (80 ml) was collected directly from the chemostat, washed twice with and resuspended in 20 ml carbon-free medium. Substrate (DCM, acetate or SS) stimulated specific oxygen uptake rates were recorded at 30°C in a glass Clark-type oxygen electrode (Rank Brothers, Great Britain). The total volume of the assay was 3.0 ml, consisting of 2.8 ml of cell suspension plus 0.2 ml of a 0.1 M substrate solution. Sampling and assay times were standardized. All assays were preformed in triplicate. Quantification of biomass. Biomass was quantified by measuring dry weight or optical density. Dry weight (Dwt) was obtained by filtering cells through 0.2 µm pore size polycarbonate filters (Nuclepore, Pleasanton, CA, U.S.A.) which together were washed with distilled water to remove salts. Filters were dried at 105°C overnight to constant weight. Optical density was determined at 480 nm in 1 cm cuvettes using a Uvikon 860 spectrophotometer (Kontron, Zurich, Switzerland). Acetate analysis. Acetate was measured by high pressure ion exclusion chromatography as described by Schneider et al. (24). The detection limit was 1 mg l-1 acetate carbon. DCM measurements. Samples (2.5 ml) were taken directly from the chemostat, acidified with 1 drop of concentrated HCl, centrifuged for 7 min at 20,000g, with supernatant removed and stored at 4°C in teflon- Chapter3 63 lined crimp vials (Supelco, Inc., Bellefonte, Pa., USA). Loss of DCM during sampling (approximately 10 seconds), due to consumption ( < 0.001 %) before stabilization by acidification, was deemed acceptable given the analytical detection limits. DCM measurements were performed by gas chromatography (Hewlett Packard 5892 series II, U.S.A.) using a 60 m long, 0.32 mm diameter glass capillary column (J&W DB624), fitted with an ECD detector, by direct analysis. The injector and detector temperatures were maintained at 250°C. The oven temperature was maintained at 30°C with helium as the carrier gas. Total flow rate was 32 ml min-I. The detection limit was 0.1 mg 1-l DCM carbon. Dissolved organic carbon (DOC). Culture liquid (20 ml) from the chemostat was filtered through 0.2 µm pore size membrane filters (Nuclepore, Pleasanton, CA, USA) pre-rinsed with carbon-free distilled water. The filtrate was acidified with concentrated HCI, dissolved C02 stripped with N2 gas and DOC measured with a TOCOR2 total organic carbon analyzer (Maihak AG, Hamburg, Germany). Immunoquantification of the DCM dehalogenase. Culture suspension (2 ml) was collected from the bioreactor and cells were immediately harvested by centrifugation at 20,000g for 7 min. The pellet was resuspended in 0.2 ml sample buffer ( 17), boiled for 2 minutes, immediately frozen and stored at -20°C. Detection of the DCM dehalogenase proceeded similar to that described by Bally and Egli (3). Detection limit was approximately 0.1 ng of protein. Protein concentrations were measured by the method of Bradford (4) using bovine serum albumin as a protein standard. Purified DCM dehalogenase from strain DM4 and anti-DCM dehalogenase was kindly provided by Dr. S. Vuilleumier, ETH-Ziirich. Confirmation of pure cultures. lnoculum and continuous cultures were tested for contaminants by immunodetection with a method Chapter3 64 similar to that described by Bally and Egli (3). Polyclonal antibodies raised against strain DM4 were prepared after established proceedures (13) with heat inactivated whole bacterial cells. Antisera were purified by cross-reaction depletion against related and non-related organisms.

RESULTS

Growth in DCM limited chemostat culture. In this study we examined biomass, bacterial output, yield, and residual substrate concentration in DCM limited chemostat cultures of Methylobacterium strain DM4 by incremently adjusting the dilution rate (D) from a low of 0.03 hr-1 to nearly that of µmax at 0.08 hr-1. The fact that the culture continued to grow at D>µmax (0.074 hr-1) was an indication of undetected wall growth or that, perhaps due to toxic effects, µmax is not reached in batch systems at high concentrations of DCM (Fig. la). In addition to biomass concentration, substrate induced excess specific oxygen uptake rates (qsexcess) was examined. As a result of higher growth rates, substrate specific excess oxygen uptake rates for DCM increased from 26 nmol 02 (mg Dwt)-1 min-1 to 34 nmol 02 (mg Dwt)-1 min-1 while that of acetate remained constant at 2 nmol 02 (mg Dwt)-1 min-I (Fig. lb). Transient shifts from acetate to DCM as a single substrate. In order to investigate the time frame necessary to induce the DCM metabolizing enzyme system in the Methylobacterium, strain DM4 was grown in continuous culture (D= 0.05 hr-1) on acetate as the sole source of carbon and energy. During establishment of steady-state conditions, it was observed that the color of the culture shifted from pink to that of a yellowish-tan. Immunodetection confirmed this culture to be Chapter 3 65

0.7 0.20 0.07 a)

0.6 0.06

,...., 0.15 0.003 .-.. "';' 0.5 .-.. 0.05 'Z L..c: ...cc :s ...... _,t\1:1 ... .-.. .:..:i '" ~ .Cl"' 0.4 ... ~ 0.04 ._,C)I) rr.i= .::= ._,C)I) ~ .Cl 0.10 0.002 j ... "' ~ it: 0.3 rr.i= 8= 0.03 0= Q -; Q ca ~ "O ~ ·c "O = Cl ·m ~ ;:;; 0.2 ~ 0.02 0.05 0.001 i:i::i=

0.1 0.01

o.o 0.00 0.00 .l-...----111--..-111-=;:=9:::;:::!t:::::!.._J.o.ooo 0.02 0.09 D Dilution Rate (hrl) c

b) _____ ...... ----·------.

O+---.~""T""___,,...... -.-~..-...... ~-.---.~-.---1 0.03 0.04 0.05 0.06 0.07 0.08

Dilution Rare (hr-1)

Figure 1. a) Relationship between bacterial concentration, substrate concentration,

bacterial output and yield in steady-state cultures of Methylobacterium strain

DM4, at differing dilution rates in a DCM limited chemostat. The ordinates are as follows: C•1 residual DCM concentration (g 1-1) in the culture vessel; [e] biomass (g 1-1 ); [A] bacterial output (g 1-l hr-1 ); [O] yield [g Dwt (g Substratet 1]. The abscissa: Dilution rate (hr-1) and De, calculated critical

dilution rate of 0.074 hr-1. b) Excess DCM specific oxygen uptake C•1 and excess acetate specific oxygen uptake [A]. Cluipter 3 66 strain DM4. After steady-state conditions were reached, the feed was switched to a medium containing DCM as the sole source of carbon and energy. For the first 20 hrs biomass, as indicated by optical density of the culture, decreased with an unexplainable lag following theoretical wash- out patterns. After this point, biomass began to increase and reached a new steady-state after 50 hrs (Fig. 2a). The yield coefficient for DCM (Y x/DCM= 0.33 g dry biomass [g DCM]-1 is slightly lower than that of acetate (Yx/Ac = 0.35 g dry biomass [g acetate]-1 thus explaining the difference in biomass from the start point to that of the endpoint of the experiment. Measurement of residual DCM concentrations in the chemostat indicated an accumulation of DCM to a maximum concentration of approximately 2 mg 1-1 DCM carbon at 14 hrs decreasing to below detection after around 20 hrs. Theoretical wash-in concentrations of DCM at 14 hrs if no utilization were to occur would be 101 mg 1-1 DCM carbon. This corresponds to a 98% removal efficiency of DCM at this point (Fig. 3). DCM specific oxygen uptake rates indicate a two stage induction pattern. Up to 6 hrs after the switch from acetate to DCM, the excess specific oxygen uptake rates for DCM remained around 0.5 nM 02 (mg dwt)~ 1 min-1 which corresponds to the basal levels seen in the previous experiment. After this point, DCM specific oxygen uptake rates increased to 2 nM 02 (mg dwt)-1 min-1 and remained so for the next 20 hrs. At around 29 hrs the DCM specific excess oxygen uptake rates increased to a maximum of approximately 24 nM 02 (mg dwt)-1 min-1 and remained so for the duration of the experiment (Fig. 4a.). During the course of the experiment, the DCM specific utilization rates (qDCM), remained relatively constant [0.15 g-C (g dwt)-1 hr-1] except for a slight hump attributed to lower biomass (Fig Sa). By the end of the experiment, culture color returned to the normal pink color. Increase in levels of Chaprer3 67

Figure 2. Growth of Methylobacterium strain DM4 in carbon-limited continuous culture (dilution rate 0.05 hr-1) as a result of switching carbon sources. Temperature was held at 30°C with pH regulated at 6.8. a) Switch from acetate to dichloromethane (200 mg C 1-1 each). b). Switch from dichloromethane to acetate (200 mg C 1-1 each). Changes in biomass, as optical density, are shown as [e] while the theoretical wash out of biomass is shown as [O] .

3 105 I ...... --- u I "O ~ 2 8 I!( c:P- ~~ - -c 100 ~ '-' ::::: lf)- .- ~ uJF- - :;J u ~ Q u -; Q 1 I'.\ 95 "O= I ' -=41 -~ I '.. ~ ml ~ "' ' 0 • ' 90 0 20 80 100 Time tbrs)

Figure 3. Residual dichloromethane (DCM) concentration in carbon-limited culture of Methylobacterium strain DM4 at D = 0.05 br-1 during carbon shift from acetate to DCM. [•]represents residual DCM in culture vessel (mg C 1-1 ). [O] represents percentage DCM utilized compared to that of the influent (200 mg C 1-1 ). ( ···)signifies the theoretical wash in curve of DCM. ~ a) b) ~= Q> .lll: = ..... c. • ~ -.... 3 • - e= = ..... ~ ...... • • ....~ 0 ~ y -t> • a= "CJ ·c:i ~ Q> c...... e VJ. N ~ 0 f -Q .cIll § -:I VJ. Ill - ~ ~ ~

20 40 60 80 100 0 10 20 30 40 50 60 Time (hrs) Time (hrs)

Figure 4. Substrate induced excess specific oxygen uptake rates in carbon-limited culture of Methylobacterium strain DM4 during carbon substrate shift. a) Shift from acetate to dichloromethane (200mgC1-I each). b) Shift from dichloromethane to acetate (same as above). [.A] represents acetate excess specific oxygen uptake. £•1 represents dichloromethane (DCM) excess specific oxygen uptake rate. [O] represents theoretical wash out of DCM degrading activity while [£\]the theoretical wash out of acetate degrading activity. ....~ ~ b) ~ a) =0 ....~ ,-.. ·- .-I N i.. ·--.... .c 0.2 ·-~ .-I Cj ....I ~ =Cj "O ------·-- ·4-~... ~---~-----~--- ~ bl) ---·-- ·-c. rJ:J. u ....~ bll 0.1 f '-" ....

0 20 40 60 80 1000 10 20 30 40 50 60 Time (hrs) Time (hrs)

Figure 5. Substrate specific utilization rates in carbon-limited culture of Methylobacterium strain DM4 during carbon substrate shift. a) Shift

from acetate to dichloromethane (200 mg C 1-1 each). b) Shift from dichloromethane to acetate (same as above). [•]represent

acetate specific utilization rate (qacetate) while [e] represents dichloromethane specific utilization rate (qocM). Chapter3 70

DCM dehalogenase were proportional to DCM excess oxygen uptake rates (Fig. 6a). Transient shifts from DCM to acetate as a single substrate. Strain DM4 was initially cultivated in a carbon-limited chemostat culture at a constant dilution rate of 0.05 hr-1 with DCM supplied in the feed. After the establishment of steady-state conditions, the feed was switched to an acetate containing medium. Biomass as shown from optical density increased with time after the substrate shift which is in accordance to the higher yield coefficient of Mb. strain DM4 for acetate compared to that of DCM (Fig. 2b ). DCM specific excess oxygen uptake rate of the cells began to decrease after two hours. This decrease followed the theoretical wash-out curve of DCM activity (Fig. 4b ). A basal DCM specific excess oxygen uptake of approximately 0.5 nM 02 (mg dwt)-1 min-1 was always observed, it did not decrease to zero after > 100 hrs. Acetate specific utilization rate (qAc), discounting variances in biomass, remained around 0.14 g-C (g dwt)-1 hrl (Fig. 5b). Analysis of residual acetate concentrations indicates that acetate was immediately utilized after the substrate shift. Similarly, decreases in the level of DCM dehalogenase were proportional to DCM specific excess oxygen uptake rates (Fig. 6b ). Color of the culture shifted from the characteristic pink pigmentation to that of a tan-yellow. Use of indirect labelling techniques with specific surface antibodies raised against strain DM4 indicated that the tan-yellow culture was also immunogenically positive and not a contaminant. Transient shifts from synthetic sewage to a synthetic sewage containing 10% DCM. Much of the DCM which is found in aquatic environments first enters into wastewater treatment systems before being released into surface· waters. In order to see the time necessary for DCM induction in a model municipal wastewater, strain 25 a) b)

en ...... 20 ~ C'. '\ ~ -~ ,1~------1 \ 0 Q 15 -.c ._,~ " Q ~\ =Q> ~ ·-0 10 1-c /{~ u ~- -.l Q ...... ~ I l-~?~ ...... e s ,,,. ,,,{ ~------0 ------0 20 40 60 80 1000 10 20 30 40 50 60

Time (hrs) Time (hrs)

Figure 6. Dichloromethane (DCM) dehalogenase concentration [O] in carbon-limited culture of Methylobacterium strain DM4 during carbon substrate shift a) Shift from acetate to dichloromethane (200 mg C 1- l each). b) Shift from dichloromethane to acetate (same as above). Chapter3 72

DM4 was first cultivated in a carbon-limited chemostat culture (total carbon 200 mg 1-1) at a constant dilution rate of 0.05 hr-1 with synthetic sewage supplied in the feed. After the establishment of steady-state conditions, the feed was switched to a synthetic sewage containing medium with 10% DCM. Measurement of DCM concentrations demonstrate a transient increase in residual DCM to 1 mg 1-1 as carbon centered around 8 hrs. DOC measurements also showed a slight increase in residual DOC centered around 10 hrs (Fig. 7a). A similar pattern with the acetate to DCM shift experiment was seen with the exception that maximal induction for the DCM degradative system occurred approximately 10 hrs sooner after the shift to synthetic sewage with 10% DCM (Fig. 7b). An eighteen fold increase in DCM specific excess oxygen uptake rate above a constitutive level of 1 nM 02 mg-1 dwt min-1 was observed. Synthetic sewage consumption rates initally followed that of theoretical washout until remaining constant approximately 25 nM 02 (mg dwt)-1 min-1. DCM dehalogenase levels were not measured but, as with the previous experiment, can be expected to proportionally increase with the DCM specific excess oxygen uptake rates. Biomass remained constant during the experiment. No color change was seen in these experiments.

DISCUSSION

Due to the lack of carbon/energy in many aquatic and terrestrial ecosystems, starvation/survival is the normal "lifestyle" for most heterotrophic microorganisms. Most of the organic matter found in these Chapter3 73

20 ...... ---.. - r-/· -- . 10 j .19 o'1- ...

b)

.... • 20 ...... ·--·------...

10

Time (hrs)

Figure 7. Growth of Methylobacterium strain DM4 in carbon-limited continuous culture

(dilution rate 0. 05 hr 1) as a result of switching carbon sources from

synthetic sewage to 10% dichloromethane in synthetic sewage (total carbon

200mgC1-l). Temperature was held at 30°C with pH regulated at 7.5. a) [•l represent dichloromethane excess specific oxygen uptake rates while [O] represents synthetic sewage excess specific oxygen uptake rates. b) £•] represents the residual dichloromethane concentration and £•] represents the residual dissolved organic carbon (DOC) levels in the culture vessel. Chapter3 74 ecosystems are not readily available and, therefore, low microbial growth rates are typically measured (21). Derepression of many catabolic pathways occur under carbon-limitation and the ability to utilize many substrates is considered normal and not an anomaly (9). It is not expected that heterotrophic microorganisms would use pollutants as single substrates but rather in combination with other naturally available carbon sources. It is believed that a few percent pollutant carbon in relationship to available natural carbon sources is necessary to fully induce pollutant degrading systems (3, 26). In these two previous studies, constitutive levels of pollutant degrading enzymes could be measured. However, nature is not in steady-state. On the contrary, it is always m a state of flux where many carbonaceous substrates, including pollutants, are only transiently available. Adequate metabolic flexibility is necessary for organisms to compete and survive in the environment. The ability to utilize organic pollutants may allow some organisms a selective advantage. Unfortunately in many cases the ability to degrade pollutants is not constitutive but (2, 26). However, if these organisms are to be harnessed for waste stream processing or if the nature in which pollutant removal in the environment is to be truly understood, the factors which influence the expression of these inducible systems must be examined. Enzyme concentration and enzyme activity at reduced growth rates: responses to decreasing substrate concentrations. Microorganisms are versatile and adapt themselves very efficiently to the imposed nutrient deprived conditions. Adjusting the dilution rate from near µmax (approximately 0.1 hr-1 in our case) to well below 0.01 hr-1 does result in nutrient availability from near-saturation levels to far below Ks values of the organism. In response to such dramatic decreases in substrate availability, microorganisms maintain the highest possible rate of metabolism and growth by increasing their nutrient uptake potential Chapter3 75 through 1) increases in the V max of the uptake system or 2) by using a transport or catabolic system with a higher affinity or 3) internal enzyme content adjustment (5,12). Accordingly, several fold increased levels and activities have been observed in many microorganisms for many different intracellular enzymes involved in the initial steps of the catabolism of the growth limiting nutrient at decreasing dilution rates (2, 6, 8). The opposite relationship was seen in the Methylobacterium. At higher dilution rates, increased levels and activity of the DCM dehalogenase were measured. A plausible explanation is that since transport of DCM is thought not to be regulated, increased levels of DCM in the cell may be fatal. Detoxification by dehalogenation converts DCM to formaldehyde, a toxic metabolite whose intracellular levels are tightly regulated in certain methylotrophs (1). Enzymes similar to DCM dehalogenase from the Theta class of glutathione transferase have been isolated in blood from humans exposed to DCM and in liver tissue from rats exposed to DCM indicating a similar detoxifying function (19). Enzymatic response to transient growth behavior. The authors are only aware of a few published reports concerning actual experiments with the transient behavior of microbial cultures using different carbon sources (3, 25, 27). A key paper on this type of experimental approach is that by Standing et al. (25) where the effect of switching medium feed from glucose (constitutive enzymes) to xylose (inducible enzymes) in continuous cultures of Escherichia coli was investigated. The only shortcoming of that study was that the specific activities of the respective enzymes were not examined. A recent paper by Bally and Egli (3) concerning transient responses of the nitrilotriacetate (NTA) degrading Chelatobacter heintzii to carbon switch in the medium demonstrates similar trends with the Standing et al. paper with respect to biomass and residual substrate concentration. In addition, Chapter 3 76 the activities of glucose metabolizing enzymes (constitutive) and NTA metabolizing enzymes (inducible) were monitored. The decrease in biomass during the switch from glucose to NTA was attributed to the time needed for induction of the NT A metabolizing enzymes. The same response was also reported for Comamonas testosteroni, a p- toluenesulfonate (TS) degrader, when the feed was switched from acetate to TS (27) and Methylobacterium strain DM4 from the present study. A period of 50 hrs was necessary for full induction of the DCM metabolizing enzymes when the feed was switched from acetate to DCM. A decrease in the residual DCM concentration with subsequent increases in both the activities of the DCM degrading system and concentration of the DCM dehalogenase protein signaled the induction of the DCM metabolizing enzymes. In previous studies, the addition of other easily utilizable substrates decreased the time necessary for induction of the pollutant degrading enzymes by making available more energy for the rearrangement of cellular metabolism (3, 27). This trend was seen with strain DM4 when medium containing synthetic sewage was switched to synthetic sewage with 10% DCM. A 33% quicker induction of the DCM degrading system was seen compared to a switch from acetate to DCM. These similarities indicate a common response to transiently available substrates which require inducible enzyme systems in four distinctly related strains with three chemically distinct pollutants. Why does color of the culture vary in response to carbon type? Not much is known about why Methylobacterium strain DM4, a member of the pink-pigmented facultative methylotrophs (PPFMs), produces its characteristic pink pigment when grown on DCM or . other factors which influence its pigmentation. It has been reported that the pink pigment found in PPFMs are oxo-carotenoids similar to rhodoxanthin and serves as a prophylactic against UV radiation m Chapter3 77

stationary-state cells (7,14). Cultures of Methylobacterium extorquens (formerly P. extorquens NCIB 9399), a close relative to strain DM4, grown on ethanol (batch) produced a yellow pigmented culture while those grown on methanol (batch) exhibited a normal pink pigmented culture. It was thought that ethanol or a metabolic intermediate may inhibit the oxidation of the a-carotene causing an accumulation of a yellow a-carotene precursor (7). Similarly, in our study, transient carbon shifts from DCM to acetate produced yellow-tan pigmented cultures of strain DM4 in continuous culture. However, when strain DM4 was grown on acetate in continuous culture and the carbon source was switched to DCM, the characteristic pink pigmentation returned.

