Enumeration studies on methanogenic bacteria

Water Research Pergamon Press 1968. Vol. 2, pp. 545-554. Printed in Great Britain

ENUMERATION STUDIES ON METHANOGENIC BACTERIA M. L. SIEBERT,D. F. TOERIEN and W. H. J. HATTINGH National Institute for Water Research, South African Council for Scientific and Industrial Research P.O. Box 395, Pretoria, Republic of South Africa (Received 22 February 1968) Abstraet~Various media for the enumeration of methanogenic bacteria were compared statistically. It is suggested that a reduced medium containing mineral salts, vitamins, small amounts of fatty acids, carbon dioxide (20 per cent) and hydrogen (80 per cent) should be used for the enumeration of methanogenic bacteria from natural habitats. The addition of rumen fluid and digester fluids to media seemed to have stimulated the growth of more colonies, some of which may have been non-methanogenic in origin. Repressive effects of formate and acetate on colony counts in certain circumstances were observed. INTRODUCTION IN QUANTITATIVEecological studies of microbial ecosystems, methods for the enumeration of the microbial populations in the ecosystems are required. Since methane is one of the major end-products in anaerobic disgestion, quantitative ecological studies on anaerobic digestion require amongst others, methods for the enumeration of methanogenie bacteria. Various methods have been proposed for the enumeration of methanogenic bacteria of various habitats. HEUKELEKIANand HEINEMAN(1939) proposed a method based on most probable numbers. The same approach was followed by SIEBERTand HATTINGH (1967), but it was suggested by THIEL et al. (1968) that this method was probably unsatisfactory. MYLROIE and HUNGATE (1954) developed a method in which methanogenic bacteria were enumerated usingcarbon dioxide and hydrogen as substrates and where palladium was incorporated into the medium as reduction catalyst. They obtained culture counts ranging from 105 to l0 s per ml. SMITH and HUNGATE (1958) enumerated rumen methanogenic bacteria on carbon dioxide (20 per cent) and hydrogen (80 per cent) as substrates in a medium supplemented with 30 per cent clarified rumen fluid. The redox potential of the medium was poised by addition of sodium pyruvate and an Escherichia coli culture. They obtained culture counts of 2 x 10 s methanogenic bacteria per ml of rumen contents. These results were confirmed by BRYANT (1965). SMITH (1965, 1966) enumerated methanogenic bacteria of raw sewage sludge digestion using a procedure similar to that of SMITHand HUNGATE (1958). He obtained counts exceeding 1 x 104 methanogenic bacteria per ml in various media. The purpose of this investigation was to compare various media for the enumeration of methanogenie bacteria, on a statistical basis, in order to find a suitable medium for enumeration during ecological studies of anaerobic digestion. M A T E R I A L S AND METHODS Anaerobic digester A digester receiving a synthetic substrate (HATTINGH et al., 1967) was used as source of inocula during these studies. 545

Mineral solution I KH2PO4 Mineral solution II KH2PO4 NaC1 NH4C1 MgCI2.7H20 CaC12.2H20 COC12.6H20 Na2MoO4.2H20 Ferric citrate Fatty acid mixture and

22 78 37.4 37'4 1.0 1-0 5'0 1'0 3.1 200 Nil 10 Nil Nil 17 143 20 555

99 1.0 37.4 37.4 1.0 1'0 5-0 1'0 3.1 200 Nil 10 Nil Nil 17 143 20 555

B

22 78 37'4 37'4 1"0 1.0 5"0 1"0 3-1 Nil Nil 10 Nil Nil 17 143 20 755

C 22 78 37-4 37"4 1'0 1.0 5"0 1.0 3.1 Nil 200 10 Nil Nil 17 143 20 555

D 99 1'0 37"4 37-4 1.0 1'0 5'0 1.0 3.1 Nil 200 10 Nil Nil 17 143 20 555

E 22 78 37"4 37.4 1-0 1"0 5"0 1.0 3.1 200 Nil Nil Nil Nil 17 143 20 555

F 22 78 37.4 37"4 1"0 1"0 5-0 1'0 3.1 Nil Nil Nil Nil Nil 17 143 20 755

G 22 78 37.4 37.4 1"0 1"0 5"0 1"0 3.1 Nil 200 Nil Nil Nil 17 143 20 555

H 22 78 37'4 37"4 1-0 1"0 5"0 1"0 3.1 200 Nil Nil Nil 10 17 143 20 555

I 99 1"0 37-4 37.4 1-0 1"0 5-0 1.0 3.1 200 Nil Nil Nil 10 17 143 20 555

22 78 37.4 37.4 1"0 1'0 5"0 1.0 3.1 Nil Nil Nil Nil 10 17 143 20 755

22 78 37"4 37"4 1'0 1.0 5"0 1"0 3"1 Nil 200 Nil Nil 10 17 143 20 555

99 1'0 37.4 37.4 1-0 1"0 5"0 1.0 3.1 Nil 200 Nil Nil 10 17 143 20 555

Medium designation J K L M 22 78 37"4 37"4 1"0 I-0 5-0 1.0 3'1 200 Nil Nil 10 Nil 17 143 20 555

