A Study of Mixed Continuous Cultures of Sulfate-Reducing and Methane-Producing Bacteria

TH. E. CAPPENBERG

Limnological Institute "Vijverhof," Nieuwersluis, The Netherlands

Abstract
Ecological relationships between sulfate-reducing and methane-producing bacteria in mud of Lake Vechten have been studied by continuous culture studies using the chemostat technique. The m a x i m u m specific growth rate (/-~max) and saturation constant (K~) were, respectively, 0.36 hr -1 and 0.047 mM for lactate-limited growth of Desulfovibrio desulfuricans and 0,011 hr -~ and 0.17 mM for acetate-limited growth of Methanobacterium sp. Calculated values for the true molar growth yields(Ya ) and maintenance coefficients (m) were 30.6 g bacterial mass/mole of lactate and 0.53 g substrate/g dry wt hr for D.desulfuricans and 37.8 g bacterial mass/mole of acetate and 0.54 g substrate/g dry wt hr for Methanobacterium. No growth of Methanobacterium was observed at a pS z- value (the hydrogen sulfide potential) of more than 11 and there was no effect on the growth a t p S 2- values above 13. In mixed continuous culture experiments the concentration of acetate decreased in the secondstage growth vessel, whereas that of methane increased stoichiometrically. If the substrate concentration in the reservoirs (St) was increased from 0.1 to 0.5 mg/ml, the population of Desulfovibrio increased and that of Methanobacterium was washed out of the culture vessel, since the concentration of hydrogen sulfide reached a pS 2- value of 10.5. From the mixed continuous culture experiments a commensalism between the two species can be described, i.e., the acetate-fermenting Methanobacteriura benefits from the acetate released by Desulfovibrio which is, in turn, not affected in the presence of the former.

Introduction
Previous investigations indicated an ecological interrelationship between sulfate-reducing and methane-producing bacteria in mud of Lake Vechten [ 4 - 6 ] . It was demonstrated that maximum numbers of the two groups occur at different layers in the bottom deposits. It was suggested that this was due to sensitivity of methane producers to hydrogen sulfide produced by sulfate-reducing bacteria. A substrate relationship between the two groups of bacteria was proposed, since acetate produced by sulfate reducers may be subsequently used by methane producers. Further data on this relationship can be obtained from continuous culture studies. The chemostat provides a model system which can be 6O
MICROBIAL ECOLOGY, Vot. 2, 6 0 - 7 2 (1975) 9 1975 by Springer-Verlag New York Inc.

Mixed Continuous Cultures of Bacteria

61

a p p l i e d w i d e l y in e c o l o g i c a l a n d p h y s i o l o g i c a l s t u d i e s ( f o r a r e c e n t r e v i e w , see Veldkamp and Jannasch [18]). If different growth-limiting substrates are used by different organisms, coexistence would occur, since these organisms then occupy different ecological niches. The influence of the acetate-producing bacterium, Desulfovibrio desulfuricans, on the fermentation of limiting amounts of acetate by a methane-producing bacterium, Methanobacterium sp., was investigated in mixed continuous cultures. A case of commensalism t h u s d i s c o v e r e d is d e s c r i b e d . T h i s s i t u a t i o n i s , however, complicated by the release of hydrogen sulfide by D.desulfuricans, which inhibited growth of the other organism.

Methods and Materials

Microorganisms and Media. A Desulfovibrio desulfuricans strain, isolated from mud of Lake Vechten, was used throughout this study. Enrichment and isolation of sulfatereducing bacteria were performed according to the methods of Postgate (1965). The organism was identified as Desulfovibrio desulfuricans according to the criteria proposed by Campbell and Postgate [12]. Contamination tests for aerobes and anaerobes were performed by the methods described by Postgate [11]. The liquid medium was a modification of that used previously [3], that is, by omitting Fe(NH4)2SO4 and additing Na-ethylenediaminetetraacetate (0.0005 % v/v) as chelating agent. A Methanobacterium sp. was isolated from the mud of Lake Vechten according to enrichment methods proposed by Barker [2]. The inoculum was transferred to a glassstoppered bottle, completely filled with the medium as described by the author in a recent paper [4] and incubated at 30~ for appropriate periods. Organisms from this enriched culture on acetate were isolated by serial dilution according to the roll-tube technique of Hungate [8] with an agar medium under an oxygen-free N2 atmosphere. A strain was obtained as a colony from the highest dilution (10 -r) showing growth and methane production. The organism produced methane from acetate and from hydrogen and carbon dioxide. The organism was a nonmotile, nonsporeforming, Gram-variable rod and was not indentical to the types of methane bacteria described by Wolfe [19] and Zeikus and Wolfe [20]. The strain, however, resembled that in the enrichment culture of Pretorius [13] using acetate as sole energy source. Morphologically, these bacteria were identical to the rod-shaped bacteria found by Mylroie and Hungate [9] in the acetate-enrichment cultures.
Lactate and acetate were added to the media in the concentrations as indicated in the text.