CONCLUSIONS

Rarely are pollutants the sole source of carbon and energy in the natural environment or engineered systems. Fluxes in the spectrum and quantity of alternative substrates can affect the degradation of pollutants in these systems. From the results a better understanding of the factors which influence the degradation of DCM have been presented and may be applied. In technical systems, transient "slugs" of DCM may actually inhibit the other microorganisms found in the activated sludge resulting in decreased performance. From our experiments it can be surmised that addition of easily utilizable substrates can decrease the time necessary for induction of pollutant degrading systems and that the use of pre-induced organisms can reduce the levels of pollutants such as DCM in wastewater treatment systems before they are released into the aquatic environment. Chapter] 78

ACKNOWLEDGEMENTS

The authors are indebted to the Swiss National Science Foundation (Project No. SPP 5001.35285) who financed the work of A. Tien. We would also like to acknowledge Prof. A. M. Cook for his imput and critical discussions and the working group of Prof. Th. Leisinger for use of their laboratory facilities. We appreciate the technical assistance of F. Junker, Dr. S. Vuilleumier and the graphical expertise of Dr. S.-C. Ko.

REFERENCES

1. Attwood, M. M., and J. R. Quayle. 1984. Formaldehyde as a central intermediary metabolite of methylotrophic metabolism. pp. 315-323. In R. L. Crawford and R. S. Hanson (ed.). Microbial Growth on C -Compounds. American Society for l Microbiology.Washington, D.C. 2. Bally, M. 1994. Physiology and Ecology of Nitrilotriacetate Degrading Bacteria in Pure Culture, Activated Sludge and Surface Waters. Diss. ETH Nr. 10821. Ph.D. Swiss Federal Institute of Technology Zurich. 3. Bally, M., and T. Egli. 1996. Dynamics of substrate consumption and enzyme synthesis in Chelatobacter heintzii during growth in carbon-limited continuous culture with different mixtures of glucose and nitrilotriacetate. Appl. Environ. Microbiol. 62: 133-140. 4. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72: 248-254. Chapter 3 79

5. Button, D. K. 1985. Kinetics of nutrient-limited transport and microbial growth. Microbiol. Rev. 49: 270-297. 6. Dean, A. C. R. 1972. Influence of environment on the control of enzyme synthesis. J. Appl. Chem. Biotechnol. 22: 245-259. 7. Downs, J., and D. E. F. Harrison. 1974. Studies on the production of pink pigment in Pseudomonas extorquens NCIB 9399 growing in continuous culture. J. Appl. Bact. 37: 65-74. 8. Duetz, W., A., S. Marques, C. De Jong, J. L. Ramos, and J. G. Van Andel. 1994. Inducibility of the TOL catabolic pathway in Pseudomonas putida (p WWO) growing on succinate in continuous culture: Evidence of carbon catabolite repression control. J. Bacteriol. 176: 2354-2361. 9. Egli, T. 1995. The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates. p.305-386. In J. Gwynfryn Jones (ed.) Advances in Microbial Ecology, Plenum Press, New York. 10. Egli, T., H ... u. Weilenmann, T. El-Banna, and G. Auling. 1988. Gram-negative, aerobic, nitrilotriacetate-utilizing bacteria from wastewater and soil. system. Appl. Microbiol. 10: 297-305. 11. Galli, R., and T. Leisinger. 1988. Plasmid analysis and cloning of the dichloromethane-utilization genes of Methylobacterium sp. DM4. J. Gen. Microbiol. 134: 943-952. 12. Gottschal, J. C. 1992. Continuous Culture. pp. 559-572. In J. Lederberg (ed.). Encyclopedia of Microbiology. Academic Press, London. 13. Harlow, E., and D. Lane. 1988. Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, New York. 726 p. 14. Ito, H., and H. Iizuka. 1971. Part XII. Taxonomic studies on a radio-resistant Pseudomonas. Agric. Biol. Chem. 35: 1566-1571. Chapter3 80

15. Klecka, G. M. 1982. Fate and effects of methylene chloride in activated sludge. Appl. Environ. Microbiol. 44: 701-707. 16. Kohler-Staub, D., S. Hartmans, R. Galli, F. Suter, and T. Leisinger. 1986. Evidence for identical dichloromethane dehalogenases in different methylotrophic bacteria. J. Gen. Microbiol. 132: 2837-2843. 17. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685. 18. LaPat-Polasko, L. T., P. L. McCarty, and A. J. B. Zehnder. 1984. Secondary substrate utilization of methylene choride by an isolated strain of Pseudomonas sp. Appl. Environ. MicrobioL 47: 825-830. 19. Leisinger, T., R. Bader, R. Hermann, M. Schmid-Appert, and S. Vuilleumier. 1994. Microbes, enzymes and genes involved in dichloromethane utilization. Biodegradation. 5: 237-248. 20. Morita, R. Y. 1993. Bioavailability of energy and the starvation state. pp. 1-23. In S. Kjelleberg (ed.). Starvation in bacteria. Plenum Press, New York. 21. Morita, R. Y. 1992. Low-nutrient environments. pp. 617-624. In J. Lederberg (ed.). Encyclopedia of microbiology. Academic Press, London. 22. OECD. 1994. Risk reduction monograph no. 2: Methylene chloride. Environment Directorate. OECD Environment Monograph Series. 96. 23. Pirt, S. J. 1985. Principles of Microbe and Cell Cultivation. Blackwell Scientific Publications, Oxford. 271 p. 24. Schneider, R. P., F. Zurcher, T. Egli, and G. Hamer. 1988. Determination of nitrilotracetate in biological matrices using ion exclusion chromatography. Anal. Biochem. 173:278-284. Chapter3 81

25. Standing, C. N., A. G. Fredrickson, and H. M. Tsuchiya. 1972. Batch- and continuous-culture transients for two substrate systems. Appl. Microbiol. 23: 354-359. 26. Tien, A. J., and T. Egli. 1996. Regulation of pollutant degradation in the presence of an alternative substrate by the para- toluenesulphonate utilizing Comamonas strain T-2 and the dichloromethane utilized Methylobacterium strain DM4 during growth in batch and continuous culture. Appl. Environ. Microbiol. (submitted) 27. Tien, A. J., F. Junker, A. M. Cook, and T. Egli. 1996. Dynamics of substrate consumption and enzyme activity in response to carbon substrate shifts by Comamonas testosteroni strain T-2 m carbon-limited continuous cultures. Biodegradation. (submitted) 28. Veldkamp, H., and H. W. Jannasch. 1972. Mixed culture studies with the chemostat. J. Appl. Chem. Biotechnol. 22: 105-123. Chapter4 82

Dynamics of Substrate Consumption and Enzyme Activity by Comamonas testosteroni strain T-2 in Response to Carbon Substrate Shifts in Carbon-Limited Continuous Cultures

Albert J. Tien, Frank Junker, Alasdair Cook, and Thomas Egli

Key words: Acetate, carbon substrate shifts, Comamonas sp., continuous culture, toluenesulfonate

ABSTRACT

Comamonas testosteroni strain T-2 utilizes p-toluenesulfonate (TS) as a sole source of carbon and energy for growth via p-sulfobenzoate and protocatechuate. Regulation of TS degradation was examined in continuous culture at a dilution rate of 0.05 h-1 starting from carbon- limited conditions where the feed (200 mg 1-1 as carbon) was switched from a medium containing the pollutant as the sole carbon and energy source to one containing an alternative carbon source (acetate) and vice versa. Replacing TS with acetate resulted in the rapid decrease in toluenesulfonate methylmonooxygenase system (TSMOS) activity, measured by excess TS specific oxygen uptake. When the feed was switched from acetate to TS, a sigmoidal induction pattern in TSMOS activity was seen. The first level of induction occurred rapidly after 2 hrs and lasted approximately 10 hrs with the second level reaching a maximum after about 15 hrs. The response to a switch from acetate to different mixtures of acetate plus TS was investigated. Strain T-2 was able to simultaneously utilize TS and acetate. Replacing acetate with 10% TS/90% acetate and 40% TS/60% acetate resulted also in a sigmoidal Chapter4 83 induction pattern with maximal TSMOS activity around 3.5 hrs. and 5.5 hrs, respectively. Simultaneous utilization of alternative substrates decrease the lag period before induction occurred.

INTRODUCTION

Toluenesulfonate (TS) is the least substituted member of the linear alkylbenzesulfonate (LAS) group of compounds. It is typically used as a cloudpoint depressant in many liquid detergents. Up until nearly the 1950's, TS was considered to be non-biodegradable (7). As it has been the case for many other "recalcitrant" compounds, the biodegradability of TS has been elucidated under laboratory conditions with pure cultures that can utilize TS as a sole source of carbon and energy. One organism, Comamonas testosteroni strain T-2, has been particularly well studied [for more information on the metabolic pathways and history of these strains refer to references (12, 15, 20)]. Growth on TS as the only carbon source at high concentrations, however, contrasts with the prevailing conditions found in the environment as well as in wastewater treatment systems, where pollutant-degrading bacteria rarely encounter such compounds at high concentrations or as sole carbon sources (8). Studies with steady-state continuous cultures of Cs. testosteroni strain T-2 demonstrate that under carbon-limited conditions, TS and alternative substrates were simultaneous utilized (23). In related studies, it was observed for nitrilotriacetate and dichloromethane degrading organisms that enzyme expression of these pollutant degrading enzyme systems may be dependent on the ratio of pollutant carbon in the total available carbon pool (2, 24 ). Chapter4 84

Rarely, however, are conditions considered steady-state in many natural systems and wastewater treatment facilities. Often bacteria experience periods of "feast or famine" and therefore have evolved the ability to respond to environmental changes by altering or adjusting their metabolic equipment to meet these challenges. Metabolic activities such as the biodegradation of recalcitrants have to be carried out over a range of temperatures, oxygen partial pressures, pH values, redox potentials and, most importantly, variations in the spectrum and concentrations of existing alternative substrates. Transient environmental conditions and the resulting lag phases due to adaptation mechanisms can and will affect the efficiency of biodegradation over time. From a practical sense it is necessary to know how rapidly microbes are able to adapt to the input of a pollutant which potentially may be used as a substrate whether in wastewater treatment systems or ·in the natural environment. In order to further understand the dynamic behavior of pollutant degradation under transient nutrient conditions, Cs. testosteroni strain T-2 was used in a model system to study the time scale involved in the induction of TS metabolism during changing feed composition.

MATERIALS AND METHODS

Microbial strains and cultivation conditions. Comamonas testosteroni strain T-2 (DSM 6577) was used in all experiments. Precultivation conditions and individual medium compositions used in the different transient experiments have been previously described (23). Carbon concentration in the feed medium from TS, acetate or mixtures of both was held at 200 mg i-1. When switching the feed from one medium type to another, a sterile air gap was used to prevent mixing of the two Chapter4 85 media. The bioreactor (MBR, Wetzikon, Switzerland, 1.8 1 working volume) was aerated at 0.5 1 min-1 while stirred at 1,500 rpm and pH controlled at 7 .5 with H3P04 (1 M) and KOH/NaOH (0.5 M, each). The temperature was maintained at 30°C. Determination of excess TS and acetate specific oxygen uptake rate of cells. Culture liquid (80 ml) was collected directly from the chemostat, washed twice with and resuspended in 20 ml carbon- free medium. Excess substrate specific oxygen uptake rates (qols,excess and q02Acetate,excess) were recorded at 30°C in a Clark-type oxygen electrode (Rank Brothers, Great Britain). The total volume of the assay was 3.0 ml, consisting of 2.8 ml of cell suspension with a known optical density plus 0.2 ml of a 0.1 M substrate solution. Sampling and assay times were standardized. All assays were preformed in triplicate. Quantification of biomass. Biomass was quantified by measuring dry weight (dwt) or optical density. Dry weight was obtained by filtering culture liquid through 0.2 µm pore size polycarbonate filters (Nuclepore, Pleasanton, CA, U.S.A.). Cells retained on the filter were subsequently washed with distilled water. Filters were dried at 105°C overnight to constant weight. Optical density was determined at 480 nm using a Uvikon 860 spectrophotometer (Kontron, Ziirich, Switzerland). Analysis of substrates. Residual concentrations of acetate were measured by high pressure ion exclusion chromatography as described by Schneider et al. (21). The detection limit was 1 mg 1-1 acetate carbon.

Determination of TS concentration was performed by high pressure liquid chromatography after Locher (1991). The detection limit was 0.1 mg 1-1 TS carbon. Chapter4 86

RESULTS

Shift from acetate to TS. To determine the time necessary to induce the TS degradative enzymes, strain T-2 was grown by continuous culture with acetate as the sole source of carbon and energy. When steady-state was achieved, the feed was changed to TS as the sole carbon and energy source. Analysis of residual TS concentrations indicates that a gradual build up of TS (maximum of 5.5 mg I-1 TS-carbon) occurred, decreasing to around 1 mg 1-1 TS-carbon after 18 hrs. Two hours after the shift from acetate to TS, nearly 85-90% of the supplied TS was utilized and by eight hours nearly 98% of this compound was utilized (Fig. 1). During the initial 10 hrs biomass, as indicated by change in optical density, decreased slightly faster that of the theoretical wash-out profile. A possible explanation for this may be the "detergent" effect of accumulating TS leading to partial cell lysis. However, after some 10 hrs the culture recovered, growth resumed and biomass reached steady-state values after 45 hrs and was higher than the initial biomass level. This can be attributed to the higher yield of strain T-2 during growth with TS (Y x/s=0.36) as compared to growth with acetate (Y x/s=0.32) (Fig. 2a).

The q02TS,excess indicate that induction of the TS degradative enzymes followed a sigmoidal pattern. The inital qo2TS.excess was approximately 1 nM 02 (mg dwt)-1 min-1 and within 1 hr, increased to about 14 nM 02 . (mg dwt)-1 min-1 and remained so for the next 8 hrs. At 15 hrs the final step was attained, with an activity of approximately 30 nmoles 02 (mg dwt)-1 min-1. Repression of qo2Acetate,excess exhibited a decreasing trend and it is thought that accumulating TS may have affected the uptake system for acetate which resulted in the inital scattering of the

q02Acetate,excess data (Fig. 3a). The TS specific utilization rate increased 6 I 100 I Q I ~ ~ 5 I 90 ""! I -i::.. "O .-. I Qi ~ N I .... 4 I 80 -.... u-I I ;:;;, ~ I TS rJJ.- e • E-4 I percent TS 00 3 70 -E-4 I utilized -=Qi • ~ ~ I Theoretical Qi - --- Washin ~ ...."O= I Cll 2 I 60 00 ~ -.....1

• 50 ~~

0 40 0 5 10 15 40 60 80 100 120 Time (hrs)

Figure l. Residual toluenesulfonate (TS) concentration in carbon-limited culture of Comamonas testosteroni during carbon shift from acetate to

TS. C•) represents residual TS in culture vessel (mg C l-1 ). C•) represents percentage TS utilized compared to that of the influent (200 mg C l-1 ). (---) signifies the theoretical wash in curve of TS. 0.5 a) b) • Change in OD due to transient _...... - 0 Theoretical change in OD due to washout a 0.4 4) .,...... = / ------..- i e/ _,...... "2' \ I _.. 0.3- ,o ~.... .1 r,,.:i I °'b ·--~ oo I 0.2- 0 Q \ ob 0 -u= ' 0 Q. ' . ·- 0.1- ... 0- 0 0 0 00 oo 00 0.0 I • • I I 0 10 20 30 40 50 600 10 20 30 40 50 Time (hrs) Time (hrs)

Figure 2. Growth of Comamonas testosteroni in carbon-limited continuous culture (dilution rate 0.05 hr- I) as a result of switching carbon sources.