N 99 1-0 37-4 37.4 1'0 1.0 5"0 1"0 3.1 200 Nil Nil 10 Nil 17 143 20 555

O 22 78 37.4 37-4 1'0 1"0 5"0 1.0 3'1 Nil Nil Nil 10 Nil 17 143 20 755

P 22 78 37.4 37.4 1.0 1-0 5"0 1-0 3.1 Nil 200 Nil I0 Nil 17 143 20 555

Q

S

T

99 22 99 1'0 78 1.0 37-4 37.4 37'4 37-4 37.4 37,4 1-0 1"0 1'0 1-0 1'0 1-0 5'0 5'0 5'0 1.0 1.0 I'0 3'1 3"1 3-1 Nil Nil Nil 200 Nil Nil Nil 5-0 5"0 10 Nil Nil Nil 5.0 5'0 17 17 17 143 143 143 20 20 20 555 755 755

R

H WERE USED

22 78 37.4 37"4 1-0 1.0 5"0 1'0 3.1 Nil Nil 5"0 5"0 Nil 17 143 20 755

U

99 1'0 37'4 37.4 1-0 1"0 5'0 1'0 3.1 Nil Nil 5-0 5.0 Nil 17 143 20 755

V

• .~

z =

.~

.~

per cent (w/v) NaHCO3 solution 0.3 29.9 g NaHCO3 dissolved in 500 ml distilled water 7. per cent (w/v) Phenosafranine solution 0-3 0"2 g phenosafranine dissolved in 100 ml distilled water 0.6 Resazurine solution 0.6 0-2 g resazurine dissolved in 20 ml 0'1 N NaOH and diluted to 500 ml with 0.06 distilled water 0.06 Digester fluid and reducing solution were prepared as described by TOERIEN 0.02 and SIEBERT (1967) 0.015 Rumen fluid was prepared according to the procedure for the clarified rumen 0.015 fluid No. 2 of BRYANT and ROBINSON(1961) Vitamin solutions A and B were prepared according to TOERIEN and SIEBERT(1967)

CO2 per cent H2 per cent Mineral solution I (ml) Mineral solution 1I (ml) Vitamin solution A (ml) Vitamin solution B (ml) Resazurine solution (ml) Phenosafraninesolution(ml) Fatty acid mixture(ml) Digester fluid (ml) Rumen fluid (ml) Sodium formate (g) Sodium acetate (g) Glucose (g) Reducing solution (ml) NaHCO3 solution (ml) Bacto-Agar (g) Dist. H20 (ml)

A

IN EXPERIMENT NO. 1 A N D MEDIA A TO V WERE USED IN EXPERIMENT NO. 2

TABLE 1. COMPOSITION OF MEDIA USED TO ENUMERATE METHANOGENIC BACTERIA~ MEDIA A TO

Enumeration Studies on Methanogenic Bacteria

547

Outlay of experiments Two experiments were carried out. Both were completely randomized designs. In the first experiment eight treatments (media A - H , TABLE l) with five replications of each treatment, and in the second experiment twenty-two treatments (media A-V, TABLE 1) with five replicates of each treatment were compared. The results (colony counts) of the first experiment on the 8th day of incubation and the results of the second experiment on the 9th day of incubation were statistically analysed by means of the nonparametric method of KRUSKAL and WALLIS (1952). The formula used, was:

"= where N = nl = i = N = Rt = k =

[

12

1

R;2

Z .-7--

3(N+l)

total numbers of observations, numbers of observations in the ith treatment, 1,...,k, Z, ni, sum of the ranks in the ith treatment, number of treatments,

The calculated H values in both experiments were compared with Z2o.os ( k - 1) and when the null hypothesis was rejected, it was concluded that the treatments differed significantly. In both experiments, pair-wise comparisons of the treatments were made by means of the multiple range test developed by REINACH (1966): The least significant difference for pair-wise comparisons of the rank totals of the treatments, is given by:

,sa =/I-. x!L

12

÷')-I _j. q,.,. oo

where n = number of observations in each treatment (all equal), N = total number of observations, q,,, k, O0 = value from studentized range tables at ~ per cent level, k treatments and oo degrees of freedom for error variance. The treatments were arranged in increasing order of magnitude of the Ri and the pairs which differed significantly calculated. To evaluate the relative rates by which colonies developed for each treatment, the areas covered by graphs of the results of each treatment over the periods in both experiments, were calculated by means of the rule of Simpson (GRANVILLEet al., ! 957). The formulae used were: Uneven number of observation periods Area = at (Uo +4U1 + 2U 2 + 4 U 3 + ... +2U2n- 2 + 4U2n_ 1 +

U2n)

Even number of observation periods Area = at ( U o + U1)+ at(U~ +4U2 +2U3 + . . . + 4 U , _ t + U2.) where

548

M. L. SIEBERT,D. F. TOERIENand W. H. J. HATTINGtt

Uo, U1, U2... un l

denote the corresponding observations at the 1st, 2 n d , . . . nth periods = length between any two successive periods. Since the exact length is of no importance but only the relative lengths, I was substituted by 1 in the above formulae, because the interval periods were equal.