Apparatus. Single stage continuous culture experiments were performed using a Microferm Fermentor and Continuous Culture Console (New Brunswick Scientific Co., Inc., New Brunswick, U.S.A. ). A 2-liter growth vessel made of Pyrex glass and having three extra ports for inserting electrodes was used. Autoclavable glass and gold electrodes (Leeds & Northrop Company, U.S.A.) were used, respectively, to measure the pH and redox potential. The pH and redox potential were measured simultaneously employ'ing an autoclavable calomel reference electrode with a pH- and redox-transminer (Electrofact N.V., the Netherlands). The transmitter is equipped with two current alarm units, which can be preset at the desired values of pH and redox potential in the growth vessel, and with solenoid valves (Radiometer type MNV 2, Denmark) to deliver HCI (0.02 N), NaOH (0.02 N), perchloric acid (0.1% v/v), and Na~S'9 H20 (0. l % v/v) in the vessel. A two-pen potentiometric recorder was used for continuous registration.

62

Th. E. Cappenberg

The apparatus for mixed culture experiments consisted of three all-glass, water-jacked culture vessels fitted with multi-socked flat-flanged lids and Quick-fit glass components (as proposed by Baker [1]). Glass tubing was preferably used, and connections were made with heavy-wall butyl rubber tubing. The operating volume in the vessels could be controlled by adjusting the overflow. The fl~w rates between the reservoirs and the culture "vessels were controlled by peristaltic pumps (Vario Perpex 12000, LKB Producten AB, Sweden). Seven sockeds in the lid provided connections to the reservoirs and the buffering solutions, and fitted an overflow, an inoculation and sampling tube, electrodes, and gas inlet. Electrodes were autoclavable combined glass and reference electrodes (Radiometer, Denmark or Leeds & Northrop, U.S.A.). Redox- and pS z electrodes were designed and calibrated as described previously [4]. By simultaneous measurements of the pH and t h e p S 2 (the hydrogen sulfide potential) the H~Sg,~ concentration can be calculated from the equilibrium constant for the reaction H-,S~,,s -~- 2 H+aq + S~.a~. = 10 ~z~'~ The methane-producing bacteria were cultured at constant pH and redox potential, and the sulfate reducing bacteria at constant pH. In mixed cultures only the redox potential was kept constant. The pH could not be kept constant but was fbund not to change significantly. T h e p S z both in culture of sulfate-reducing bacteria and in mixed culture was measured with a pS z electrode. No " p o i s o n i n g " of the electrodes by culture constituents has been noted by runs lasting a few months. The electrodes were connected to a Titrator TTT 2 (Two-channel Titrator module + Two way pH-stat module, Radiometer, Denmark). Meter and electrode drifts were corrected, if necessary, after checking the values of the routine dally samples externally. Each fermentor was connected to a nitrogen tank, the pressure being maintained at 1.1 aim, and was stirred by a Teflon-covered magnet. Traces of oxygen in the nitrogen gas were removed by leading it through a column of BASF catalyzer R3- 11 at 150~ The gas was sterilized by a micro-flow filter (Microflow Ltd., England). All liquids were thoroughly treated with this gas before they were added to the system. Resazurin in the medium remained reduced throughout the experiments. The apparatus was maintained at a temperature of 30 ~ -+ 0.01"C with a thermostat (TE 9, P.M. Tamson N.V., the Netherlands). The glassware, tubes, and liquids were sterilized by autoclaving (20 rain, 118~ except those for the energy and carbon sources, which were sterilized by filtration through a Sartorius filter (0.22/z). If wall-growth occurred in the vessels with Methanobacterium after runs of over 100 days, as happened occasionally, the experiment was immediately suspended. The rate at which the buffering solutions was added to the cultures was about 4% of the medium flow rate; dilution rates are given as the actual medium flow rate. For theoretical considerations on continuous cultures and for symbols used, reference is made to Tempest [17]. Analyses. The concentrations of acetate and lactate in supernatant fluids of the cultures were assayed spectrophotometrically by specific enzymatic methods as described by Cappenberg [5]. Sulfide was determined colometrically using the N,N-dimethyl-p-phenylenediamine method and sulfate turbidimetrically with a 30% BaCIz " 2 H20 solution as proposed by van Gemerden [7]. The concentration of methane in the gas phase of the cultures was assayed using gas chromatography [3]. The bacterial dry weight was determined by filtering 10 ml samples through a prewashed, dried, and preweighed Sartorius membrane filter (0.22/.t). The sample was rir~sed twice with 5 ml membrane-filtered water, and dried at 115~ for I hr and weighed.