Temperature was held at 30°C with pH regulated at 7.5. a) Switch from acetate to toluenesulfonate (200mgC1-l each). b). Switch

from toluenesulfonate to acetate (200 mg C 1- l each). Changes in biomass, as optical density, are shown as (•) while the theoretical

wash out of biomass is shown as (0). 4 a) b) -A- Acetate stimulated oxygen uptake -4-- TS stimulated oxygcn uptake • - - Thooretiail wash out of actiate activity ------Thooraiail wash out of TS activity 3 •

1 ...... '~"' A. ... -- ... ------· 10 20 30 40 500 20 40 60 80 100 120 Time (hrs) Time (hrs)

Figure 3. Excess substrate specific oxygen uptake rates in carbon-limited culture of Comamonas testosteroni during carbon substrate shift. a)

Shift from acetate to toluenesulfonate (200 mg C l- l each). b) Shift from toluenesulfonate to acetate (same as above). (A) represents

q02Acetate,excess. (•)represents qo2TS,excess. (--)represents theoretical wash out of TS degrading activity while(· - ·)the theoreti- cal wash out of acetate degrading activity. Chapter4 90 immediately from zero to almost the final steady-state level of 2 g-C (g dwt)-1 hr 1 during the shift from acetate to TS because also in the initial phase most of the TS entering the reactor was utilized by the culture (Fig. 4a). Shift from TS to acetate. In order to elucidate the effect of carbon replacement on TS degradation capacity by strain T-2, a steady- state culture provided with TS in the feed was supplied with a medium containing acetate as the sole carbon and energy source. Residual TS concentrations indicate that even as acetate was being washed in, residual TS was being consumed as indicated by the steeper slope as compared to the theoretical wash-out of the TS (data not shown). Acetate concentrations during the experiment were below detection limits. This indicates that both the remaining TS and acetate were used simultaneously. During the first 5 hrs the culture biomass paralleled that of the predicted theoretical washout curve. This can be attributed to the fact that the acetate utilization systems had to be induced and although it was utilized to completion - the cells were not able to build it into biomass. Steady-state biomass values (15 hrs) were lower than the initial values with TS due the lower growth yield on acetate.(Fig. 2b). The q02TS,excess decreased with the switch to acetate, followed the theoretical washout profiles and remained at 1-2 nmoles 02 (mg dwt)-1 min-1. Initial q02Acetate,excess was approximately 1 nmole 02 (mg dwt)-1 min-1 and reached a maximum of around 20 nmoles 02 mg-1 dwt min-1 after 16 hrs. This level was maintained through the duration of the experiment (Fig. 3b). Acetate specific utilization rates remained constant at a level of 1.5 g-C (g dwt) -1 hr 1 except for a short increase that was mainly due to decreasing biomass (Fig. 4b). Shift from acetate as a single substrate to mixtures of TS and acetate. Under carbonJenergy limited conditions, heterotrophic 0.10 b) i:,; a) - -A - qAcetate =i:,; fl. --11-QTS ·- Q,) =...... I'\ r./} -..c Q,) ~= - - -~ ------"1 \ • -- - .... e= .... · \ f -~ · fl) .... ·- .... Q 0.05 .,Q ·- ~'D:----ll.~------~-- =·~= u r./} $ Ctl• I ;;;;> '-' CJ ;.< i:,;i;;i

0.00 0 4 60 80 1000 40 60 80 100 120 Time (hrs) Time (hrs)

Figure 4. Substrate specific utilization rates in carbon-limited culture of Comamonas testosteroni during carbon substrate shift. a) Shift from

acetate to toluenesulfonate (200mgC1-l each). b) Shift from toluenesulfonate to acetate (same as above). (A) represent acetate

specific utilization rate (qacetate) while(•) represents toluenesulfonate specific utilization rate (qTs). Chapter4 92 microorganisms are able to simultaneously utilize different carbon sources as with the case of strain T-2 cultivated under carbon-limited conditions with different mixtures of TS and acetate (23). It has been reported recently that the presence of a secondary carbon/energy source may have a positive effect on the time required to induce new enzyme systems (1). Therefore, the availability and effects of a second easily utilizable substrate such as acetate and its influence on the induction of TSMOS in strain T-2 were investigated in continuous culture at a constant dilution rate (D=0.05 hr-1 ). For this the feed was switched from acetate as the sole source of carbon to different mixtures of TS (10% and 40%

TS carbon) and acetate. The qo2TS,excess of cells taken from the chemostat was followed until the rate began to increase, marking the start of induction, and followed until a maximal rate was achieved. The start of induction was defined as an increase in q02TS,excess over that of the constitutive level of 4 nmoles 02 (mg dwt)-1 min-1. During the transient experiments with the mixtures, residual TS concentrations increased at a rate less than that of the theoretical wash-in curve indicating that TS was utilized. Similar two step induction patterns as with that of the replacement of 100% acetate carbon with 100% TS carbon experiment was observed (Fig. 5a and 5b ). The time necessary for induction to occur, time of maximal induction and maximal TS specific oxygen uptake rates are plotted against the fraction of TS carbon in the feed (Fig. 6). From the data, it can be seen that induction of TSMOS proceed quicker when the medium composition was switched from 100% acetate carbon to mixtures of TS and acetate as compared to 100% TS carbon. Furthermore, the time necessary for maximal induction and also the rate of enzyme synthesis seemed to proceed faster with lowering the proportion of TS in the feed. Nevertheless, the time needed until Chapter4 93

a) 9---Q ,.. I \ I I \ I -10 I \ I ---·-· I \ ~ .... I \ / .:.. I 0.,~/ -8 /5 /o'--o---o. 10- / I ' -6

/ /f I ·-·-· '0---0 / I -4 ,....0 0- .... .I I - - •--TS stimul:ted 2 I oxygen uptake 11------·-·I --o--TS O· _..o 0 0 1 2 3 4 5

nme (hrs)

30 20 b)

.....~ ... ,.------/ = 15 ·5= /a...... • '1.- 20 ..... =!J ... / ' ..... u ...... 0 'Q I)() s ~ / \ Q. / / ::> Q \ / • - •-TS stimul:ted '-'e I)() 0 \ / 10 I \ / oxygen uptake r:n ~ ~ =I)() e I Q / --0--TS ;;.., N I -; ~ 0 I 10 w---•\ 0 Cl'J I - "O= ~ I \ Cl'J .. b- __ .o. __ ... Cl'J I I 5 ~ ~ 0 ...... CJ I • - '0',_.0------o-- ~ e = ~ '-'= I -•-•" I 0 0 0 2 4 6 8

Time (hrs)

Figure 5. Residual toluenesulfonate concentration and excess toluenesulfonate

stimulated specific oxygen uptake with carbon-limited continuous culture

of Comamonas testosteroni (dilution rate 0.05 hr-1) as a result of

switching carbon sources from acetate to varying proportions of

toluenesulfonate (TS) in acetate (total carbon 200 mg C 1-1 ). Temperature

was held at 30°C with pH regulated at 7.5. a) Shift from acetate to 10% TS in acetate. b) Shift from acetate to 40% TS in acetate. <•) represent

q0 ?S,excess while (e) represents the residual TS concentration. Chapter4 94 induction started did not significantly change and was always in the range of 1 hr.

30 15 2 - - / q -- / > - - / ll """'-I \ / C,.l ... ·= 10 8 ·oIC ~"""'" S 20 \/ - / QJ QJ I - .:::.-- / g...:.: ... +' ' ?; \.. t:l.'.l .e ~ / .:- .: ~ c.Q I / -QJ -~ ' / I ...... e E:; a ' ~ C,.l QJ :tJ ' s f:':; 4 = ~ CIJ - 10 "{/- ~ .... 0 I ,,.,.. 11< E - - 0 = I ., ------/..... - - ... - - - - 0 0 0 0 20 40 60 80 100

Fraction of TS Carbon in Feed(%)

Figure 6. Level of maximal induction, start of induction (hrs) and time of maximal

induction in carbon-limited continuous culture of Comamonas testosteroni

(dilution rate 0.05 hr-1) as a function of toluenesulfonate carbon in the feed medium during carbon switch experiments. <•) represent time after shift until the maximum rate of induction was measured. (•)represent time after

shift until the maximal level of induction was reached. (.&.) represents the

level of induction based upon excess toluenesulfonate specific oxygen uptake

measurements and (0) represent rate of induction.

DISCUSSION AND CONCLUSIONS

Carbon fluxes and pollutant degradation. Most of the carbon found in the environment is not biologically available. Dissolution Chapter4 95 and decomposition of particulate and polymeric organic materials is the rate-limiting step over great periods of time in aquatic ecosystems, sediments, and soils despite the presence of high concentrations of organic matter. This results in a situation where microorganisms in nature usually grow under carbon-limited conditions in the presence of complex mixtures of potential substrates (8, 9, 11, 16, 18). Use of continuous culture systems represent excellent tools for studying microorganisms under carbon limitation since rigorously controlled environmental conditions ensure that the physiology of these cells are properly adapted to the chosen conditions. Gottschal ( 10) advocated the use of continuous culture systems in a controlled discontinuous mode in order to study microbial adaptability and flexibility. Very little exists in the literature concerning actual experiments using continuous cultures to study carbon fluxes (1, 3, 22, 24). The first of such experiments using transient carbon replacement was performed with sugars and not pollutants (3, 22). The results demonstrated that microorganisms need time to readjust internal metabolic pathways to handle new carbon sources. Similar patterns have been seen with pollutant degrading organisms (1, 24). When the NT A degrading Chelatobacter heintzii growing in continuous culture with glucose was challenged with a sudden replacement of glucose with mixtures of glucose and NT A in the inflowing medium, a decrease in the time necessary for the induction of the NTA degrading pathways was seen with decreasing proportions of NT A to glucose in the feed. Unfortunately, the relationship was not clear with respect to the time until induction started and the rates of induction (1). Similar results were seen in the present study with the TS degrading Comamonas testosteroni and varying mixtures of TS and acetate. The reasoning behind these results is that with mixtures of substrates, microoganisms can draw on one carbon source for energy while rearrangement of cellular metabolism for the Chapter4 96 synthesis of the pollutant degrading enzymes. At the same time the carbon-limited nature of growth in the continuous culture avoids that the cells experience the phenomenon of carbon catabolite repression by acetate or other easily degradable carbon sources used. Therefore, under environmental conditions, if organisms with the proper metabolic pathways are present, the presence of other easily utilizable substrates may actually be beneficial and support the induction process and decrease the time necessary for the induction of pollutant degrading systems. Factors which influence induction. Bacteria adapted to carbon/energy limited environments possess the ability to scavange substrates from limiting concentrations in the environment (5, 17). The exact factors which influence the derepression of degradative pathways during carbon-limitation are still unclear. Bally and Egli ( 1) demonstrated that the absence of inducer rather than the presence of a repressor controls NT A degradation by Chelatobacter heintzii and that probably not the absolute concentration but the proportion of pollutant carbon comprising the total available carbon triggers induction. Conversely, in Escherichia coli ML30, an induction threshold of approximately 5 mg 1-1 3-phenyl propionic acid was observed with continuously grown cultures fed mixtures of 3-phenyl propionic acid and glucose (13). These differing results in the two systems may be resolved by understanding the mode of transport for the two substrates. To date it is not certain how TS is transported into the cell; however, we believe a mechanism similar to the vectorial partitioning mechanism described by Button (4) may explain the two-step induction pattern seen in carbon shift experiments with strain T-2 [for more on transport mechanisms, refer to references (4, 6)]. In the environment or technical systems, microorganisms are confronted with potential carbon sources that vary in concentration, and Chapter4 97 quality. Fluxes in the spectrum and quantity of alternative substrates can affect the degradation of pollutants in these systems. From the results a better understanding of the factors which influence the degradation of TS have been presented and may be applied. Use of easily utilizable carbon sources in addition to organic pollutants by specialized microorganism decreases the time necessary for induction of pollutant degrading systems. At high concentrations of alternative carbon sources diauxic/repressor effects of inducible systems are often observed but this is not seen under carbon-limited growth conditions. It may be possible that the ability for certain microorganisms to utilize organic pollutants gives these microorganisms a selective advantage at least on a kinetic basis [see reference ( 14)]. In addition, little is known concerning the presence of other microorganisms competing for available carbon upon pollutant degrading systems. Competition experiments with defined mixed cultures under continuous cultivation may clarify these questions.

ACKNOWLEDGEMENTS

The authors are indepted to the Swiss National Science Foundation (Project No. SPP 5001.35285) which financed the work of A. Tien and F. Junker.

We would like to thank Prof. Th. Leisinger for the use of his laboratory facilities and continued interest in this project; Ms. A. Schafer for technical assistance with TS measurements; and Dr. S.-C. Ko for graphical assistance. Chapter4 98

REFERENCES

1. Bally, M., and T. Egli. 1996. Dynamics of substrate consumption and enzyme synthesis in Chelatobacter heintzii during growth in carbon-limited continuous culture with different mixtures of glucose and nitrilotriacetate. Appl. Environ. Microbiol. 62: 133-140. 2. Bally, M., E. Wilberg, M. Kiihni, and T. Egli. 1994. Growth and regulation of enzyme synthesis in the nitrilotriacetic acid (NT A) degrading Chelatobacter heintzii ATCC 29600. Microbiol. 140: 1927-1936. 3. Baloo, S., and D. Ramkrishna. 1991. Metabolic regulation in bacterial continuous cultures II. Biotech. Bioeng. 38: 1353-1363. 4. Button, D. K. 1985. Kinetics of nutrient-limited transport and microbial growth. Microbiol. Rev. 49: 270-297. 5. Button, D. K. 1991. Biochemical basis for whole-cell uptake kinetics: specific affinity, oligotrophic capacity, and the meaning of the michaelis constant. Appl. Environ. Microbiol. 57: 2033-2038. 6. Button, D. K. 1993. Nutrient-limited microbial growth kinetics: overview and recent advances. Antonie van Leeuwenhoek 63: 225- 236. 7. Czekalowski, J. W., and B. Skarzynsky. 1948. The breakdown of phenols and related compounds by bacteria. J. Gen. Microbiol. 2: 231-238. 8. Egli, T. 1995. The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates. pp. 305-386. In J. Gwynfryn Jones (ed.) Advances in Microbial Ecology, Plenum Press, New York. Chapter4 99

9. Fenchel, T. M., and B. B. Jorgensen. 1977. Detritus food chains of aquatic ecosystems: the role of bacteria. Adv. Microb. Ecol. 1: 1-58. 10. Gottschal, J. C. 1992. Continuous culture, 559-572. In J. Lederberg (ed.). Encyclopedia of Microbiology. Academic Press, London. 11. Jannasch, H. W., and R. I. Mateles. 1974. Experimental bacterial ecology studied in continuous culture. Adv. Microb. Physiol. 11: 165-212. 12. Junker, F., E. Saller, H. R. Schliifli Oppenberg, P. H. M. Kroneck, T. Leisinger, and A. M. Cook. 1996. Degradative pathways for p-toluate and p-toluenesulfonate and their multicomponent monooxygenases in Comamonas testosteroni strains PSB-4 and T-2. Microbiology (in press). 13. Kovarova, K., V. Chaloupka, and T. Egli. 1996. Threshold substrate concentrations required for induction of the catabolic pathways for 3-phenylpropionic acid in Escherichia coli. Appl. Environ. Microbiol. (for submission) 14. Lendenmann, U., M. Snozzi, and T. Egli. 1996. Kinetics of simultaneous utilization of sugar mixtures by Escherichia coli in continuous culture. Appl. Environ. Microbiol. 62: 1493-1499. 15. Locher, H. H. 1991. Bacterial Degradation of p-Toluenesulfonate and Related Aromatic Sulfonic Acids: Characterization of Degradative Pathways and Enzymes. Ph.D. Thesis no. 9434. Swiss Federal Institute of Technology Ztirich. 16. Matin, A. 1979. Microbial regulatory mechanisms at low nutrient concentrations as studied in chemostat. pp. 323-339. In M. Shilo (ed.) Strategies of Microbial Life in Extreme Environments. Verlag Chemie, Weinheim. Chapter4 100

17. Morita, R. Y. 1988. Bioavailability of energy and its relationship to growth and starvation in nature. Can. J. Microbiol. 43: 436-441. 18. Morita, R. Y., and C. L. Moyer. 1989. Bioavailability of energy and the starvation state, 75-79. In T. Hattori, Y. Ishida, Y. Maruyama, R. Y. Morita and A. Uchida (ed.). Recent Advances in Microbial Ecology. Japan Scientific Societies Press, Tokyo. 19. Morita, R. Y. 1992. Low-nutrient environments, 617-624. In J. Lederberg (ed.). Encyclopedia of Microbiology. Academic Press, London. 20. SchHifli-Oppenberg, H. R., G. Chen, T. Leisinger, and A. M. Cook. 1995. Regulation of the degradative pathways from 4- toluensulphonate and 4-toluenecarboxylate to protocatechuate m Comamonas testosteroni T-2. Microbiology 141: 1891-1899. 21. Schneider, R. P., F. Zurcher, T. Egli, and G. Hamer. 1988. Determination of nitrilotracetate in biological matrices using ion exclusion chromatography. Anal. Biochem. 173:278-284. 22. Standing, C. N., A. G. Fredrickson, and H. M. Tsuchiya. 1972. Batch- and continuous-culture transients for two substrate systems. Appl. Microbiol. 23: 354-359. 23. Tien, A. J., and T. Egli. 1996a. Regulation of pollutant degradation in the presence of an alternative substrate by the para- toluenesulfonate utilizing Comamonas strain T-2 and the dichloromethane utilized Methylobacterium strain DM4 during growth in batch and continuous culture. Appl. Environ. Microbiol. (submitted). 24. Tien, A. J., and T. Egli. 1996b. Growth of Methylobacterium strain DM4 in continuous culture: effects of dilution rate and response to substrate shifts during continuous cultivation. Appl. Environ. Microbiol. (submitted). Chapter5 101

Dynamics and Regulation of Mixed Pollutant Degradation by a Four-Membered Defined Bacterial Consortium in Continuous Culture

A. J. Tien and T. Egli

Key words: continuous culture, defined mixed culture, mixed pollutant degradation, regulation, synthetic sewage

ABSTRACT

The effects of mixed substrate growth and transient carbon substrate availability upon pollutant degrading activities and population dynamics/composition were examined using a model system consisting of the nitrilotriacetate (NT A) degrader Chelatobacter heintzii, the p-toluene sulfonate (TS) degrader Comamonas testosteroni strain T2, the dichloromethane (DCM) degrader Methylobacterium strain DM4, and the non-pollutant degrading Escherichia coli strain ML30 cultivated in a carbon-limited continuous culture on a synthetic sewage. In order to track individual populations and activities, cells were quantified by fluorescent antibodies, enzyme expression by the oxygen specific uptake rate in the presence of excess substrate and degradation was monitored by chemical analysis. During batch growth with the four strains in a synthetic sewage containing the specific pollutants, E. coli strain ML30 outnumbered its nearest competitor, Comamonas testosteroni strain T-2, approximately 2: 1. Mb. strain DM4 and Cb. heintzii strain 29600 were present at concentrations less than half that of Cs. test. strain T-2. However, under Chapter5 102

continuous cultivation (D=0.05 hr 1) a steady-state population of the four strains was established with the following composition: 15% Mb. strain DM4, 15% Cb. heintzii strain 29600, 15% Cs. test. strain T-2, 55% E. coli strain ML30. When NT A, TS or DCM was omitted from the feed, total cell counts of the three pollutant degrading strains were only slightly reduced. with no significant changes in the ratio of individual strains. However, the pollutant-degrading activity of the specific strains decreased. When NT A, TS or DCM was added again in the feed, induction of the specific pollutant degrading system was observed after a lag period. No enrichment in the population of the specific pollutant degrading strain was observed. The stability of the four-membered defined bacterial culture used in our experiments can be attributed to the ability of the pollutant degrading strains to utilize not only their specific pollutants but also synthetic sewage (mixed substrate utilization) under the carbon-limited conditions imposed. The data demonstrates in this case induction of the necessary enzyme systems and not enrichment of pollutant degrading strain is the primary mechanism that leads to pollutant removal in the environment.

INTRODUCTION

The treatment of wastewater and subsequent removal of many pollutants by activated sludge systems is one of today's most important biotechnological processes. Although much effort has been directed towards process engineering, activated sludges are still treated as black boxes for wastewater processing. Knowledge about community structure and function is limited. Activated sludges are composed of a consortium Chapter5 103 of microorganisms, many of which are unidentifiable or unculturable (24, 38, 39). Much of what has been elucidated concernmg the carbonaceous pollutant degrading abilities of heterotrophic microorganisms in sludges has been obtained in the laboratory with pure cultures under batch conditions. However, microorganisms found in the environment are rarely confronted with these pollutants under the conditions used in the laboratory or as sole sources of carbon and energy. In most natural environments, nutrient-deficiencies, especially the lack of available carbon compounds limits the growth of heterotrophic microorganisms. Cycles of starvation and survival are the typical lifestyle for heterotrophic microorganisms (26). Under such conditions derepression of many catabolic pathways has been observed and simultaneous utilization of available substrates may be the norm (4). Additionally, there are indications, although not systematically tested, that the spectrum of carbon sources utilized might become wider as the degree of starvation increases at decreasing growth rates (25). Furthermore, the steady-state concentrations of individual carbon substrates were reduced during growth with mixtures as compared to growth on single substrates. The later property of heterotrophic microorganisms allows relatively fast growth at low individual carbon substrate concentrations characteristic in most environments (7, 19, 21). In industrialized countries pollutant carbon contributes to a significant proportion of the available carbon pool which makes the ability for heterotrophic microorganisms to degrade pollutants all the more important. Pollutant as well as other carbon substrate concentrations often fluctuate in ecosystems such as wastewater treatment plants. Little is known about the effects of competition on the survival and function of pollutant degrading organisms or the effects of transient Chapter5 104 carbon (pollutant) shifts in such an environment. The question exists: Would the ability of a microorganism to utilize a specific compound, for example a pollutant, in addition to easily degradable natural carbon substrates allow these organisms to compete against other members of the microbial community? In order to address this, a model system consisting of the nitrilotriacetate (NT A) degrader Chelatobacter heintzii, the p- toluene sulfonate (TS) degrader Comamonas testosteroni strain T2, the dichloromethane (DCM) degrader Methylobacterium strain DM4, and the non-pollutant degrading Escherichia coli strain ML30 was assembled and cultivated in a carbon-limited continuous culture on a synthetic sewage. Regulation of the key enzymes was first studied in pure culture to see if the repression/derepression patterns could be extrapolated to a more complex situation (3, 34, 35, 36). In our present study, some of the principles regulating growth and stability of mixed cultures were addressed. The effects of mixed substrate growth and transient carbon substrate availability upon pollutant degrading activities and population dynamics/composition were examined, giving insight into how microbial populations survive and function on a normal basis in natural or technical systems.