Mixed Continuous Cultures of Bacteria

63

Bacterial concentrations are expressed either as units of optical density (2 cm cuvet, filter Hg 365, Zeiss Elko lid or as numbers of cells per milliliter obtained by differential counts of Desulfovibrio and Methanobacterium by microscopic examination (Biirker-Tiirk counting chamber, depth 0.01 ram). The chemicals used were all of reagent grade quality and obtained from commercial sources.

Results
Batch Culures. The d o u b l i n g time (t a) and specific growth rate (/x) o f Desulfovibrio and M e t h a n o b a c t e r i u m were d e t e r m i n e d for batch cultures with various c o n c e n t r a t i o n s of lactate and acetate, r e s p e c t i v e l y (Table 1). The specific growth rate was calculated from the f o r n m l a , /~ = ( 1 / x ) ( d x / dt), and the doubling time from the formula, ta = ln2//x.
The results show that the doubling time o f Methanobacterium is always very long, about 70 hr. In both o r g a n i s m s the d o u b l i n g time increases at limiting substrate c o n c e n tr a ti o n s .

Continuous Cultures. In a continuous culture the dilution rate ( D ) was ne v er al l o wed to e x c e e d the critical dilution rate (De), because this would result in gradual wash out o f all the m i c r o o r g a n i s m s . The rate of e x c h a n g e

Table 1
Specific Growth Rates (It) and Doubling Times (td) o f D. desulfuricans and Methanobacterium sp. at Various Substrate Concentrations for Batch Cultures a

Substrate added (mg/ml)

D. desul furicans ~
6 3 1 0.1 0.05 0.5 0. I 0.025

Substrate used (mg/ml)

6 3 1 0.1 0.05 0.43 0.1 0.025

la

td

(hr- 1 )
0.26 0.14 0.04 0.034 0.015 0.011 0.011 0.008

(hr)
2.7 5.0 17.5 20.3 46.2 65.0 65.0 86.5

Methanobacterium sp. c

aCultures were grown at 30~ The optical density of the cultures was continuously recorded in a recording b io p h o t o m e te r for 2 and 14 days, respectively. bLactate added as substrate CAcetate added as substrate.