MATERIALS AND METHODS

Microbial strains and cultivation conditions. In experiments with synthetic sewage and mixtures of dichloromethane (DCM), toluene sulfonate (TS), nitrilotriacetic acid (NT A), Methylobacterium strain OM4 (10), Comamonas testosteroni strain T-2 [DSM 6577, (32)], Chelatobacter heintzii strain ATCC 29600 (I) and Escherichia coli strain ML30 (DSM 1329) was used as a model system to Chapter5 105 investigate pollutant degradation in simulated municipal wastewaters. Conditions for culture maintenance and batch growth have been previously described (9, 16, 22). Table 1 gives maximal specific growth rates of these bacterial strains with various substrates. For continuous culture, a synthetic sewage was used containing per liter 0.14 g peptone, 0.1 g meat extract, 0.009 g urea, 0.006 g NaCl, 0.18 g Na-acetate, 0.3 g MgS04·7H20, 0.02 g CaCl2·2H20, 0.14 g KCl, 1.07 g NH4Cl, 0.02 g (NH4)2S04, 2.46 g Na2HP04, 5 ml of trace elements (Egli et al., 1988), 1.8 ml H3P04 (85%) and 10 mg silicon antifoam (Fluka, Buchs, Switzerland). Total carbon in the medium was 275 mg-C 1-1, 175 mg-C from the synthetic sewage, 25 mg-C 1-1 from EDTA in trace elements and 25 mg-C 1-1 each from DCM, TS, and NTA. The bioreactor (MBR, Wetzikon, Switzerland, 2 liter working volume) was aerated at 0.1 1 min- 1 while stirred at 1,500 rpm. The pH was controlled at 7 .5 by automatic addition of H3P04 (1 M) and KOH/NaOH (0.5 M, each). The temperature was maintained at 30°C.

Table 1. Maximal specific growth rates with different substrates

Strain specific substrate acetate synthetic sewage

Cb. 29600 0.18 hr-1 (NTA) 0.095 hr-1 0.130 hr-1

Cs. T-2 0.290 hr-1 (TS) 0.231 hr-1 0.321 hr-1

Mb.DM4 0.079 hr-1 (DCM) 0.078 hr-1 0.106 hr-1

E. coli l\1L30 0.211 hr-1 0.429 hr-1

Growth rates measured in batch culture at pH 7.5 and 30°C while stirred at 200 rpm. Chapter5 106

Determination of excess substrate specific oxygen uptake rates. Culture liquid (80 ml) was collected directly from the chemostat, washed twice with and resuspended in 20 ml carbon-free medium. Excess substrate (DCM, TS, NTA, acetate or synthetic sewage) specific oxygen uptake rates were recorded at 30°C in a glass Clark-type oxygen electrode (Rank Brothers, Great Britain). The total volume of the assay was 3.0 ml, consisting of 2.8 ml of cell suspension plus 0.2 ml of a 0.1 M substrate solution. Sampling and assay times were standardized. All assays were preformed in triplicate. Quantification of biomass. Biomass was quantified by measuring dry weight or optical density. Dry weight (Dwt) was obtained by filtering culture liquid through 0.2 µm pore size polycarbonate filters (Nuclepore, Pleasanton, CA. U.S.A.) which together were washed with distilled water to remove salts. Filters were dried at 105°C overnight to constant weight. Optical density was determined at 480 nm in 1 cm cuvettes using a Uvikon 860 spectrophotometer (Kontron, Ztirich, Switzerland). Analysis of substrates. Headspace analysis of DCM was performed by gas chromatography (Hewlett Packard 5892 series II, U.S.A.) using a 60 m long, 0.32 mm diameter glass capillary column (J&W DB624), fitted with an ECD detector. The injector and detector temperatures were maintained at 250°C while the oven temperature was maintained at 30°C. Helium was used as the carrier gas at a total flow rate was 32 ml min-1. The detection limit was 0.1 mg 1- l DCM carbon. Determination of TS concentrations were performed by high pressure liquid chromatography after Locher et al. (22). The detection limit was 0.1mg1-1 TS carbon. Chapters 107

NT A was measured by high pressure ion exclusion chromatography as described by Schneider et al. (30). The detection limit was 1 mg 1-1 NTA carbon. Dissolved organic carbon (DOC) was measured as following: 20 ml of culture from the chemostat were filtered through 0.2µm pore size membrane filters (Nuclepore, Pleasanton, CA. USA) pre-rinsed with carbon-free distilled water. The filtrate was acidified with concentrated HCl, dissolved C02 stripped with N2 gas, and DOC measured with a TOCOR2 total organic carbon analyzer (Maihak AG, Hamburg, Germany). A baseline DOC value of approximately 25 mg I-1 as carbon was consistantly measured and could be attributed to the Na-EDTA (20 mg 1-1 as carbon) used to complex trace elements and the organic additives ( 1 mg 1-1 as carbon) in the silicon antifoam agent used in the medium. Surface antibodies against specific strains. Polyclonal antibodies against Methylobacterium strain DM4, Comamonas testosteroni strain T-2 and Escherichia coli strain ML-30 were prepared after established procedures (15) with heat inactivated whole bacterial cells. Antisera were purified by cross-reaction depletion against related and non-related organisms. Specific surface antibodies prepared against outer membrane fractions of strain 29600 were generously provided by M. Bally and described previously (2). All surface antibodies used in this study were found to be specific and did not cross-react with other bacterial strains used in the defined mixed culture. Chapter5 108

RESULTS

Establishment of the steady-state mixed culture. A 2 l working volume chemostat containing synthetic sewage (175 mg 1-1 as carbon), and the pollutants (each 25 mg 1-1 as carbon) was inoculated with OD adjusted inocula of the four strains (approximately 25% each) and operated under batch growth conditions. Increase in biomass of the mixed culture during batch conditions and continuous growth conditions are shown in Fig 1a. Population dynamics of the four strains during batch cultivation demonstrate E. coli strain ML30's competitive superiority over the other three strains (Fig. 1b ). At the end of batch growth, E. coli strain ML30 (5.8 x 109 per ml) outnumbered its nearest competitor, Cs. test. strain T~2 (2.9 x 109 per ml), approximately 2: 1. Mb. strain DM4 and Cb. heintzii strain 29600 were present at concentrations less than 2.0 x 109 per ml. Calculation of the specific . µmax of the individual strains within the defined mixed culture indicated similar growth rates for Mb. strain DM4, Cs. test. strain T-2 and Cb. heintzii strain 29600 as compared to batch experiments (pure cultures) and suprisingly a lower growth rate for E. coli strain ML30 (µ=0.377 hr-1) than that of µ=0.429 hr-1, measured in pure culture. Whereas cell numbers for all strains increased during batch growth, the relative proportions of Mb. strain DM4 and Cb. heintzii strain 29600 decreased. Only E. coli strain ML30 was able to make significant gains. Furthermore, the ratio of Cs. test. strain T-2 in the mixed culture remained relatively stable (Fig. lb and le). After 20 hrs the bioreactor was switched to operation under continuous mode (D=0.05 hr-1 ). Steady- state conditions (stable biomass, population composition, DOC values) were achieved after four residence times (80 hrs). Total cell counts were in 3.87x 109 cells ml-1. The steady state populations of the four strains Chapter 5 109

0.6 a) Bat:h : ContirvouSJ=0.051T-l 0.5 Ei #'Q, = p I 'o - Cb- ---ifO. - ...,.=QC 0.4 --o ... I I ~ I I ~ I I ·i;; 0.3 p I Q;= Q I I -; 0.2 ~ I .i= I c. I 0 0.1 I I I 0.0

b) l.40e+10 Bath ContinuousD=0.051'11'1 1.20e+10 . 1.00e+lO i ~ 8.00e+9 Q; .t::l Ei 6.00e+9 z= 'il 4.00e+9 u 2.00e+9

O.OOe-+9

60 Bath c) I I ,,...... =0 ...... · - ~ 50 I .-· ----·--·-·-· c. ,f----· =0 , ... c:i. 40 , I -; , I I ContinuousD=0.05f'1rl ~ I .... I 0 30 I ' I s~ =Q; 20 ..,.... i:t 10 0 20 40 60 80 100 Time (hrs)

Figure 1. Growth of a four-membered defined mixed culture under batch and continuous cultivation conditions. Vertical dashed line indicates the transition from batch to continuous cultivation at D=0.05 hr-1. a) Biomass of mixed culture as measured by optical density is represented by (0). b) Increase in cell numbers; (0) represents total cell counts of the defined mixed culture. Cell counts of individual strains within the defined mixed culture are as follows: (0) Chelatobacter heintzii strain 29600, (~) Comamonas testosteroni strain T-2, (..6.) Methylobacterium strain DM-4, and C•) Escherichia coli strain ML30. c) Individual strains as a percentage of total population. Symbols identical as above. Chapters 110

were as follows: 5.8 x 108 cells m1-l Mb. strain DM4, 5.9 x 108 cells m1-l Cb. heintzii strain 29600, 6.0 x 108 cells m1-l Cs. test. strain T-2, 2.1 x 1o9 cells m1- l E. coli strain ML30 (Fig. 1b ). The residual concentrations of NTA, DCM and TS were below the detection levels of the methods used and the residual DOC from 60 hrs on remained at approximately 25 mg 1-1 as carbon and can be attributed to the Na-EDTA (25 mg 1-1 as carbon) used to complex trace elements and the organic additives (1 mg 1-1 as carbon) in the silicon antifoam agent used in the medium. Transient removal of NT A. Steady-state cultures containing all the pollutants were perturbed by removal of NT A from the feed. After four residence times a new steady-state was achieved. Total cell counts (3.5 x 109 cells per ml) were slightly lower than the initial steady- state values. No changes in the ratio of individual strains were measured however cell counts of individual strains decreased (approximately 10%) because of the lower DOC. The start of the experiment began with the addition of NT A (25 mg 1- l) back into the feed. Interestingly, no peak in residual NT A concentration was seen before the onset of increased NT A degrading activity. The residual concentrations of NT A, DCM and TS were below the detection levels of the methods used and the residual DOC levels remained constant at 25 mg as carbon 1-1. Approximately 96 hrs were necessary before an increase in the activity of the NT A degrading enzymes could be measured. Values for excess substrate specific oxygen uptake rates for the TS were constant at 1 nmol 02 (mg Dwt)-1 min-1 while that of DCM increased from 0.2 nmol 02 (mg Dwt)-lmin-1 to 0.5 nmol 02 (mg Dwt)-1 min-1 indicating that with respect to Mb. strain DM4, steady-state DCM specific oxygen uptake rates may not been achieved (Fig. 2). Synthetic sewage specific oxygen uptake rates remained at 2 nmol 02 (mg Dwt)-lmin-1 (Fig. 2). Total cell counts Chapter5 111

2

/ 1 / / / /

/ / / ODDIJ..-r---_,..,n-_,.--11-i=-=:;.;.=.,.-....--,-...... --.--..---1---d 0 20 40 60 80 100 120 140 160 Time (hrs)

Figure 2. Growth of the four-membered defined mixed culture under continuous

cultivation (D=0.05 hr-1) after transient omission and replacement of NTA.

Excess substrate specific oxygen uptake rates after transient replacement of

NTA into feed reservoir. Approximately 100 hrs were necessary before

induction over constitutive levels was observed. (0) represent excess NTA

specific oxygen uptake rates,(~) represent excess TS specific oxygen uptake

rates, (.&) represent excess DCM specific oxygen uptake rates, and (e)

represent excess synthetic sewage (SS) specific oxygen uptake rates.

slightly increased from 3.5 xl09 to approximately 4 x 109 cells ml-1 and may be attributed to the additional carbon when NTA was added back into the feed. However, no enrichment in the population of strain 29600 was observed. The population composition of the defined mixed culture remained stable at 55% strain ML30 + 5.5%, 15% strain 29600+ 1.5%, 15% Cs. test. strain T-2± 1.5%, and 15% strain DM4± 1.5%. Transient removal of TS. As in the previous experiment, steady-state cultures containing all the pollutants were perturbed, in this case, by removal of TS from the feed. After four residence times a new Chapter 5 112 steady-state was achieved. Total cell counts (3.4 x 109 cells per ml) were similar to that when NT A was removed from the feed and population ratios of the mixed culture remained constant. This new steady-state was reperturbed with the reintroduction of 25 ppm of TS carbon into the feed. Measurement of TS concentrations in the culture vessel indicated that all TS was utilized. NT A and DCM concentrations remained below the analytical detection limit and further indicated that the pollutants were utilized by the defined mixed culture. Residual DOC levels remained constant at 25 mg as carbon 1-1. Approximately 3-4 hrs was necessary for induction of the TS degradative enzymes. Excess NT A specific oxygen uptake rates and excess DCM specific oxygen uptake rates remained at a nearly constant level of 1.2 nmol 02 (mg Dwt)-lmin-1. Excess synthetic sewage specific oxygen uptake rates fluctuated between 1.5 to around 2 nmol 02 (mg Dwt)-lmin-1 (Fig. 3). During the time period within which this experiment was conducted (70 hrs plus), total cell counts gradually increased to 4.0x 1o9 cell per ml. The readdition of TS into the feed did not result in the enrichment of Cs. test. strainT-2. The population composition did not vary and remained stable. Transient removal of DCM. In order to see the effect of the transient removal and replacement of DCM, steady-state cultures containing the three pollutants were fed an identical medium with DCM omitted. After four residence times (new steady-state) total cell counts were slightly lower at 3.6xl09 cells per ml but the population profile remained constant at 55% strain ML30 + 5.5%, 15% strain 29600+ 1.5%, 15% Cs. test. strain T-2± 1.5%, and 15% strain DM4± 1.5%. When DCM (25 mg I-1 DCM carbon) was introduced in the feed, DCM, TS and NTA concentrations in the cultivation vessel remained below the detection limits of the techniques used. Residual DOC levels were constant at 25 mg carbon 1-1. Approximately 25 hours were necessary to induce the Chapter5 113

,,0, - . ' 2 ,o,,'' ,, 'o------0 ----~:::.-.:&:'~b.------6--&--

1 I' I I ~ I I 0 0 20 40 60 80 Time (hrs)

Figure 3. Growth of the four-membered defined mixed culture under continuous

cultivation {D=0.05 hr-1) after transient omission and replacement of TS.

Excess substrate specific oxygen uptake rates after transient replacement of TS

into feed reservoir. Approximately 4 hrs were necessary before induction over

constitutive levels was observed. (D.) represent excess TS specific oxygen

uptake rates, (0) represent excess NTA specific oxygen uptake rates, (.&.)

represent excess OCM specific oxygen uptake rates, and (e) represent excess

synthetic sewage (SS) specific oxygen uptake rates.

activity of the DCM degrading enzymes (Fig. 4). The excess TS and NT A specific oxygen uptake rates decreased slightly from about 1.2 nmol 02 (mg Dwt)-1 min-1 to around 1 nmol 02 (mg Dwt)-1 min-1. Unexpectedly, synthetic sewage specific oxygen uptake rates increased from 1.3 nmol 02 (mg Dwt)-1 min-1to2.5 nmol 02 (mg Dwt)-1 min-1. This increase in the excess synthetic sewage specific oxygen uptake rates may indicate that the mixed culture was not actually in steady-state or that the DCM induced cells may have a higher oxygen uptake rate. As with Chapter5 114

--o---0 ------o---o 00-0- __ ..__ .. 0'00000' --- 2 .-----

1

o... 1'.1111-11 .. .,.,..11*-,---...---r--...---,---.....-,--....---i 0 10 20 30 40 so 60 Time (hrs)

Figure 4. Growth of the four-membered defined mixed culture under continuous cultivation (D=0.05 hr-1) after transient omission and replacement of DCM. Excess substrate specific oxygen uptake rates after transient replacement of DCM into feed reservoir. Approximately 20 hrs were necessary before induction over constitutive levels was observed. (8)

represent excess TS specific oxygen uptake rates, (0) represent excess

NTA specific oxygen uptake rates, (.A.) represent excess DCM specific

oxygen uptake rates, and (e) represent excess synthetic sewage (SS) specific oxygen uptake rates.

the other two experiments, total cell counts increased approximately 10% with no enrichment in the specific pollutant degrading strain during the transient addition of the specific pollutant. The population composition of the defined mixed culture did not vary and remained constant throughout these experiments at 55% strain ML30 ± 5.5%, 15% strain 29600+ 1.5%, 15% Cs. test. strainT-2+ 1.5%, and 15% strain DM4± 1.5%. Chapter5 115

DISCUSSION

In order to study how pollutant degrading orgamsms surv1 ve and function in the environment, a model system containing three distinct pollutant degrading strains and a non-pollutant degrading strain were used. This system was designed to test techniques developed for autecological studies in more complex systems such as wastewater treatment plants. Combining methods such as population and activity specific assays for autecological studies has been previously advocated (27). In our study the use of specific surface antibodies and excess substrate specific oxygen uptake rates were used to track population dynamics and shifts in pollutant degrading activity in response to transient nutrient (pollutant) conditions found so often in technical or natural systems. Microorganisms used in this study are phylogenetically distinct as revealed from searching 16s rRNA data banks developed at the Technical University Munich by W. Ludwig. Furthermore, much data concerning the genetics and degradative pathways for these organisms have already been determined (2, 5, 6, 20, 22, 23, 31). Baseline studies with pure cultures have indicated that many common trends exist with the diverse pollutant degrading organisms used in our study and may hold true for many natural microorganisms: 1) Mixed substrate utilization is the rule under carbon limited conditions, 2) a few percent pollutant carbon is necessay to induce pollutant degrading systems and 3) simultaneous utilization of pollutant with easily utilizable carbon substrates often decreases induction times during transient carbon shifts (3, 34, 35, 36). Induction vs. enrichment. The usual understanding, especially with wastewater engineers, is that degradation of pollutants in a system follows the enrichment of microorganisms capable of utilizing this Chapter5 116

pollutant as a nutrient. This is in contrast to what Bally (2) discovered which was that the induction of NTA-monoxygenase was the major mechanism for the degradation of NT A in activated sludges. In our defined consortium and natural ecosystems, pollutant degrading strains may be present but not fully induced. However, low levels of pollutant degrading activity were measured in the absence the inducing pollutant which allowed pollutant degraders to utilize low fluxes of pollutant carbon when they become available. When the numbers of uninduced pollutant degrading strains are high, this combined constitutive activity can amount to a considerably large pollutant degrading capacity of the natural or technical systems. There are now several reports that have demonstrated that during the simultaneous utilization of pollutant together with alternative carbon substrates only a few percent pollutant carbon is necessary to induce pollutant degrading systems (3, 34, 35, 36). Full induction was observed already when pollutant carbon contributed to 5-10% of the available carbon for Methylobacterium strain DM4 and Comamonas testosteroni strain T-2, but not for Chelatobacter heintzii strain 29600. In addition, data suggests that it is the ratio of "pollutant" to readily utilizable carbon rather than absolute concentration of "pollutant" which triggers induction (2, 8). However, other mechanisms for induction of degradative pathways may exist. Kovarova et al. ( 17) report that a threshold substrate . concentration of 5 mg 1-1 was necessary for induction of the 3- phenylpropionic acid catabolic pathway in E. coli strain ML30. In our experiments individual pollutants contributed to some 10% of the total available carbon in the feed and full induction of the pollutant degrading pathways were expected. From the population composition one can extrapolate that the specific pollutant degrading strain utilized some 5- 10% of the synthetic sewage carbon in the absence of its specific pollutant. Chapter5 117