64

Th. E. Cappenberg

in the growth vessel is the flow rate in volume units per hour (f) against the working volume of the vessel ( V ) : f / V = D. During steady state conditions the concentration of microorganisms is constant, in other words D = /z. Continuous cultures were performed under conditions corresponding to those in the batch cultures with substrate concentrations which were shown to be growth-limiting (Table 1). The practicable values found were a dilution rate of 0.013 hr -1 with a substrate concentration in the reservoir (S~) of 0.1 mg/ml lactate for Desulfovibrio, and a dilution rate of 0.01 hr-: and a substrate concentration of 0.1 mg/ml acetate in experiments for Methanobacterium . In order to determine optimal values of pH and redox potential for growth, the bacteria were cultured under varying pH or redox potential values (Table 2). The results indicate pH values of 7.4 and 7.1, and of redox potentials of - 1 4 0 mV and - 3 8 0 mV, respectively, for optimal growth of Desulfovibrio and Methanobacterium. Consequently, in all continuous culture experiments these values of pH and redox potential were maintained. Maximum specific growth rates (/-/,max) and saturation constants (K~) for lactate- and acetate-limited growth, respectively, of Desulfovibrio and Methanobacterium were estimated graphically as double reciprocal plots of specific growth rate against substrate concentration. The results obtained are given in Fig. I. The extrapolated lines indicate a /,/,max of 0.36 hr -1 and a K.~ for lactate-limited growth of 4.23 mg/liter for Desulfovibrio, and a /Zma of 0.011 hr -1 and a K~ for acetate-limited growth of 10.2 rag/liter for Methanobacteriurn . Besides the fall in growth rate with decreasing substrate concentration, the yield of organisms also decreased. To explain such a relation between yield and growth rate, Pirt [10] proposed the relationship m /x where Y is the observed growth yield, Ya is the " t r u e " growth yield, i.e., the yield that would be obtained in the absence of a maintenance requirement, and m is the maintenance coefficient (g substrate/g dry wt hr).
I I

Y(;

Plotting observed values of I/Y against 1//x (for specific growth rates in the range of 0.01 to 0.005 hr -1) for Methanobacterium sp., a straight line and values of 0.63 g/g and 0.54 g/g hr for Ya and m, respectively, were obtained. Calculated values for Ya and m for D.desulfuricans were 0.34 g/g and 0.53 g/g hr, respectively (Fig. 2). The values obtained for the mainte-

Table 2

Effect o f Varying the pH or the Redox Potential (Eh in m V) Values on the Dry Weight (mg/liter) Values by Continuous Cultivation of D. d e s u l f u r i c a n s ( D = 0. O13 hr-1, Sr = 0.I mg/ml Lactate) and M e t h a n o b a c t e r i u m sp. ( D = 0.01 hr -1, S r = 0 . 1 mg/ml Acetate) D. desulfuricans
(dry wt mg/liter) 26 63 76 84 65 41 -380 -400 -

D. desulfu~cans
(dry wt rag/liter) 32 0 -50
1 0 0

Methanobacterium sp.
Eh (mV)

Methanobacterium sp.
(dry wt mg/liter)
e~

pH

(dry wt mg/liter)

6.8 68 78 56
1 4 0

7.0

38

7.1

7.2 28
- 1 8 0

50

=__ 35 57 68 76 82 78

7.4 -250 -300 -350 -200

82

7.5

70

7.8

22

66

Th. E. Cappenberg

9!

ta

200

5
100

/
' I

' I/11'
-0.2 0

I i I
0.2 0,4 1 mg/I Lactate 1 Km

--0.4
1

-0.1

I/'

0.1

0,2

1 mg/I Acetate

Km

Fig. 1. Double reciprocal plots of specific growth rate (/,z, hr -1) of Desu!fovibrio desulfuricans (A) and Methanobacterium sp. (B) at different substrate concentrations (g, rag/liter). Data obtained in steady-state conditions, which were assumed to have been reached when there had been no significant change in cell number for a minimum of four volume changes.

I 5O

100

150

50

100

150

l/Specific Growth Rate (HR - 1 )

Fig, 2. Double reciprocal plots of yield (g bacterial mass/g substrate)of Methanobacterium (A) and Desulfovibrio (B) at different specific growth rates (~, hr-'). Intercepts at the ordinates are the reciprocal values of Yc (g/g), slopes of the lines are the values of m (g substrate/g dry wt hr).

Mixed Continuous Cultures of Bacteria Table 3

67

Effect of Varying the Hydrogen Sulfide Potential Values ( p S 2 - ) on the Dry Weight (rag~liter) Values by Continuous Cultivation of M e t h a n o b a c t e r i u m sp. (D = 0.01 hr-1, S r = 0.1 mg/mlAcetate) Methanobacterium sp.
pS 2 15 (dry wt mg]liter) 95

Methanobacterium sp. pSZ12.5 (dry wt mg/liter) 59 28 17 14

14.5 14
13.5 13

92 96
95 91

12 11.5
"11

(--X~x~X--'L--x~X~x~X
2 _a
0 1 _z~--z~z~

X~X~X----X~x~>

~,/

~,.----. . ~ , ~ z ~ ' CH4

z~ 14 if
3

~:

~o

Acetate

0! t=0.01 HR - 1

D =0.01 HR - 1

10

~0

~E

~o ~
0 o.