It was shown by specific fluorescent antibody enumeration that although total cell numbers increased, there was no enrichment of the specific pollutant degrading strain, relative to the other three strains, during the transient reintroduction of its specific pollutant (25 mg 1-1 as carbon) into a synthetic sewage. Calculations using yield coefficients for the various pollutants indicate that the additional biomass (specific pollutant degrading strain) produced would be insignificant. For example, the addition of TS (25 mg 1-1 as carbon) into steady-state cultures cultivated in the absence of this pollutant would contribute to less than a 10% increase in total dryweight. In most ecosystems where the availability of nutrients, including carbon sources, frequently oscillates (28), it is a conclusion from our experiments that induction rather than enrichment would play a greater role in the degradation of pollutants given that organisms capable of these compounds are present. Stability of mixed populations. Gottschal (13) remarked that the coexistence of competing species is the rule rather than the exception in most natural habitats. However, from numerous laboratory investigations with pairs of orgamsms which were cultivated by continuous culture (competition for similar resources or the coexistence of distinct microorganisms), the general conclusion is that during competition for the similar resources, there will be a winner and there will be a loser (13, 14, 37). However, under conditions of multiple substrate limitation and discontinuous or alternating supply of nutrients, coexistance of microorganisms has been frequently observed (11, 13, 18). In addition, if multiple substrates are available which different strains can utilize, the coexistance of mixed cultures was predicted to occur (29). Confirming this, Thumheer et al. (33) described the stable coexistance of four sulfonated aromatic degrading microorganisms in continuous culture fed with media containing five differing sulfonated aromatic compounds. Clulpter 5 118

Unfortunately, due to the close relationship of the parent strains, new strains were detected after seven months, presumably from horizontal gene transfer. Our results generally validate the hypothesis of Pirt (29) which stated that a multiplicity of nutrient sources such as carbon, in the presence of excess amounts of essential elementary nutrients (i.e. phosphate, potassium, and magnesium) would support a maximum diversity of microorganisms. The stability of the four-membered defined bacterial culture used in our experiments can be attributed to the ability of the pollutant degrading strains to utilize not only their specific pollutants but also synthetic sewage (mixed substrate utilization) under the carbon- limited conditions imposed. (Table 2). No accumulation of substrates were detected indicating that the low constitutive degradative capacity was obviously enough to ensure the degradation of transiently available pollutants until induction occured. Moreover, the time frames for induction were similar with pure culture experiments (35, 36). With our model system, some of the principles which govern the survival of pollutant degrading microorganisms were demonstrated. The ability for heterotrophic pollutant degrading strains to utilize mixed substrates (pollutant and synthetic sewage) under carbon-limitation may explain how these organisms survive and compete with other microorganism in natural or engineered systems. This ability for distinct pollutant degrading microorganisms to coexist may be exploited for the creation of "designer" mixed cultures to degrade known industrial wastestreams. Perhaps, the addition of easily utilizable substrates to industrial wastestreams could promote increased stability of the mixed culture during fluxes in type and concentration of pollutants. However, further experiments should be performed to test the stability. of such mixed culture systems under other variable environmental conditions such as temperature, pH and dissolved oxygen levels. Chapter 5 119

Table 2. Estimation of biomass produced from pollutants and synthetic sewage

during growth of a four-membered continuous culture at D=0.05 hr-1

Strain Specific Substrate 1 Theoretical 2 Calculated3

Cb. 29600 48 mg Dwt (NTA) 9.5 mg Dwt (NTA) 22mgDwt

Cs. T-2 72 mg Dwt (TS) 9.0 mg Dwt (TS) 22 mgDwt

Mb. DM4 44 mg Dwt (DCM) 8.3 mg Dwt (DCM) 22mgDwt

E. coli l\1L30 123.5 mg Dwt (SS) 109 mg Dwt (SS) 80.7 mg Dwt

1 Obtained from batch experiments where specific substrate supplied at 200 mg 1- l as carbon.

SS=Synthetic Sewage

2 Calculated from 25 mg 1- l as carbon for each specific pollutant (NTA, TS, DCM) and 175 mg 1- l as

available carbon from synthetic wastewater with YXJNTA =0.38 (Cb. 29600), Yxrrs =0.36 (Cs. T-2),

YXJDCM =0.33 (Mb. DM4) and Yxtss =0.62 (E.coli ML30) assuming each strain would utilize their

specific carbon source within the pollutant synthetic sewage mixture.

3 Calculated using cell numbers obtained from specific antibody direct counts and drywt relationships

derived from batch experiments

ACKNOWLEDGEMENTS

The authors are indepted to the Swiss National Science Foundation (Project No. SPP 5001.35285) which financed the work of A. Tien.

We would like to thank Prof. A. M. Cook for critical discussions and help; Prof. Th. Leisinger and staff for the use of their laboratory facilities; A. Schafer and F. Junker for technical assistance with TS measurements; M. Nay for technical assistance with DCM measurements; Chapter5 120

H.-U. Weilenmann for technical assistance with DOC measurements and Dr. S.-C. Ko for graphical assistance.

REFERENCES

I. Auling, G., H.-J. Busse, T. Egli, T. El-Banna, and E. Stackbrandt. 1993. Description of the gram-negative, obligately aerobic, nitrilotriacetate (NT A)-utilizing bacteria as Chelatobacter heintzii, gen. nov., and Chelatococcus asaccharovorans, gen. nov., sp. nov. System. Appl. Microbiol. 16: 104-112. 2 • Bally, M. 1994. Physiology and Ecology of Nitrilotriacetate Degrading Bacteria in Pure Culture, Activated Sludge and Surface Waters. Diss. ETH Nr. 10821. Ph.D. Swiss Federal Institute of Technology Zurich. 3. Bally, M., and T. Egli. 1996. Dynamics of substrate consumption and enzyme synthesis in Chelatobacter heintzii during growth in carbon-limited continuous culture with different mixtures of glucose and nitrilotriacetate. Appl. Environ. Microbiol. 62: 133- 140. 4. Egli, T. 1995. The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates. p.305-386. In J. Gwynfryn Jones (ed.) Advances in Microbial Ecology, Plenum Press, New York. 5. Egli, T. 1994. Biochemistry and physiology of the degradation of nitrilotriacetic acid and other metal complexing agents. pp. 179-195. In C. Ratledge (ed.) Biochemistry of Microbial Degradation. Kluwer Academic Press, Dordrecht. Chapter5 121

6. Egli, T., M. Bally, and T. Uetz. 1990. Microbial degradation of chelating agents used in detergents with special reference to nitrilotriacetic acid (NTA). Biodegradation 1: 121-132. 7. Egli, T., U. Lendenmann, and M. Snozzi. 1993. Kinetics of microbial growth with mixtures of carbon sources. Antonie van Leeuwenhoek 63: 289-298. 8. Egli, T., N. D. Lindley, and J. R. Quayle. 1983. Regulation of enzyme synthesis and variation of residual methanol concentration during carbon-limited growth of Kloeckera sp. 2201 on mixtures of methanol and glucose. J. Gen. Microbiol. 129: 1269-1281. 9. Egli, T., H.-U. Weilenmann, T. El-Banna, and G. Auling. 1988. Gram-negative, aerobic, nitrilotriacetate-utilizing bacteria from wastewater and soil. system. Appl. Microbiol. 10: 297-305. 10. Galli, R., and T. Leisinger. 1988. Plasmid analysis and cloning of the dichloromethane-utilization genes of Methylobacterium sp. DM4. J. Gen. Microbiol. 134: 943-952. 11. Gerritse, J., F. Schut, and J. C. Gottschal. 1990. Mixed chemostat cultures of obligately aerobic and fermentative or methanogenic bacteria grown under oxygen limiting conditions. FEMS 11icrobiol. Lett. 66: 87-93. 12. Gerritse, J., F. Schut, and J. C. Gottschal. 1992. Modelling of mixed chemostat cultures of an aerobic bacterium, Comamonas testosteroni, and an anaerobic bacterium, Veillonella alcalescens- Comparison with experimental results. Appl. Environ. Microbiol. 58: 1466-1476. 13. Gottschal, J. C. 1993. Growth kinetics and competition-some contemporary comments. Antonie van Leewenhoek. 63: 299-314. Chapter5 122

14. Grover, J. P. 1990. Resource competition in a variable environment: phytoplankton growing according to Monod's model. Am. Nat. 136: 771-789. 15. Harlow, E., and D. Lane. 1988. Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, New York. 726 p. 16. Kohler-Staub, D., S. Hartmans, R. Galli, F. Suter, and T. Leisinger. 1986. Evidence for identical dichloromethane dehalogenases in different methylotrophic bacteria. J. Gen. Microbiol. 132: 2837-2843. 17. Kovarova, K., V. Chaloupka, and T. Egli. 1996. Threshold substrate concentrations required for induction of the catabolic pathway for 3-phenylpropionic acid in Escherichia coli. Appl. Environ. Microbiol. (submitted). 18. Kuenen, J. G., and J. C. Gottschal. 1982. Competition among chemolithotrophs and methylotrophs and their interactions with heterotrophic bacteria. pp. 153-187. In A. T. Bull and J. H. Slater (eds.) Microbial Interactions and Communities. Academic Press, London. 19. Law, A. T., and D. K. Button. 1977. Multiple-carbon-source- limited growth by a marine coryneform bacterium. J. Bacteriol. 129:115-123. 20. Leisinger, T., R. Bader, R. Hermann, M. Schmid-Appert, and S. Vuilleumier. 1994. Microbes, enzymes and genes involved in dichloromethane utilization. Biodegradation. 5: 237-248. 21. Lendenmann, U., M. Snozzi, and T. Egli. 1996. Kinetics of simultaneous utilization of sugar mixtures by Escherichia coli m continuous culture. Appl. Environ. Microbiol. 62: 1493-1499. 22. Locher, H. H., T. Leisinger and A. M. Cook. 1989. degradation of p-toluenesulphonic acid via sidechain oxidation, Chapter5 123

desulphonation and meta ring cleavage m Pseudomonas (Comamonas) testosteroni T-2. J. Gen Microbiol. 135: 1969-1978. 23. Loc'her, H. H., C. Malli, S. W. Hooper, T. Vorherr, T. Leisinger, and A. M. Cook. 1991. Degradation of p-Toluic acid (p-toluenecarboxylic acid) and p-sulphonic acid via oxygenation of the methyl sidechain is initiated by the same set of enzymes m Comamonas testosteroni T-2. J. Gen. Microbiol. 137: 2201-2208. 24. Manz, W., M. Wagner, R. Amann, and K-H. Schleifer. 1994. In situ characterization of the microbial consortia active in two wastewater treatment plants. Wat. Res. 28: 1715-1723. 25 Matin, A. 1979. Microbial regulatory mechanisms at low nutrient concentrations as studied in chemostat. pp. 323-339. In M. Shilo (ed.) Strategies of Microbial Life at Extreme Environments. Verlag Chemie. Weinheim. 26. Morita, R. Y. 1992. Low-nutrient environments, 617-624. In J. Lederberg (ed.). Encyclopedia of Microbiology. Academic Press, London. 27. Mills, A. L., and P. E. Bell. 1986. Determination of individual organisms and their activities in situ, 27-60. In R. L. I. Tate (ed.) Microbial Autecology- A Method for Environmental Studies. John Wiley & Sons, New York. 28. Munster, U. 1993. Concentrations and fluxes of organic carbon substrates in the aquatic environment. Antonie van Leeuwenhoek 63: 243-264. 29. Pirt, S. J. 1985. Principles of Microbe and Cell Cultivation. Blackwell Scientific Publications, Oxford. 271 p. 30. Schneider, R. P., F. Zurcher, T. Egli, and G. Hamer. 1988. Determination of nitrilotracetate in biological matrices using ion exclusion chromatography. Anal. Biochem. 173:278-284. Chapter5 124

31. Schlafli-Oppenberg, H. R., G. Chen, T. Leisinger, and A. M. Cook. 1995. Regulation of the degradative pathways from 4- toluenesulphonate and 4-toluenecarboxylate to protocatechuate m Comamonas testosteroni T2. Microbiol. 141:1891-1899. 32. Thurnheer, T., A. M. Cook, and T. Leisinger. 1988. Co- cultures of defined bacteria able to degrade seven sulfonated aromatic compounds: efficiency, rates and phenotypic variations. Appl. Microbiol. Biotechnol. 29: 605-609. 33. Thurnheer, T., T. Kohler, A. M. Cook, and T. Leisinger. 1986. Orthanilic acid and analogues as carbon sources for bacteria: growth physiology and enzymatic desulphonation. J. Gen. Microbiol. 132: 1215-1220. 34. Tien, A. J., and T. Egli. 1996a. Regulation of pollutant degradation in the presence of an alternative substrate by the para- toluenesulphonate utilizing Comamonas testosteroni strain T-2 and the dichloromethane utilized Methylobacterium strain DM4 during growth in batch and continuous culture. Appl. Environ. Microbiol. (submitted) 35. Tien, A. J., and T. Egli. 1996b. Growth of Methylobacterium strain DM4 in continuous culture: Effects of dilution rate and response to substrate shifts during continuous cultivation. Appl. Environ. Microbiol. (submitted) 36 Tien, A. J., F. Junker, A. M. Cook, and T. Egli. 1996. Dynamics of substrate consumption and enzyme activity by Comamonas testosteroni strain T-2 in response to carbon substrate shifts in carbon-limited continuous cultures. Biodegradation. (submitted) 37. Van der Hoeven, J. S., M. H. de Jong, P. J. M. Camp, and C. W. A. van den Kieboom. 1985. Competition between oral Chapter5 125

Streptococcus species in the chemostat under alternating conditions of glucose limitation and excess. FEMS Microbiol. Ecol. 31: 373-379. 38. Wagner, M., R. Amann, H. Lemmer, and K-H. Schleifer. 1993. Probing activated sludge with oligonucleotides specific for proteobacteria: Inadequacy of culture-dependent methods for describing microbial community structure. Appl. Environ. Microbiol. 59: 1520-1525. 39. Wallner, G., R. Erhart, and R. Amann. 1995. Flow cytometric analysis of activated sludge with rRNA-targeted probes. Appl. Environ. Microbiol. 61: 1859-1866. Chapter6 126 Conclusions

CONCLUSIONS

It is often difficult to extrapolate results, obtained under defined laboratory conditions (optimal temperature, pH, redox conditions, growth nutrients etc.) from single bacterial isolates or strains which degrade one type of organic pollutant in a well-defined medium, to natural environments or to wastewater treatment facilities. In such habitats mixtures of many different microorganisms and nutrients are present. Under these conditions, the classical interpretation is that degradation of organic pollutants may be repressed when other more readily utilizable nutrients are available (catabolite repression). Furthermore, it has been frequently suggested that the absence or low rate of degradation of certain organic pollutants in natural environments is directly related to the lack of the appropriate microbial strain (1, 12, 20, 22) and that successful degradation requires the enrichment of such microorganisms.

Catabolite Repression Catabolite repression by definition is the phenomenon whereby the presence of a rapidly metabolizable nutrient in the growth medium of a microorganisms inhibits, at the transcriptional level, synthesis of carbon catabolic enzyme systems and related proteins. As other forms of catabolite repression exist, in the following section when the topic of catabolite repression is brought up, we generally speak of "carbon catabolite repression". Three kinetic and mechanistically independent phenomenon fall under the category of catabolite repression; permanent repression, transient repression, and inducer exclusion. Permanent repression, which is often equated with catabolite repression, describes the phenomenon where repression occurs as long as the repressing carbon source is present in the medium. Transient repression is a temporal, Chapter6 127 Conclusions although severe form of repression which occurs following the addition of a carbon source to a bacterial culture actively growing on a different carbon source. Inducer exclusion is defined as the exclusion of the inducing compound of a catabolic operon from a cell upon the addition of the repressing carbon source (most often carbohydrates) to the culture medium (18). One common factor linking these different processes, which are often grouped as "catabolite repression", is the role of bacterial cyclic adenosine-3',5'-monophosphate (cAMP). Both permanent and transient repression have been shown to be promptly reversed by the addition of cAMP to the culture medium, and after extended growth in the presence of cAMP plus an inducer, the phenomenon of inducer exclusion was also eliminated. It is now known that cAMP and its receptor protein (CRP), which is also known as the catabolite activator protein (CAP), can mediate catabolite repression in Gram-negative bacteria (21). However, cAMP independent mechanisms of catabolite repression have been found in • 17 Gram-negative, Gram-positive, and eukaryotic m1croorgamms• . Nevertheless, it appears that the energy charge (the ratio of the adenylates) somehow regulates the activities of catabolic enzymes (19). The synthesis of many bacterial proteins and organelles, associated either directly or indirectly in carbon and energy utilization, are subject to catabolite repression. These include enzymes involved in the extracellular degradation of carbon containing macromolecules, chemoreceptors, permeases, and enzymes involved with the catabolism of exogenously supplied carbon compounds which enter into amphibolic, catabolic or anabolic pathways. For a comprehensive summary of bacterial functions subject to catabolite repression, the reader is kindly referred to Saier (17) and Saier and Fagan (18). Chapter6 128 Conclusions

Substrate utilization under carbon-excess conditions. Catabolite repression is a well known phenomenon in batch cultures of microorganisms such as pseudomonads and enterics which results in the diauxic utilization of two carbon sources when they are present in the growth medium at high initial concentrations (9, 14). In addition, repression of catabolic pathways for aromatic compounds has also been observed in batch cultivation or in continuous culture, where the repressor was supplied at a non-limiting rate (5, 10, 23). However, in all studies, the effects of precultivation conditions on the substrate utilization profiles were not investigated. It was seen from our investigations that varying precultivation conditions (Chapter 2) usually the simultaneous utilization (although perhaps somewhat unbalanced and not every substrate was utilized at the maximal possible rate) results. Preculture conditions can also influence substrate utilization profiles as seen in Chapter 2 where in one case diauxic growth was observed and in the other case simultaneous utilization was observed for the same microoganism. No catabolic repression was seen with Comamonas testosteroni (acetate and TS) or Chelatobacter heintzii (glucose and NT A) when grown with substrate mixtures, irregardless of precultivation conditions. Unexpectedly, no enhancement of µ was observed in the case of C. testosteroni (Chapter 2) in contrast to the case of C. heintzii where enhanced growth rates were measured (8). The experimental data suggests that the presence of acetate partially repressed the utilization of TS by C. testosteroni. However, the extent of this repression and how perhaps also the ratio of acetate to TS affects this repression remains to be elucidated. The results also demonstrate that many microorganisms are able to utilize many substrate combinations simultaneously (6) and that often the specific growth rate during simultaneous utilization is at least as high as with single carbon compounds or often higher (4 ). This should Chapter6 129 Conclusions confer a competitive advantage to an organism capable of mixed substrate growth. The results also point to the importance of precultivation conditions and that the catabolic potential of cells is very much influenced by the cultivation conditions. Carbon-limitation and catabolic repression. Most natural environments are limited by the availability of carbon; in spite of this fact, bacteria seem not only to survive but prosper in ecosystems (see Chapter 1). Hirsch et al. ( 11) predicted that a "model heterotrophic bacterium" living in a low nutrient (carbon) environment must possess certain characteristics which facilitate the simultaneous utilization of substrates. These predictions included that such an organism would express large proportions of catabolic enzymes which are inducible and that the carriers would be constitutive, constantly capable of uptake, and that the uptake systems are of high affinity. Considerable evidence for the derepression (synthesis of a protein in the absence of an obvious inducer) of catabolic enzymes has been shown with cells grown under carbon/ energy-limited conditions at low dilution rates in continuous culture (7, 13, 15). Release from catabolite repression during carbon-limited or starvation conditions when the potentially repressive substrate is present at a growth-limiting concentration is a generally occuring phenomenon (9). Measurement of cAMP levels in carbon-limited cultures at low dilution rates indicate that catabolite repression should not operate at low growth rates (13). The influence of an alternative substrate on pollutant degradation in carbon-limited continuous cultures was investigated (see Chapter 2). During growth with mixtures of acetate and pollutant derepression or release from catabolite repression was observed for all the bacteria used in this study (Chapter 2 and Bally et al. [3]). For instance, when C. testosteroni was cultivated by continuous culture at a constant dilution Chapter6 130 Conclusions rate, with all mixtures tested, both TS and acetate were consumed to completion (Chapter 2). Similar patterns have been observed with steady- state cultures of Methylobacterium strain DM4 with mixtures of acetate and DCM (Chapter 2) and with C. heintzii with mixtures of glucose and NTA (3). It has been reported that not only the carriers, but many catabolic pathways are derepressed or show a low constitutive level under carbon- limitation (6). In Chapter 2, during acetate-limited growth at low dilution rate, low levels of pollutant degrading activity were measured in both Methylobacterium strain DM4 and C. testosteroni. Similarly, low levels of NT A degrading enzymes were expressed during growth in glucose- limited chemostat cultures at low dilution rates (3). During carbon replacement experiments (Chapter 3 and 4 ), low levels of specific pollutant degrading activity were measured during acetate-limited growth or synthetic sewage-limited growth for the three degraders. These results further confirm that release from catabolite repression occurs during carbon-limited growth.

Induction versus enrichment The "classical" understanding, especially with wastewater engineers, is that degradation of pollutants in a system follows the enrichment of microorganisms capable of utilizing this pollutant as a nutrient. This is in contrast to what Bally et al. (3) discovered which was that the induction of NTA-monoxygenase was most likely the major mechanism for the degradation of NTA in activated sludges. In natural ecosystems or recreations there of (our model system), pollutant degrading strains may be present but not be fully induced. However, low levels of ·pollutant degrading activity were measured even in the absence of the inducer (pollutant). This allowed these microorganisms to utilize low fluxes of Chapter6 131 Conclusions pollutant carbon when it (pollutant) became available. Although the pollutants were utilized when present below 1% of carbon in the mixture, the effects on the expression of the enzyme systems has not yet been investigated in detail due to limitations in the present method. However, no detectable expression was observed below approximately < 1% pollutant carbon in the substrate mixture in the feed. This is an important field that is still open and information here would allow the assessment/estimation of the catabolic potential of ''uninduced" cells. When the numbers of uninduced pollutant degrading strains are high, this combined constitutive activity can amount to a considerably large fraction of the pollutant degrading capacity of the natural or technical systems. In most ecosystems, nutrient availability including carbon sources, frequently oscillates (16). In experiments detailed in Chapter 5, an individual pollutant contributed to some 10% of the total available carbon in the feed and full induction of the pollutant degrading pathways was expected. During growth with mixtures of synthetic sewage, extrapolation from population composition of the defined mixed culture indicated that the specific pollutant degrading strain utilized some 5-10% of the synthetic sewage carbon in the absence of its specific pollutant (Chapter 5). In addition, use of direct counts with specific fluorescent antibodies indicated that although total cell numbers increased, no enrichment of the specific pollutant degrading strain was observed, relative to the other three strains, during the transient reintroduction of its specific pollutant (approximately 10% of the total supplied carbon) into a synthetic sewage. Calculations using yield coefficients for the various pollutants indicate that the additional biomass (specific pollutant degrading strain) produced would be insignificant. For example, in our experimental system the addition of DCM (25 mg 1-1 as carbon) into steady-state cultures cultivated in the absence of this pollutant would Chapter6 132 Conclusions contribute to less than a 10% increase in total dry weight. This leads to the conclusion that induction rather than enrichment would play a role in the degradation of pollutants when ratios of pollutant carbon to available carbon are low (Chapter 5).

Practical uses of defined bacterial mixtures for the biological treatment of waste containing mixtures of pollutants

The difficulty of extrapolating results obtained under defined laboratory conditions to natural environments or to wastewater treatment facilities is well known. Understanding these complexities, an effort was made to "bridge the gap" by constructing a model system (Chapter 1 and 5) to see if patterns for pollutant degradation with mixed cultures would be similar as with pure culture systems. Results obtained with continuous pure cultures of C. heintzii (2), C. testosteroni, and Methylobacterium strain DM4, cultivated under carbon-limitation, indicate similar patterns for mixed substrate utilization and metabolic response to transient carbon shifts (Chapter 2, 3, 4). All of these strains originated from distinct subgroups of the Proteobacteria (Chapter 1) and these findings may demonstrate a common trend for bacterial utilization of pollutants: usually simultaneous utilization of pollutants and alternative carbon substrates occurs; when the pollutant carbon contributes less than a few percent of the total utilized carbon, no significant induction is observed and degradation of the pollutant is brought about by a low constitutive level. If the pollutant carbon level is higher than a few percent, additional induction occurs (Chapter 2). Additionally, similar patterns for the derepression of pollutant degrading systems were seen with defined mixed cultures as with pure culture experiments (Chapter 2, 3, 4, 5). Mixed substrate utilization by microorganisms often leads to lower residual Chapter6 133 Conclusions concentrations of the individual substrates than if these microorganisms were grown only on individual carbon substrates (13). Therefore, with respect to mixtures of pollutants and alternative carbon substrates, similar results can also be expected (Chapter 5). Together, the findings of the presented research indicate that assembly of stable, defined mixed cultures, capable of degrading mixed pollutants in the presence of alternative carbon sources is feasible as long as the microbes are not competing exclusively for a single nutrient (Chapter 5). Low biodegradation rates of organic pollutants observed in natural systems have typically been attributed to a lack of the appropriate bacterial strains rather than to sub-optimal physicochemical conditions or limited bioavailability of the pollutant or other essential nutrients. The combined results of Bally et al. (3) and Chapter 5 of this thesis further support the alternative possibility that the appropriate organisms might be present, but not be fully induced and that this may lead to the inability of the system to cope with fluctuating pollutant concentrations. In the environment where carbon is often the limiting nutrient, microorganisms must utilize all available carbon sources explaining why in many cases pollutant degrading systems are expressed at low levels. Only when the fraction of pollutant relative to the total available carbon utilized becomes high enough does it become worthwhile to express these degradative systems at a higher level (Chapter 2, 3, 4). However, as shown in Chapter 4, quicker induction rates are proportional to lower fluxes of pollutant relative to the available carbon. In engineered systems, where wider fluctuations of pollutant spectrum and quantity often occur, the time necessary for induction may be too slow to protect other relevant organisms from toxic effects of the incoming pollutant(s). This may result in loss of relevant strains and a decrease in the pollutant removal efficiency of the system. Two possible Chapter6 134 Conclusions strategies may be employed: the use of easily utilizable substrates to decrease the time necessary for induction to occur or the use of pre- induced cells. Addition of specific pollutants to a concentration of a few percent of the total available carbon (constant selective pressure) m activated sludge systems may also prevent decrease or elimination of pollutant degrading activity due to loss of the catabolic plasmid where the specific genes for pollutant degradation can be encoded. Furthermore, it may be possible to use designer mixtures of pollutant degrading microorganisms to treat industrial wastestreams or to augment the biodegradative abilities of activated sludges. As increasing pressure to properly monitor effluents continues, it can be envisioned that immobilized "cassettes" of preinduced pollutant degrading organisms may be used as "biofilters" and placed into the process wastestream to eliminate problematic pollutants as soon as they are detected. In cases where industrial wastestreams are seemingly recalcitrant, use of combined treatment strategies that incorporate both chemical attack and then biological treatment as a polishing step may be used. An example of such an approach using ozonation and then biological treatment was presented in Appendix 1. Wastewater treatment systems must no longer be viewed as "black boxes" but as co-ordinated biological treatment systems where carbon removal (pollutant and natural sources), denitrification, phosphate elimination must be integrated. The requirements which wastewater treatment facilities should fulfil have been enlarged to include both organic and inorganic pollutant removal. Through a combination of physiological and molecular approaches (Chapter 1), a better understanding of community structure and function of activated sludges, and the optimization of individual processes, may lead to intelligent design Chapter6 135 Conclusions of more efficient, cost-effective and space saving wastewater treatment facilities.

Concluding Remarks

The production of wastes, unfortunately, is a reality of today's "modem society". While the industrialized nations of the world presently discuss "sustainable development" much of their "undesirable" manufacturing and basic chemicals production have been shifted to "under-developed" countries where cheaper labor due to high birth-rates and lax environmental standards exists. However, a direct consequence of "capital investment" in "under-developed" countries is an increase in the local standard of living, increased consumption of raw and manufactured materials and increased waste production. Many developing countries are rapidly sacrificing their natural resources and damaging their environment for short term economic gains under the seductive banner of "modernization". Basic forms of wastewater treatment do not exist and most domestic and industrial wastes are directly discharged into the environment. It is the obligation of the industrialized nations to develop low-cost, robust and dependable wastewater treatment technologies for both urban and rural areas in developing countries. Restoration and protection of the environment are costly propositions which only the wealthiest of nations endeavor within their own borders. Pollution and environmental crisises do not respect national boundaries. All ecosystems on this planet are interconnected. Clean water, air and soils should be basic human rights for all and not just for those who can afford it. Chapter6 136 Conclusions

REFERENCES

1. Atlas, R. 1992. Petroleum Microbiology. p. 363-369. In J. Lederberg (ed.) Encyclopedia of Microbiology. Academic Press, Inc., New York. 2. Bally, M., and T. Egli. 1996. Dynamics of substrate consumption and enzyme synthesis in Chelatobacter heintzii during growth in carbon-limited continuous culture with different mixtures of glucose and nitrilotriacetate. Appl. Environ. Microbial. 62: 133- 140. 3. Bally, M., Wilberg, E., Kiihni, and T. Egli. 1994. Growth and enzyme synthesis in the nitrilotriacetic acid (NTA) degrading Chelatobacter heintzii ATCC 29600. Microbiology 140: 1927- 1936. 4. Brinkmann, U., and W. Babel. 1992. Simultaneous utilization of heterotrophic substances by Hansenula polymorpha results m enhanced growth. Appl. Microbiol. Biotechnol. 37: 98-103. 5. Duetz, W .A., Marques, S., De Jong, C., Ramos, J .L., and J. G. Van Andel. 1994. Inducibility of the TOL catabolic pathway in Pseudomonas putida (p WWO) growing on succinate in continuous culture: evidence of carbon catabolite repression control. J. Bacteriol. 176:2354-2361. 6. Egli, T. 1995. The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates. p. 305-386. In J. Gwynfryn Jones (ed.) Advances in Microbial Ecology, Plenum Press, New York. 7. Egli, T., 0. Kappeli and A. Fiechter. 1982. Mixed substrate growth of methylotrophic yeasts in chemostat culture: Influence of Chapter6 137 Conclusions

dilution rate on the utilization of a mixture of glucose and methanol. Arch. Microbiol. 131: 8-13. 8. Egli, T., H.-U. Weilenmann, T. El-Banna, and G. Auling. 1988. Gram-negative, aerobic, nitrilotriacetate-utilizing bacteria from wastewater and soil. System. Appl. Microbiol. 10: 297-305. 9. Harder, W., and L. Dijkhuizen. 1982. Strategies of mixed substrate utilization in microorganisms. Phil. Trans. R. Soc. London B 297: 459-480. 10. Helm, V., and H. Reber. 1979. Investigations on the regulation of aniline utilization in Pseudomonas multivorans strain Anl. Eur. J. Appl. Microb. Biotechnol. 7: 191-199. 11. Hirsch, P., M. Bernhard, S. S. Cohen, J. C. Ensign, H. W. Jannasch, A. L. Koch, K. C. Marshall, A. Matin, J. S. Pointdexter, S. C. Rittenberg, D. C. Smith, and H. Veldkamp. 1979. Life under conditions of low nutrient concentrations: Group report, p. 357-372. In M. Shilo (ed.). Strategies of Microbial Life in Extreme Environments. Dahlem Konferenzen, Berlin. 12. Leahy, J.G., and R.R. Colwell. 1990. Microbial degradation of hydrocarbons. Microbiol. Rev. 54: 305-315. 13. Lendenmann, U., M. Snozzi, and T. Egli. 1996. Kinetics of the simultaneous utilization of sugar mixtures by Escherichia coli in continuous culture. Appl. Environ. Microbiol. 62: 1493-1499. 14. MacGregor, C. H., J. A. Wolff, S.K. Arora, P .B. Hylemann, and P.V. Phibbs, Jr. 1991. Catabolite repression in Pseudomonas aeruginosa, p. 198-206. In E. Galli, S. Silver, and B. Witholt (ed.), Pseudomonas: Molecular Biology and Biotechnology. ASM Press, Washington, D.C. Chapter6 138 Conclusions

15. Matin, A. 1979. Microbial regulatory mechanisms at low nutrient concentrations as studied in chemostat. p. 323-339. In M. Shilo (ed.) Strategies of Microbial Life at Extreme Environments. Verlag Chemie, Weinheim. 16. Munster, U. 1993. Concentrations and fluxes of organic carbon substrates in the aquatic environment. Antonie van Leeuwenhoek. 63: 243-264. 17. Saier, M. H. Jr. 1985. Mechanisms and Regulation of Carbohydrate Transport in Bacteria. Academic Press, Orlando, Florida. 18. Saier, M. H. Jr., and M. J. Fagan. 1992. Catabolite repression. p. 431-442. In J. Lederberg (ed.) Encyclopedia of Microbiology. Academic Press, Inc., New York. 19. Schlegel, H.G. 1993. General Microbiology. Cambridge University Press, Cambridge. 20. Timmis, K. N., Steffan, R.J., and R. Unterman. 1994. Designing microorganisms for the treatment of toxic wastes. Ann. Rev. Microbiol. 48: 525-557. 21. Ullman, A., and A. Danchin. 1980. Role of cyclic AMP in regulatory mechanisms of bacteria. Trends in Biochem. Sci. 5:95- 96. 22. Vecchioli, G.I., Del Panno, M.T., and M.T. Painceira. 1990. The use of selected autochtonous soil bacteria to enhance degradation of hydrocarbons in soil. Environ. Pollut. 67: 249- 258. 23. Zylstra, G. J., R. Olsen, and D.P. Ballou. 1989. Cloning, expression, and regulation of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase. J. Bacteriol. 171: 5907-5917. Appendix 1 139

Combined Ozonation and Biological Treatment for the Removal of a Model Refractory Compound, Toluenesulfonate, from Simulated Industrial Wastewater

Albert J. Tient, Cristina Maria2, Wan Xil, Thomas Eglil and Elmar Heinzle2* 1Department of Microbiology, EA WAG, Diibendoif, Switzerland 2vepartment of Chemical Engineering, Swiss Federal Institute of Technology, Zurich, Switzerland

In preparation for submittal to Water Science Technology

Corresponding Author : *E. Heinzle Department of Chemical Engineering, Safety and Environmental Technology Group, Swiss Federal Institute of Technology (ETH), CH-8092, Ztirich, Switzerland

ABSTRACT

During the industrial production of chemicals, wastewater streams are pro- duced which often are recalcitrant to biological degradation. Selective ap- plication of the ozonation process has been suggested for the oxidation of refractory compounds which are unable to be degraded by cheaper, bio- logical means. Although use of ozonation for the treatment of drinking water is widely accepted, it is generally considered too expensive for large-scale Appendix 1 140

treatment of wastewaters. A model refractory compound, toluenesulfonate (TS), was used in all experiments and its degradation compared in sludge reactors, solid-liquid fluidized bed reactor (biological system) and a com- bined ozonation and biological (combined system) treatment system. In order to simulate TS degradation in wastewater treatment facilites, experi- ments with a sludge reactor were performed. Switching feed from a syn- thetic sewage to a synthetic sewage containing 10% TS in this system re- sulted in approximately a 5 hr lag period before induction of the TS degrad- ing systems. As expected, populations of the relevant TS degrading strains remained constant. Experiments were performed to compare the efficiency of TS removal in the combined system and the biological system. The initial, quicker, decrease in TS concentrations in the combined system has been attributed to the production of various alcohols, aldehydes, and other organic acids during the non-selective oxidation (ozonation) of the feed, which in turn, due to the lower overall concentrations of the various com- pounds (mixed substrate availablity), decreased the time necessary for in- duction of the TS degrading systems of the relevent TS utilizing organisms. Effluent concentrations of TS were 20 to 30 percent lower in the combined treatment system as compared to that of the biological system. However, within 30 to 40 days the effluent TS concentrations were similar in the two systems indicating greater adaptation of the relative strains for TS removal. · Steady-state operations of the biological system were perturbed by switch- ing the feed from TS and organic acids, to acetate or glucose, and then to TS. No enrichment or loss of the relevent TS degrading strain was ob- served which indicates induction plays an important part in TS removal in attached biofilm systems Appendix I 141

SELECTED REFERENCES

1. Bally, M., and T. Egli. 1996. Dynamics of substrate consumption and enzyme synthesis in Chelatobacter heintzii during growth in car- bon-limited continuous culture with different mixtures of glucose and nitrilotriacetate. Appl. Environ. Microbiol. 62: 133-140. 2. Egli, T. 1995. The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates. p.305-386. In J. Gwynfryn Jones (ed.) Advances in microbial ecol- ogy, Plenum Press, New York. 3. Junker, F., E. Saller, H. R. Schlafli Oppenberg, P.H. M. Kroneck, T. Leisinger and A. M. Cook. 1996. Degradative pathways for p- toluate and p-toluenesulfonate and their multicomponent monooxygenases in Comamonas testosteroni strains PSB-4 and T-2. Microbiology. (in press) 4. Lendenmann, U., M. Suozzi and T. Egli. 1996. Kinetics of simulta- neous utilization of sugar mixtures by Escherichia coli in continuous culture. Appl. Environ. Microbiol. 62: 1493-1499. 5. Locher, H. H., C. Malli, S. W. Hooper, T. Vorherr, T. Leisinger and A. M. Cook. 1991. Degradation ofp-Toluic acid (p-toluenecarboxylic acid) and p-sulphonic acid via oxygenation of the methyl sidechain is initiated by the same set of enzymes in Comamonas testosteroni T-2. J. Gen. Microbiol. 137: 2201-2208. 6. Pirt, S. J. 1985. Principles of microbe and cell cultivation. Blackwell Scientific Publications, Oxford. 271 p. Appendix 1 142

Stockinger, H., E. Heinzle, and 0. M. Kut. 1995. Removal of chloro and nitro aromatic wastewater pollutants by ozonation and biotreatment. Environ. Sci. Technol. 29: 2016-2022. 8. Tien, A. J. and T. Egli. 1996c. Dynamics of substrate consumption and enzyme activity by Comamonas testosteroni strain T-2 in response to carbon substrate shifts in carbon-limited continuous cultures. Bio degradation. (submitted) 9. Stern, M., E. Heinzle, 0. M. Kut and K. Hungerbiihler. 1996. Removal of substituted pyridines by combined ozonation/fluidized bed biofilm treatment. Wat. Sci. Technol. (submitted) Appendix 2 143

Microbially Mediated Formation of Struvite by Methylobacterium strain DM4: Biotechnological Applications

Albert J. Tienl and Suz-Chung Ko*2

1 Department of Microbiology, EAWAG, CH-8600 Dubendorf, Switzerland 2 Geotechnical Engineering Institute, Laboratory for Clay Mineralogy, Swiss Federal Institute of Technology (ETH), Sonneggstrasse 5, CH-8092 Zurich, Switzerland

For Submission to Geomicrobiology Journal

Corresponding Author: *Suz-chung Ko Geotechnical Engineering Institute, Laboratory for Clay Mineralogy, Swiss Federal Institute of Technology (ETH), Sonneggstrasse 5, CH-8092 Ziirich, Switzerland

Keywords: anti-corrosive coatings, biomineralization, continuous culti- vation, Methylobacterium, phosphate removal, struvite. Appendix2 144

ABSTRACT

Struvite, NH4Mg(P04)-6H20, a complex phosphate mineral of insular struc- ture was produced in batch (carbon excess) and continuous cultures (car- bon-limited) of Methylobacterium strain DM4. Identification of this min- eral was confirmed by X-ray diffraction (XRD), infrared analysis, and de- structive thermal analysis. Formation of struvite occurred as a by-product during growth of this organism on acetate, dichloromethane or synthetic sewage. It is thought that during growth on these substrates, strain DM4 shifted the equilibrium of the dissolved constituents and initiated the depo- sition of struvite. From SEM analysis it appears if strain DM4 serves as nucleation centers for crystallization. Results of this experiment combined with other investigations further support that struvite formation is microbially induced. Biotechnologically, this process may be exploited to aerobically remove phosphates from wastewater or form anti-corrosive protective coat- ings on metal surfaces.