5
_...___...i

10 ~ ,
I I

A I
0

2 3 4 Time (HR X 100)

Fig. 3. Population levels o f MethanobacteHum (M) and Desulfovibrio (D), and concentrations of acetate, m e t h a n e , and pS'-'- values in first-state (A) and secondstage (B) continuous cultures. The effect of dilution on the start of the second-stage continuous cultures was elirninated by multiplying the numbers of bacteria by a factor of 2. In the first stage the working volume of the continuous cultures was 300 ml; in the second stage, 600 ml.

68

Th. E. Cappenberg

nance coefficient were in the same order of magnitude as those quoted by Pirt [10] for anaerobic growth of various bacterial species. Previous investigations [4, 5] indicated that existence at different depth in the mud of the maximum numbers of sulfate-reducing and methane-producing bacteria may be due to sensitivity of the latter group of bacteria to hydrogen sulfide produced by the former group. To confirm this the sensitivity of Methanobacterium sp. to hydrogen sulfide was tested in a continuous culture experiment by altering the sulfide concentration in the growth vessel. Dry weight (rag/liter) and pS 2- values (the hydrogen sulfide potential measured in the growth vessel at pH 7.1) are given in Table 3. For calculations of the actual sulfide ion strength in the medium, see Cappenberg [4]. No growth was observed at a p S 2- value beyond 11 and no effect on the growth at pS z- values greater than 13 was detected.

Mixed Continuous Cultures. In order to examine the behavior of the two bacterial species together a number of steady-state mixed-population levels were established. The two first-stage fermentors were operated over a range of dilution rates (D < Dc = 0.36 hr -~ forDesulfovibrio a n d D < D,. = 0.011 hr -1 for Methanobacterium) to provide a source of bacteria for the second-stage fermentor. Soon after the start of the mixed continuous culture

pS=
X -X

Acetate

o
E

\
~z,

o/

14

CH4

D =0.01 HR - 1 Sr =0.1 MG/ML 10

D =0.01 HR - 1 S r =0.5 MG/ML

tO

.g.~

g~ 5
E 0 9~ 0 E ro

10 ,

'c~ r~

0 0

5 6 7 Time (HR X 100)

10

Fig.4. Effect of increasing the substrate concentrations (Sr) o n the population levels of Methanobacterium (M) and Desulfovibrio (D), and concentrations of acetale, methane, and pS 2- values in mixed continuous cultures.

Mixed Continuous Cultures of Bacteria

69

an adjustment in the population levels occurred until a new steady-state was reached, It was assumed that steady-states was reached when there had been four volume changes during which time bacterial and substrate concentrations did not change significantly. The results of concentrations of bacteria and substrates are shown in Fig. 3. The population of Methanobacterium strongly increased, whereas that of Desulfovibrio showed a relatively small increase. Moreover, the concentration of acetate decreased, whereas that of methane increased. These results confirm that acetate produced by Desulfovibrio is indeed used by Methanobacterium. If the substrate concentration in the reservoir was increased to 0.5 mg/ml, an inhibitoy effect on the growth of Methanobacterium was observed (Fig. 4). Further, when the concentration of hydrogen sulfide produced by D.desulfuricans reached a pS 2- value of 10.5, the Methanobacterium organisms were washed out of the culture vessel. Consequently, the concentration of acetate increased and the production of methane came to a standstill,