INTRODUCTION Struvite was first described and subsequently named after the Russian dip- lomat Struve, who discovered the magnesium ammonium phosphate crys- tals in manure piles This mineral, formerly known as guanite, has been found associated with degrading organic materials such as guano deposits, dung heaps and old graveyards (Robinson, 1889, Moore, 1984 and Nelson et. al., 1991). Struvite has also been found in over fertilized soils. Over fertilization of agricultural lands can result in decreased productivity due to the formation of nearly insoluble mineral complexes that were previously thought to be caused by soil-fertilizer reactions (Lindsay & Vlek,' 1977). Struvite is a major component in human or mammalian kidney and urinary Appendix 2 145

stones (Bennett, et al., 1994, Dumanski, et al., 1994, Escolar et al., 1990). The first description that struvite may be bacterially produced was in 1889, when Robinson suggested that excess ammonium from bacterial nitrogen metabolism combined with magnesium and phosphate lead to formation of the mineral. Production of struvite has been documented in many genera and strains of bacteria (Omar, et al., 1994). Most of the research on bacte- rial struvite has been performed in conjunction with understanding the physi- cochemical properties which contribute to kidney and urinary tract stone formation. (Westbury, 1974, Griffith, 1978, Bennett et al., 1994, Dumanski et al., 1994) Findings by Rivadeneyra et al. (1983) suggest that although ammonium release is necessary for microbial struvite in a medium contain- ing magnesium and phosphate. The ability of heat-killed cells to precipitate struvite indicate that the mechanism of struvite formation involves the ab- sorption of magnesium and phosphate ions, the release of ammonium ions on or in the vicinity of the cell surface (Rivadenerya et al., 1992) Previous studies have described the formation of struvite on the surface of bacterial colonies grown on or in solid media containing agar/silica gel or in batch cultures with viable or heat-killed cell, all under carbon-excess conditions. This study describes struvite formation by Methylobacterium strain DM4 under batch conditions with various substrates (carbon-excess) and carbon- limitation in continuous culture and discusses the possible biotechnological uses of struvite forming organisms.

MATERIALS AND METHODS

Bacterial strain and maintenance conditions. Methylobacterium strain DM4 (Galli, 1988) was used in all experiments. Appendix 2 146

Microorganisms were grown and maintained as previously described (Kohler-Staub et al., 1986).

Batch culture media A synthetic media containing per liter 3.2 g Na2HP04, 2.38 g KH2P04, 1.0 g MgS04·7H20 and 1.0 g (NH4)2S04, 5.0 mL of a trace element solution (Egli, 1988) with either 0.716 g of sodium acetate or 1.0 mL of DCM as the sole source of carbon and energy. Synthetic sewage was based upon a receipe detailed by Painter (1986) containing per liter 0.14 g peptone, 0.1 g meat extract, 0.009 g urea, 0.006 g NaCl, 0.18 g NaAc, 0.3 g MgS04·7H20, 0.02 g CaCl2·2H20, 0.14 g KCl, 1.07 g NH4Cl, 0.02 g (NH4)2S04, 3.2 g Na2HP04, 2.38 g KH2P04 and 1 ml trace elements (Egli et al., 1988). Dissolved organic carbon levels were maintained at 200 mg 1-1. When DCM was used, organisms were grown in erlenmeyer flasks with screw caps which were teflon lined to prevent leakage of the DCM due to its low boiling point and high vapor pressure.

Continuous culture media For continuous cultures, a modification of the media detailed above was used where phosphates were substituted for 2.46 g Na2HP04, 1.8 ml H3P04 (85%), 5 ml of trace elements, and 10 mg silicon antifoam (Fluka, Buchs, Switzerland). The bioreactor (MBR, Switzerland, 2L working volume) was aerated at 0.1 L min while stirred at 1,500 rpm with pH automatically con- trolled at 7 .5 with H3P04 (1 M) and KOH/NaOH (0.5 M, each). The tem- perature was maintained at 30°C. Appendix 2 147

Analysis of Stmvite To identify the struvite crystals X-ray powder diffraction (XRD), Infrared absorption spectroscopy (IR), thermal analysis (DTA, DTG, TG, and MS- EGA) and scanning electron microscopy (SEM) are used. Sample prepara- tions and the equipments used are described below.

XRD Crystals which were freshly collected from the chemostat were air dried, pulverized in an agate mortar, and packed into a shallow sample holder to form a flat surface containing randomly oriented crystals. The X-ray diffraction (XRD) pattern was obtained with a Philips APD 1900 diffractometer, using monochromed CuKcx. radiation at 35kV and 40mA. Scans were recorded from 2 to 65° of20, with a scan speed of0.4° of20per minute; sensitivity of 1o3 and a time constant of 2.

IR The infrared (IR) absorption spectrum was carried out by a Perkin Elmer Fourier Transform Infrared spectrometer (2000-FT-IR). Mixture of 2 mg finely grinded sample and 200 mg potassium bromide (KBr) powder were dried in the oven at 105°C overnight. Under a pressure of 12 ton/cm2 and vacuum, a 13 mm in diameter, transparent disc was pressed. Wavelength of IR between 25 and 5 µm (wavenumber between 400 and 1800 cm-1) was used in present investigation. Absorption band was recorded according to · the stretching and bending during the molecular vibrations.

TA Thermal analysis including differential thermoanalysis (DTA), differ- ential thermogravimetry (DTG), thermogravimetry (TG), and analysis for selected masses of evolved gases (MS-EGA) were performed in a system which combines a Mettler thermobalance (TG2) and a Balzers quadruople Appendix 2 148

mass spectrometer (QMG 420) with a heated steel capillary. The investiga- tion of 10-50 mg of sample were done at a heating rate of 10°C per minute in streaming dry air (1.9 liter per hour) within a temperature range from 25 to 1000°C. With dry air as a flush gas, H20, N2, NO, 02, C02 and S02 could be observed by the mass spectrometer.

SEM For scanning electron microscopy observation, crystal aggregates and single crystal samples were coated with gold by under vacuum. Sec- ondary electron images were produced by a JOEL JSM 840 SEM at work- ing distance of 18-19 mm, accelerating voltage of 25kV, and a beam current of 10-lO to 10-ll Amp.

RESULTS

Batch cultivation During batch culture of strain DM4 on acetate, DCM or synthetic sewage, struvite formation was seen within 48 hrs of inoculation. Abiotic controls remained clear with no crystal formation observed. However, with heat- killed cells struvite formation was observed within 96 hrs. No formation of struvite was observed on the agar plates used to maintain the strain.

Continuous cultivation In continuous cultures of strain DM4, irregardless of carbon source, a thin pink mineralized biofilm was found covering the inside of the glass reactor vessel and all metal surfaces becoming thicker in low turbulent areas within the chemostat (e.g. the backside of the baffles, propeller shaft, heating rod, temperature sensors, and on certain sections of the walls) Over the next two Appendix2 149

weeks, this biofilm became thicker and mineralized (up to 2-3 mm thick). Initially, it was thought that this covering was a carbonate. However, no efferescence was seen after the addition of a drop ofHCl indicating that the mineral coating was not a carbonate. Samples were air dried and saved for further identification. Fig 1 shows crystal aggregates obtained from the chemostat with visible pink inclusions attributed to strain DM4.

Identification of struvite X-ray diffraction pattern of the collected sample from the chemostat is shown in Fig. 2. fypical struvite peaks are identical with the reference 40-0621 published by JCPDS (Joint Committee on Powder Diffraction Standard). Similar XRD pattern was given by Rivadeneyra et al. (1992) where struvite crystals were precipitated by various bacterial strains.

Fig. 3 presents the IR spectrum of the crystals grown on the wall the chemostat. Absorption peaks appear at wavenumbers of 1682 and 1615 representing the OH bending; at 1470, 1434 for NH4+ bending; at 1006, 571, 461, and 441 relating to phosphate stretching and bending; and at 894, 760, and 701 corresponding to H20 liberation. These absorption peaks are found to be the same as those of struvite documented in Farmer (1974). Thermoanaylsis were also done on the mineralized coating in the chemostat. Fig. 4 show a series of the TG, DTG, DTA, and MS-EGA curves obtained from the sample. A total 7 .889 mg of weight loss at the temperature be- tween 90 to 400 °C was recorded for the sample which had initial weight of 14.5 mg (Fig. 4a). As it is shown in the DTG curve (Fig. 4b) most of the weight loss happened below 200°C which represents the loss of the crystal water. The DTA curve shown in Fig. 4c illustrates a great endothermic peak Appendix2 150

at approximately 140°C corresponding to the dehydration of the mineral phase. Shortly before 700°C was reached, mineral phase experienced an- other exothermic reaction at 690°C (Fig. 4c) which is probably caused by the rearrangement of the crystal structure. Together with the MS-EGA curve, the above information suggest that the struvite crystals (NH4Mg(P04)·6H20) was first dehydrated and lost the 6H20 between 100 and 200°C. Following the dehydration was the release of ammonia (NH3) and a further decomposition of NH3 to NO in the gas form at around 380°C (Fig. 4d). The magnesium pyrophosphate (Mg2P207) then resulted (Duval, 1963) and probably went through reconstruction of the crystal structure at 690°C. A step.;wised decomposition reaction may be written as eq. (1): 2NH4Mg(P04)·6H20 -> 2NH4Mg(P04) + 12 H20 (1) As the molecular weight of struvite is 490.82g mo1-l, and of Mg-pyrophos- phate is 222.S7g mol-1, the theoretic total weight loss during heating up to 1000°C should be S4.6%. A perfect agreement was obtained from the TG analysis which gives a 54.4% of weight loss (Fig. 4a). A small amount of C02 was also detected in the MS-EGA curves which may result from a low content of organic matters. With SEM analysis, it could be seen that bacterial bodies were embedded in the struvite crystals. (Fig Sa and 5b). Struvite crystals were perfectly formed rhombic-pyrimidal shapes identical to that described in Klockmanns Lehrbuch der Mineralogie, 1978 (Fig. Sc). From all the above mentioned mineralogical investigations, it is confirmed that the mineralized coating on the wall of the chemostat was struvite. Appendix 2 151

DISCUSSION

Factors influencing struvite crystallization Rivadeneyra et al. 1983 determined that the specific concentration of am- monium ion, although highly relevant for biologic struvite formation, is not the only factor responsible for struvite formation. Under abiotic conditions with equimolar solutions of magnesium and phosphate ions at 25°C, single crystals of struvite could be formed at a concentration range for both ions at a range of 0.005 to 0.025 M from ammoniacal solutions (Abbona and Boistelle, 1985). In this same study it was determined that for each concen- tration there is a critical lower pH where single crystals start to precipitate and another higher pH where crystal aggregates dominate. An earlier study by Boistelle et al.(1984 ) using chemically pure solutions determined that struvite crystal formation depended on the pair pH product of the total con- centration of NH4+, Mg2+, and P043-. This finding is also applicable to biologically produced struvite. Rivadeneyra et al. 1992 hypothesize that in addition to releasing ammonium at sufficient concentration, struvite pro- ducing bacteria also may be able to concentrate Nff4+, Mg2+, and P043 ions in the vicinity of the cell or on the cell envelope. The results of our experiments especially the results of the SEM analysis of struvite crystals from the chemostat support the findings of Rivadeneyra et al. and further give evidence that the cell surface is involved with the microbially medi- ated nucleation of struvite.

Role of microorganisms in the formation of phosphate minerals Ehrlich (1981) implied that under certain conditions, microbes may pro- mote the formation of insoluble inorganic phosphates, such as calcium, Appendix 2 152

aluminum, or iron. Phosphate mineral complexes include members in the variscite group, (Al, Fe)(P04)·2H20; apatite group Ca2Ca3(P04)3(0H,F); octocal group, CagH2(P04)frSH20; struvite group, NH4Mg(P04)-6H20; minyulite group, KAl2(P04)2(0H)·4H20 and churchite group, Y(P04)·4H20. Therefore it seems realistic that microbial mediation could contribute to the formation of many phosphate minerals including struvite. The role of bacteria, however, in the formation or mediation of these phos- phate minerals in the environment has not been examined. Microbial me- diation in the formation of many low temperature minerals including car- bonates and silicates have been proposed by Folk (1993). Many struvite forming microorganisms are also capable of forming calcite (CaC03) (Boquet et al, 1973; Rivadeneira et al., 1985). Dolomite (MgCaC03) has been reported to have been produced by sulfate reducing bacteria and it was hypothesized that the bacteria formed nucleation centers for crystallization (Vasconcelos et al., 1995). Similarly, in the present study, from SEM analy- sis, bacterial bodies were seen in the struvite crystals indicating their role in the crystallization process. These results contribute to the growing body of evidence that microorganisms play a greater role than previously thought in formation of low-temperature mineral.

Biotechnological implications Much of the information about biologically mediated struvite production stems from studies examining the formation of kidney or urinary tract stones in humans and animals. Fortuitously, much of this information can also be used to understand the formation of this mineral in the natural environment. Below are presented possible biotechnological uses for struvite producing m1croorgan1sms. Appendix 2 153

Wastewater treatment Several chemical and biological mechanisms are responsible for the removal of phosphorus in wastewater treatment plants (Shapiro et al, 1967, Tetreault et al. 1986, Meganck and Paup, 1988, Egli and Zehnder, 1994): 1) Addition of precipitants such as Ca, Fe, or Al under pH influenced conditions; 2) Microbial polyphosphate removal; 3) Micro- bial phosphorus uptake; and 4) Microbial/chemical precipitation. It is not our intention to discuss the large body of literature concerning biological phosphorus removal but rather to give insight into how the use of struvite producing organisms might play a role in phosphorus removal fromwastewaters. As demonstrated from Rivadeneyra et al. (1992) autoclaved cells were able to initiate the precipitation of struvite from an organically rich media designated B-41 (4 g yeast extract per liter) or EL media (also 4 g yeast extract per liter). In our study, at carbon levels closer to domestic wastewaters (200 mg carbon 1-1, autoclaved cells of strain DM4 were also able to precipitate struvite from synthetic sewage. Use of killed struvite "nucleating" microorganisms may be utilized as an alternative to the addition of chemical precipitants for phosphorus removal. In addition, as demonstrated from our continuous culture experiments the use of living struvite producing cells in a continuous treatment system with cell recycle may also be a possible method for the removal of phosphorus from wastewaters. Further research into these topics must be carried out in order to find other alternative methods for phosphorus removal in wastewaters.

Anti-corrosive coatings Microbially influenced corrosion (MIC) of steel alloys may occur via several different mechanisms (Tantell, 1981, Stoecker, 1984, Ford and Mitchell, 1990). Most of these mechanisms rely on surface Appendix2 154

colonization by the bacteria. Addition of biocides or bioinhibitors may of- ten become cost prohibitive and in some instances microbes may become resistant to these agents. Much of the impetus to the study of MIC has been due to the threat of microbially induced corrosion in primary cooling sys- tems of nuclear reactors or nuclear storage canisters (Meregay et al., 1984; Miller et al, 1987, Miller et al. 1988). Use of microorganisms to combat biocorrosion is a relatively open field. The results of our continuous culture experiments indicate that under relatively low carbon conditions, a biomineralized biofilm of Methylobacterium coated all contacted surfaces within the chemostat. The Ksp for struvite was reported to be 7 .1 x 1o-14 indicating that this mineral is incongruently soluble (Taylor et al., 1963). Data from thermal analysis indicate that struvite is stable up to 200°C where loss of water in aqueous situations is irrelevant. Dissolution by water actu- ally results in the formation of a more stable, less soluble Mg3(P04)2. More research into the use of struvite forming microorganisms is necessary to assess its application as a biological anti-corrosive coating.

ACKNOWLEDGEMENTS The authors would like to thank Dr. Djordje Grujic who assisted us with SEM analysis and Dr. Gilnter Kahr for critical discussions.