Discussion
The main object of this investigation was to supplement the existing information on the ecological relationship between sulfate-reducing and methane-producing bacteria in the mud of Lake Vechten [ 4 - 6 ] . In this study continuous cultures of both bacterial species were used. An acetatefermenting methane bacterium was chosen, since previous studies have shown that acetate is an important precursor of methanogenesis in mud and that about 75% of the mixed population of methanogenic bacteria in Lake Vechten are of the acetate-fermenting type. Field observations showed that maximum numbers of methanogenic bacteria were recorded at depths of 3 to 6 cm in the mud, where the redox potential values of - 2 5 0 to - 3 0 0 mV and the pS 2- values were high (13.4 to 14.0). On the other hand, the maximum numbers of sulfate-reducing bacteria were found at depths of 0 to 2 cm in the mud, concomitantly with redox potential values of - 1 0 0 to - 1 5 0 mV a n d p S z- values of 11.4 to 12.2 [4, 5]. It was suggested that this difference in localization may be due to sensitivity of methanogenic bacteria to hydrogen sulfide. In a continuous culture experiment in which the sensitivity of

Methanobacterium sp. to hydrogen sulfide was tested, more evidence was

found for this hypothesis (Table 3). No growth was observed at a pS zvalue of less than 11 (hydrogen sulfide concentration of about O. 1 raM) and no effect was observed at pS 'z- values greater than 13 (hydrogen sulfide conc. of about 0.001 raM). Furthermore, when the substrate concentration was increased, the sulfide concentration increased and the methane bacteria were washed out from the mixed continuous culture (Fig. 4). From these

70

Th. E. Cappenberg

results we may conclude that the absence of methanogenic bacteria at depths in the mud where sulfate-reducing bacteria are abundant is due to the production of hydrogen sulfide by the latter to which the former are sensitive. Furthermore, since metbanogenic bacteria do not grow at a redox potential higher than - 2 0 0 mV (Table 2) this is an additional factor responsible for the different localities. As very high values of pS ~ were measured in deeper layers in the mud [4], it is likely that the hydrogen suffide is used either for the reduction of ferric to ferrous ions or is precipitated as FeS. Acetate, produced by the sulfate reducers, may diffuse and may be subsequently used as a substrate by methane producers. From the results of the mixed continuous culture experiments (Fig. 3) of the two bacterial species the existence of a commensalism can be noted. On the start of the mixed continuous culture an adjustment in the population levels occurred until a new steady-state was reached in the second stage. Particularly, a large increase in the level of the Methanobacterium population was observed. The concentration of acetate decreased, whereas that of methane increased stoichiometrically. The interaction of D.desulfuricans and Methanobacterium sp. is a true commensalism in that one organism, the acetatefermenting Methanobacterium, benefits from the products (acetate) released by Desulfovibrio, which, in turn, is effected in no way by the presence of the former. Such a substrate relationship between the two groups in the sediments of Lake Vechten has already been suggested [6], since labeled methane was detected when mud containing both methanogenic and sulfate-reducing bacteria was incubated with uniformly labeled lactate. The ecological association of the two species is, however, complicated by the release by D. desulfuricans of hydrogen sulfide which inhibited the growth of the Methanobacterium (Fig. 4). If the substrate concentration in the reservoir (St) was increased to 0.5 mg/ml, the concentration of hydrogen sulfide reached a level of a p S " - value of 10.5, consequently growth of the methanogenic bacteria is inhibited, It has already been shown by selective inhibition and by enhancement of sulfate-reducing and methanogenic processes in mud [4, 5] that SO42- is the limiting factor for the abundance of sulfate-reducing bacteria. Addition of sulfate to mud samples caused an increase in the concentration of hydrogen sulfide and inhibition of methanogenesis. Summarizing the ecological relationship between the two bacterial types in the lake mud, we find that hydrogen sulfide and acetate produced by sulfate reducers which are in the upper layers sets up two diffusion gradients--those of H2S and acetate. Hydrogen sulfide would be partly or completely oxidized depending on the concentration of ferric ions in the mud. On the other hand, the shape of the acetate gradient would be

Mixed Continuous Cultures of Bacteria

71

influenced by the depth at which methane producers are present and would also determine the concentration of acetate-fermenting methanogenic bacteria.
Last, quantifying the growth of the bacteria in the first-stage continuous cultures the yield corrected for maintenance of Methanobacterium sp. growing on acetate was high (Ya = 37.8 g/mole). Molar growth yields of D.desulfuricans growing on lactate recorded in this study are substantially higher (Y~; = 30.6 g/mole) than those reported for batch cultures by Senez [14]. Stouthamer and Bettenhausen [16] and Stouthamer [15] calculated a production of approximately 30 g bacterial mass/mole of ATP. This seems to indicate a production of about I mole of ATP per mole of acetate fermented by Methanobacterium and a somewhat higher ATP yield per mole of lactate fermented by Desulfovibrio. Acknowledgments
The skillful technical assistance of Mr. H. Korthals is gratefully acknowledged. The author is indebted to Professor A.H. Stouthamer for his critical review of the manuscript.