REFERENCES

Abbona, F., and R. Boistelle. 1985. Nucleation of struvite (MgNF14P04·6H20) single crystals and aggregates. Crystal Res. & Technol. 20: 133-140. ·Bennett, J. S.P. Dretler, J. Selengut, and W.H. Orme-Johnson. 1994. Identi- Appendix 2 155

fication of the calcium-binding protein calgarnulin in the matrix of struvite stones. J. Endourol. 8: 95-98. Boistelle, R., F. Abbona, Y. Berland, M. Grandvuillemin, and M. Olmer. 1984. Les domaines de nucleation du phosphate ammoniacomagnesien dans les urines steriles , alcalines ou acides. Nephrologie, 5: 217-221. Boquet, E., A. Boronat, and A. Ramos-Cormenzana. 1973. Production of calcite (calcium carbonate) crystals by soil bacteria is a general phe- nomenon. Nature 246: 527-529. Dumanski, A.J., H. Hedelin, A. Edin-Liljegren, D. Beauchemin, and R. J. McLean. 1994. Unique ability of the Proteus mirabilis capsule to en- hance mineral growth in infectious urinary calculi. Infect Immun 62: 2998-3003. Duval, C .. 1963. Inorganic thermo-gravimetric analysis. (Translated by R.E. Oesper) Elsevier Publishing company, New York, pp.722. Egli, T., H.-U. Weilenmann, T. El-Banna and G. Auling. 1988. Gram-nega- tive, aerobic, nitrilotriacetate-utilizing bacteria from wastewater and soil. System. Appl. Microbiol. 10: 297-305. Egli, T. and A.J. Zehnder. 1994. Phosphate and nitrate removal. Current Opinion in Biotechnology. 5: 275-284. Ehrlich, H. L. 1981. Geomicrobial transformations of phosphorous com- pounds. pp.137-146. in: Geomicrobiology. Marcel Dekker, Inc. Basel. Escolar, E., J. Bellanato, and J.A. Medina. 1990. Structure and composition of canine urinary calculi. Res. Vet. Sci. 49: 327-333. Farmer, V.C. 1974. The infra-red spectra of minerals. Mineralogical Soci- ety Monograph, 4. Adland & Son Ltd., Dorking Surrey, 539pp. Folk, R. L. 1993. SEM imaging of bacteria and nanobacteria in carbonate sediments and rocks. J. Sedim.Petrol. 63 :990-999. Appendix 2 156

Ford, T., and R. Mitchell. 1990. The ecology of microbial corrosion. Ad- vances in Microbial Ecology. 11: 231-262. Galli, R. and T. Leisinger. 1988. Plasmid Analysis and Cloning of the Dichloromethane-utilization Genes of Methylobacterium sp. DM4. J. Gen. Microbiol. 134: 943-952. Griffith, D. P. 1978. Struvite stones. Kidney Int. 13: 372-382. Kohler-Staub, D., S. Hartmans, R. Galli, F. Suter and T. Leisinger. 1986. Evidence for Identical Dichloromethane Dehalogenases in Different Methylotrophic Bacteria. J. Gen. Microbiol. 132: 2837-2843. Meganck, M.T.J., and G.M. Paup. 1988. Enhanced biological phosphorous removal from waste waters. pp. 111-203, in: Biotreatment Systems. vol. 3, D.L. Wise, Ed. CRC Press, Boca Raton, FL. Mergeay, M., M. Bourdos, W. Horsten, and R. Kirchman. 1984. Detection of microorganisms in primary cooling systems of nuclear reactors. Proc. IUR/CEU Workshop on the Role of Microorganisms in the Behavior ofRadionuclides inAquatic and Terrestrial Systems and their Transfer to Man. Brussels, Belgium: 132-138. Miller, R.L., J.H. Wolfram, and Ayers, A.L .. 1987. Accelerated laboratory testing for microbially induced corrosion of Three Mile Island (TMI)- 2 cansiter materials. United States Department of Energy. Miller, R.L., J.H. Wolfram, and Ayers, A.L.. 1988. Studies of microbially influenced corrosion on TMI canister. DOE Report No. EG&G MS T 7744. Moore, P.B .. 1984. Crystallochemistry an destructures of phosphate miner- als. In: Nriagu, J.0. andP.B. Moore (eds) Phosphate minerals. Springer- Verlag., p. 155-170. Nelson, B ., J. Struble, and G. McCarthy. 1991. In vitro production of struvite Appendix 2 157

by Bacillus pumilus. Can. J. Microbial. 37: 978-983. Omar, N.B., M. Entrena, M.T. Gonzalez-Munoz, and J.M. Arias. 1994. Ef- fects of pH and phosphate on the production of struvite by Myxococcus xanthus. Geomicrobiol. J. 12: 81-90. Painter, H. A. 1986. Technical Methods Section: Testing the toxicity of chemi- cals by the inhibition of respiration of activated sludge. Toxicity As- sessment. 1:515-524. Ramdohr, P. and H. Strunz. 1978. Klockmanns Lehrbuch der Mineralogie, Ferdinand Enke Verlag, Stuttgart. 876p. Rivadeneyra, M.A.,A. Ramos-Cormenzana, and A. Garcia-Cervignon. 1983. Bacterial formation of struvite. Geomicrobiol. J. 3: 151-163. Rivadeneyra, M.A., I. Perez-Garcia, V. Salmeron, and A. Ramos- Cormenzana. 1985. Bacterial precipitation of calcium carbonate in pres- ence of phosphate. Soil Biol. Biochem. 17: 171-172. Rivadeneyra, M.A., I. Perez-Garcia, A. Ramos-Cormenzana. 1992. Influ- ence of ammonium ion on bacterial struvite production. Geomicrobiol. J. 10: 125-137. Robinson, H. 1889. On the formation of struvite by microorganisms. Proc. Cambr. Phil. Soc. 6: 360-362. Shapiro, J., G.V. Levin, and H.G. Zea. 1967. Anoxically induced release of phosphate in wastewater treatment. J. Water Pollut. Control Fed. 39: 1810-1818. Stoecker, J.G .. 1984. Guide for the investigation of microbially induced corrosion. Materials Performance. 23: 48-55. Tatnell, R.E. 1981. Fundamentals of bacterial induced corrosion. Materials Performance 20:32-38. Taylor, A.W., A.W. Frazier, and E.L. Gurney. 1963. Solubility products of Appendix 2 158

magnesium ammonium and magnesium potassium phosphates. Transections of the Faraday Society. 59: 1580-1584. Tetreault, M.J., A.H.Benedict, C. Kaempfer, and E.F. Barth. 1986. Biologi- cal phosphorous removal: A technology evaluation. J. Water Pollut. Control Fed. 58: 823-837. Vasconcelos, C., J.A. McKenzie, S. Bernasconi, D. Grujic, and A.J. Tien. 1995. Microbial mediation as a possible mechanism for natural dolo- mite formation at low temperature. Nature. 377: 220-222. Westbury, E. J. 1974. Some observations on the quantitative analysis of over 1000 urinary calculi. Brit. J. Urol. 46: 215-227.

FIGURE CAPTIONS

Figure 1. Optical photomicrograph of struvite shows the crystal aggregates with pink inclusion attributed to strain DM4. The size of the aggregates is 2.5 mm.

Figure 2. X-ray diffraction pattern of struvite. The d-values are identical to those published by JCPDS (reference 40-0621).

Figure 3. Infrared absorption spectrum of struvite. Absorption peaks (in wavelength cm-1) at 1682 and 1615 represent the OH-bending, at 1470 and 1434 for Nff4+ bending, at 1006 for phosphate stretch- ing, at 894, 760 and 701 for H20 liberation, and 571, 461, and 441 for phosphate bending. Appendix 2 159

Figure 4. A series ofthermoanalysis curves for struvite. a) TG curve shows a total 7 .889 mg weight loss between 90 and 400°C. b) DTG curve shows that most of the weight loss occurred below 200°C corresponding to the loss of crystal water. c) OTA curve demon- strates an endothermic peak at 140°C for the dehydration of struvite and an exothermic peak at 690°C caused by the rearrange- ment of the crystal structure. d) MS-EGA curve shows the re- lease of NH3 and a further decomposition of NH3 to NO.

Figure 5. SEM photographs for struvite. a) struvite crystals are coated by bacterial bodies. b) a closer view of the above figure showing the embedded bacterial bodies. c) a struvite crystal is perfectly formed in the rhombic-pyrimidal shape. Appendix 2 160 Intensity (counts) 0 1000 2000 3000 4000 5000 SMt&l... r-::::======--'-~--'..__~_,__~...._~_,_~_._~--~---i> "T1 5' ..... CD

~ 0

6.119 - 5.892 - 5.595 5.371

4.589 - N 4.25 0 4.132 =

3.555 3.469 3.289 3.189 3.067 3.02 2.959 0 2.916 = "' 2.801 2.723 - 2.691 =S' 2.66 ~ 2.549 - -i 2.51 -~ -i 2.393 2.35 -i_., 2.3 2.253 - ~ 2.182 - 2.167 - 2.128 - 2.069 2.056 = 2.046 - 2.04 - 2.016 - 1.984 = 1.963 1.957 1.934 1.922 = 1.874 - > 1.85 );. 1.824 - gj r 1.81 - (]) 1.796 - m 1.763 :D 1.738 - :-i 1.714 :D 1.682 0 1.659 1.647 =- 1.611 1.597 - 1.59 = 0 1.556 "' 1.535 1.531 = 1.513 1.466 - 1.482 1.46 1.449

191 z :qpuaddy Fig. 3

eo.o -,

75.0 -

70.0

65.0 \ 60.0 \ (' 55.0 ..; \ I '\ I \ '- 50.0-i ~~ /~\ / \ I l -,,_ . I phosphate stretching I I I J \ \ I 45.0 - \ I \ \ 1006.B ii \ \ l ··~-- / \ ! i ~ I °"--..,.../ I I \I r.. \ 1682.5 \ i 701.5 I 35.D y\ \ I \ \ I \ / \ I 30.0 ~ 1615.3 I I \ ,/ I \ I' 894.7 I I 25.0 I OH bending / i 1470.5 \\ I 'i 20.0 \ \I I \ ' H20 liberation I; 461.4 \ v I ' \ ;' 15.0 NH4+ bending I \ 571.4I 10.0 I \ I . i phosphate bending \ / 5.0 \ i / ' -~./ !

0.0+-----~-~----, ----~·------~-·-----·~------T -----;-----,·-··-···--·-,------,--·---··--·.,----··--,------~ 'l 1600 1700 15110 1400 1300 1200 1100 900 800 500 400 CM-1 Appendix 2 163

Fig. 4a a: 17525 TRU. CYC

....

200 400 600 BOO 1000 Heat inq teroperature degree C

Fig. 4b a: 1752STRU. CYC

~ c: ... 0 .., ro b c: u "'c: 3rri c: ·~_,

0

0 200 400 600 BOO IOOO Heating teroperature degree C Appendix 2 164

Fig. 4c a: l 752STRU. CYC

c 0

0 200 400 600 800 IOOO Heating temperature degree C

Fig. 4d a: 1752STRU. CYC

0

0 200 400 600 aoo 1000 Heating temperature degree c Appendix 2 165

Fig. Sa

Fig. Sb

Fig. Sc Appendix 3 166 Letters to Nature

Mlcroblal mediation as a TABLE 1 Electron diffraction analysis of experimental carbonate Miller index Sample Dolomite Ankente Calcite possible mechanism (hkl) d(hl

220 NATURE · VOL 377 · 21 SEPTEMBER 1995 Appendix 3 167 Letters to Nature

LETIERS TO NATURE

FIG. 1 SEM secondary electron images showing: a. an overview of the surface of the quartz sub- strate used in the bacterial cultural experiment- note that the surface is partially covered with a knobbly carbonate coating that appears to be colonized by subspherlcal nanobacteria (scale bar. 111m); b. close-up of a section of the car- bonate coating that was analysed for elemental identification using the EDX attachment (see Fig. 2; scale bar, 0.1 µm; c, close-up of twinned nanobacterla that may have been Imaged in the process of reproduction via cell division (binary fission) (scale bar, 0.1 µm); d, close-up of the carbonate coating, illustrating the apparent stages In the process of its bacterial formation from detached subpherical nanobacteria encrusted by nanocrystals of carbonate to attached or embedded bodies that become rounded bumps entombed in the coating, which in turn merge to cover the surface of the substrate (scale bar, 0.1 µm). SEM images were produced by a JEOL JSM 840 SEM coupled with an energy dispersive X-ray spectrometer (TRACOR N 2000) for elemental identification.

that appears to be colonized by subspherical nanobacteria. measured from the TEM negative and corrected by a factor Figure 2 is an energy-dispersive X-ray spectrum centred on the related to the TEM characteristics, arc given in Table I, together colonized coating of the quartz grain shown in Fig. lb. The with the d(likl) values for standard dolomite, ankerite and calc- spectrum contains high-intensity peaks for Mg and Ca, as well ite. A comparison of the d(hkl) values indicates that the experi- as lesser peaks for Fe, indicating that the coating would be con- mental carbonate has a composition lying between dolomite and sistent with a Ca, Mg, Fe carbonate. X-ray diffraction analysis ankerite-in other words, a fcrroan dolomite. The identification of the aggregates shows that, apart from the quarlz substrate, of fonoan dolomite for the carbonate mineral that weakly binds they comprise predominantly rhombohcdral carbonate (Fig. 3). the aggregates of the quartz substrate used in the bacterial The sharply defined major peak, the d( 104) reflection. occurs culture experiment is consistent with the qualitative elemental at 2.90 A (30.79'' 28) in the diffraction pattern, indicating the scan (Fig. 2). The presence of Fe in the dolomite structure presence of dolomite. This position is shifted slightly from the is not inconsistent with the activity of sulphate-reducing bac- major peak for ideal dolomite, which occurs at 2.89 A teria under anoxic conditions. Also, Fe is a component of (30.99'' W). The X-ray diffrnction pattern for the experimental the culture medium. Together, our analyses provide conclusive dolomite shows secondary peaks (Fig. 3). In particular, the pres- evidence that the bacterial production of dolomite can be ence of the 015 peak is consistent with its being an ordered achieved in low-temperature anoxic conditions in a relatively 12 dolomitc • short-time. Electron diffraction analysis of the aggregates using transmis- The bacterial bodies have different sizes, with an average sion electron microscopy (TEM) was done lo determine more diameter of 0.2 µm (Fig. I b-d ). At the higher magnification, the exactly the crystallographic characteristics of the experimental nanobacteria appear to be encrusted by nanocrystals of dolo- carbonate. The d(hkl) parnmeters (in Al for the carbonate, mite, which apparently precipitates on the outer surface of the

FIG. 2 An energy-dispersive X·ray (EDX) spectrum of the surface coating on the quartz grain shown In Fig. lb. Note the intensity of the Mg, Ca and Fe peaks. the dominant elements identified in the sample (apart from Si for the quartz substrate), and Au from the gold coating used to achieve a better resolution of the SEM image. The relative peak height of Mg is less than Ca because of the attenuation of Mg X-rays in the EDX system.

101 s Energy (keV)

NATURE · VOL 377 · 21 SEPTEMBER 1995 221 Appendix 3 168 Letters to Nature

LETIERS TO NATURE

9,000 Oz Oz l!a, 8,000

Oz I\ 7,000 11

6,000 1\ Oz II -~ 5,000 I c ~ 4,000 Ii Dal Oz I 3,000 024) Oz ! 2,000 of\z Dol I 202 Ii I 1,000 Dal Dal < [ ' (~15)

I'I 0 J ~_jJLJ\1 20 25 JL30 35 40 45 50 20 l'

FIG. 3 X-ray diffractogram (20" to 55° 29) of the bulk powdered sample powder diffraction pattern clearly correspond to quartz plus a rhom- recovered from the bacterial experiment. The XRD analysis was pro- bohedral carbonate. The presence of the 'ordering reflection' 015 peak 12 duced on a STOE STADIP transmission goniometer. The peaks in the Indicates that the mineral is a fully ordered dolomite • bacterial bodies. Some of the nanobacteria may be in the process make these calibrations, which are needed to interpret conditions of reproduction by cell division (Fig. le). Figure ld shows that, of ancient dolomite formation. as the process of bacterial precipitation proceeds, the bodies no The results of this bacterial culture experiment provide strong longer occur as discrete subsphercs on the surface but become evidence for the involvement of bacteria (possibly including sul- attached or embedded in it. Finally, they become rounded bumps phate-reducing bacteria) in the precipitation of dolomite. In entombed in the coating, which in turn covers the surface of the 1928, Nadson reported results from a similar bacterial culture substrate. As the nanobacteria use the quartz surface as a growth experiment in which he used sulphate-reducing bacteria from substrate, the precipitation of dolomite in conjunction with the anoxic surface sediments in a Russian salt lake to produce very microbial activity apparently cements the quartz grains to form small amounts of an authigenic carbonatcll. A qualitative chemi- the observed aggregates. cal analysis of the carbonate led him to propose that dolomite The 8 180.,DK and o13Cpoe values of the fcrroan dolomite are might have been precipitated in association with the anaerobes; -6. 7 and -12.8%o, respectively. These values are further evi- he suggested that 'understanding the essential role by this bacter- dence that the dolomite is not a contaminant but a product of ial phenomenon may be the solution to the dolomite problem'. 18 the bacterial culture experiment. As the o 0sMOW value of the As microbial activity apparently governs dolomite precipita- water used in the experiment was -14.0'Yoo, the dolomite appears tion in Lagoa Vermelha, we propose that microorganisms may isotopically to mirror the solution from which it precipitated. be important as dolomite nucleation centres in this environment, 13 The negative o Cp08 value of the dolomite reflects an input as well as in other environments of dolomite formation such of organic carbon released as a product of sulphate reduction. as coastal sabkhas and carbonate platforms. Considering the Because it has not been previously possible to produce dolomite unequal distribution of dolomite in the geological record, we in the laboratory at Earth-surface conditions, calibration of its further suggest that the bacterial production of dolomite was far isotope fractionation, as well as minor cation distributions in more significant in the past than at present, in particular in the dolomite, could not be achieved. We suggest that future bacterial Proterozoic when the dolomite/limestone ratio was about 3: I culture experiments will furnish us with a valuable method to (ref. 14). D

Received 30 May; accepted 7 August 1995. 8. Vasconcelos, C. thesis. ETH·Zurlch (1994). 1. Machel, H.·G. & Mountjoy, E. W. fanh·Sc/. Rev. 23, 175-222 (1986). 9. Folk. R. L J. sedlm. Petro/. U, 990-999 (1993). 2. Purser. B. H.. Tucker, M. E. & Zenger, D. H. in Dolomites: A Volume In Honour of Do/omieu 10. Folk, R. L. GSA Abstr., 1993 Ann. Meeting 21, A-397 (1993). (eds Purser, B .. Tucker, M. E. & Zenger, D. H.) 3-20 (Spec. Pubis Int. Ass. Sediment. 21, 11. Postgate, J. R. The Sulphate-Reducing Bacteria 2nd edn /Cambridge Univ. Press, Blackwell Scientific, Oxford, 1994). Cambridge, UK, 1984). 3. Lippman, F. Sedimentary Carbonate Mlnera/s (Sprineer. New York, 1973). 12. Reeder, R. J. in Carbonates: Mineralogy and Chemistry (ed. Reeder. R. J.) 1-47 (Rev. 4. Land, L $. in Concepts and Models of Dolomltlzation (eds zenger, 0. H., Dunham, J. B. & Mlneralog. 11, Minoralog. Soc. Am. Washington DC. 1983). Ethington, R. L.) 87-110 (SEPM Spec. Publ. 28. Tulsa. OK, 1980). 13. Nadson, G. A. Archiv. Hydroblol. 19, 154-164 (1928). 5. Gunatllaka, A. Modern Geology :U, 311-324 (1987). 14. Garrels, R. M. & Mackenzie, F. T. Evolution of Sedimentary Rocks (Nonon, New York. 1971). 6. McKenzie, J. A. in controversies In Modern Geology: Evolution of Geological Theories in Sedimenfology, fanh History and Tectonics (eds Muller. 0. W, McKenzie. J. A. & Weissen. ACKNOWLEDGEMENTS. We thank A. Rebello Wagner, who introduced us to Lagoa Vermelha H.) 35-54 (Academic, London, 1991). and its exciting potential as a natural laboratory; G. Fruli-Green and R. Gubser. who assisted 1. Land, L. S. in Isotope Signatures and Sedimentary Records (eds Ciauer, N. & Chaudhuri. with X-ray diffractiOn analysis: A.·M. Karpoll, who provided the TEM analysis and in:erpretation: S.) 4~ (lecture Notes In Eanh Sciences 43. Sprineer, Berlin, 1992). and L. Land tor critically reviewing the manuscript.

222 NATURE · VOL 377 · 21 SEPTEMBER 1995 Curriculum Vitae

1 August, 1965 Born in Lawrence, KS, USA

1971-1975 Elementary School, Greenville, NC, USA

1975-1979 Secondary School, Winterville, NC, USA

1979-1983 High School, Albuquerque, NM, USA

1983-1987 B.S., Biochemistry, Tulane University, New Orleans, LA, USA

1988-1990 M.S., Life Sciences (Microbiology), New Mexico Highlands University, Las Vegas, NM, USA

1990-1991 Associate Scientist in the Environmental Biotechnology Unit of the Idaho National Engineering Laboratory, Idaho Falls, ID, USA

1991-1993 Scientist in the Environmental Biotechnology Unit of the Idaho National Engineering Laboratory, Idaho Falls, ID, USA

1993-1996 Doctoral studies at the Institute for Microbiolgy of the Swiss Federal Institute of Technology, Zi.irich (ETH-Z) and at the Department of Microbiology at the Swiss Federal Institute for Environmental Science and Technology (EAWAG), Dilbendorf, Switzerland