References
1. 2. 3. 4. Baker, K. 1968. Low cost continuous culture apparatus. Lab. P r a t t . 17: 817-824. Barker, H.A. 1936. Studies upon the methane-producing bacteria. Arch. Mikrobiol. 7: 420-438. Cappenberg, Th. E. 1972. Ecological observations on heterotrophic, methane oxidizing and sulfate reducing bacteria in a pond. Hydrobiologia 40:471-485. Cappenberg, Th. E. 1974. Interrelations between sulfate-reducing and methaneproducing bacteria in bottom deposits of a fresh-water lake. I. Field observations. Antonie van Leeuwenhoek 40: 285-295. Cappenberg, Th. E. 1974. lnterrehttions between sulfate-reducing and inethaneproducing bacteria in bottom deposits of a fresh-water lake. II. Inhibition experiments. Antonie van Leeuwenhoek 40: 297-306. Cappenberg, Th. E. and Prins, R.A. 1974. Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. IIl. Experiments with HC-labeled substrates. Antonie van Leeuwenhoek 40: 457-469. Gemerden van, H. 1967. On the bacterial sulphur cycle of inland waters. Thesis, Leiden. Hungate, R.E. 1969. A roll tube inethod for cultivation of strict anaerobes. In: Methods in Microbiology. J.R. Noris and D.W. Ribbons, editors. Vol. 3B, pp. 117-132. Acadelnic Press, New York. Mylroie, R.L. and Hungate, R.E. 1954. Experiments on the methane bacteria in sludge. Can. J. M i c r o b i o l . l: 55-64. Pirt, S.J. 1965. The maintenance energy of bacteria in glowing cultures. Proc. Royal Soc. B 163: 224-231.

5.

6.

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9. 10.

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11. 12. 13. 14. 15. 16.

Th. E. Cappenberg Postgate, J. 1965. Enrichment and isolation of sulfate-reducing bacteria. Zbl. Bakt. 1. Abtl., Supplementheft 1: 190-197. Postgate, J. and Campbell, L.L. 1966. Classification of Desulfovibrio species, the nonsporulating sulphate-reducing bacteria. Bact. Rev. 30: 732-738. Pretorius, W.A. 1972. The effect of formate on the growth of acetate utilizing methanogenic bacteria. Water Res. 6: 1213-1217. Senez, J.C. 1962. Some considerations on the energetics of bacterial growth. Bact. Rev. 26: 95-107. Stouthamer, A.H. 1973. A theoretical study on the amount of ATP required for synthesis of microbial cell material. Antonie van Leeuwenhoek 39: 545-565. Stouthamer, A.H. and Bettenhaussen, C. 1973. Utilization of energy for growth and maintenance in continuous and batch cultures of microorganisms. A reevaluation of the method for the determination of ATP production by measuring molar growth yields. Biochim. Biophys. Acta 301: 53-70. Tempest, D.W. 1970. The cultivation of micro-organisms. 1. Theory of the chemostat. In: Methods in Microbiology. J.R. Noris and D.W. Ribbons, editors. Vol. 2, pp. 259-276. Academic Press, New York. Veldkamp, H. and Jannasch, H.W. 1972. Mixed culture studies with the chemostat. J. Appl. Chem. Biotechnol. 22" 105- 123. Wolfe, R.S. 1971. Microbial formation of methane. Advan. Microbial Physiol. 6: 107- 146. Zeikus, J,G. and Wolfe, R.S. 1972. Methanobacterium thermoautotrophicus sp. n., an anaerobic, autotrophic, extreme thermophile. J. Bacteriol. 109: 707-713.

17.

18. 19. 20.