Growth media Composition of media. In the first experiment eight different growth media (Media A to H, TABLE1) were compared. These included media which should have been more or less selective for the growth ofmethanogenic bacteria and media which should have only supported growth of non-methanogenic bacteria. In the second experiment the number of media were increased to twenty-two (Media A-V, TABLE 1) and included media which contained carbon sources such as acetic acid, glucose, etc. Preparation of media. The procedure for the preparation of media, the preparation of the anaerobic dilution solution and digester fluid was described earlier (ToERIENand SIEBERT,1967). The rumen fluid was prepared similar to the clarified rumen fluid No. 2 described by BRYANTand ROBINSON(1961). Enumeration ofmethanogenic bacteria. The enumeration procedure of TOERIENand SIEBERT(1967) was used. Enumeration bottles were incubated at 38°C and the developed colonies counted on the 2nd, 4th, 6th and 8th days during the first experiment and on the 3rd, 6th and 9th days during the second experiment. RESULTS The colony counts and areas covered by graphs of the results of both experiments are presented in TABLES2 and 3 respectively. The results of the Kruskal and Wallis analyses, the pair-wise comparison of treatments, the lsdR values and the sum of ranks in increasing order of magnitude for both experiments, are presented in TAULE4. The pairs of treatments which differed significantly (P = 0.05) are presented in TABLE5. DISCUSSION Anaerobic digestion is brought about by complex microbial populations in which aerobic and facultative anaerobic bacteria, obligate anaerobic non-methanogenic bacteria, obligate anaerobic methanogenic bacteria as well as some other groups may occur (TOERmN and HATTINOH, 1968). The complete microbial analysis of anaerobic digestion therefore urgently needs, amongst others, methods for the enumeration of methanogenic bacteria. Since the knowledge of these bacteria is still very limited, the enumeration of these bacteria presents a complex problem in which many different facets must always be kept in mind. The methanogenic bacteria are obligate anaerobes, and any method for their enumeration should ensure rigid anaerobic conditions during the enumeration procedures. This condition was fulfilled in the investigations of SMITHand HUb/GATE(1958) BRYANT(1965), SMITH(1965, 1966) and during this investigation. All methanogenic bacteria eem to be able to use carbon dioxide and hydrogen as substrates (cf. BRYANT et al., 1967), and the logical choice for substrates during

549

Enumeration Studies on Methanogenic Bacteria

"IAnLE 2. COLONY COUNTS ( × 10 6) OF THE FIRST EXPERIMENT AND THE AREAS COVERED BY GRAPHS OF THESE RESULTS

Days of incubation Medium 2 A B C D E F G H

4

0"3 0"1 0"1 15"4 15"0 8"2 0"1 16"1

6

8

79"6

11"1 20"7 11 "8 6240"0 9220"0 48"4 87"6 23000"0

Area ( x 10~)

131"6 156"0 82"8 13480"0 25840"0 161 "0 149"2 30680"0

132"4 25"6 10440"0 17600"0 78"5 138"4 27120"0

157"8 242"4 67"8 22927" 1 38246"7 201 "5 292"8 61741"4

TABLE 3. COLONY COUNTS ( X 106) OF THE SECONDEXPERIMENTAND THE AREAS COVEREDBY GRAPHSOF THESE RESULTS Days of incubation

Areas ( x 10 6)

Medium

A B C D E F G H I J K L M N O P Q R S T U V

3

6

9

183.2 0-2 26"4 158.0 152-4 190"0 221 "6 240,0 233.2 218.8 1"3 344.4 171 "2 114-4 0-1 0.1 0"2 89.5 9"9 11 '3 0.2 0"1

832-0 9400.0 12720"0 21080.0 22040"0 19120"0 21800.0 30280"0 23360.0 2812'0 1664.0 26680-0 1425"0 36000-0 15160.0 7600.0 44800.0 21880.0 16600.0 9160.0 174-0 21.9

15480.0 13020.0 16640.0 32200.0 25820.0 25530.0 30310.0 30640.0 29620.0 13000.0 25800.0 29730.0 11110"0 83040.0 34300.0 9130-0 48600.0 33440-0 23000.0 23910.0 14930.0 7820-0

6422.3 16874"2 22528.7 38971 "7 38120'3 34161"7 39354.7 50786.7 41214.3 8210.3 10833.1 45770.2 5756"0 122815"3 31650.7 13177-4 75934'4 40394.6 29808-3 20192.7 5209.6 2636.6

359.1

Experiment 99.2, No. 2

V 25

A 55

P M B J U A C S 35.5 72.5 109.5111.5143.5155 170.5 254

C 15

B 88

F 88

D 139

E 168

H 181

H D R O N Q 397 424.5449.5450.5487.5540-0

Ri~

T F E K L I G 266.5289.5292 299.5269-5369'5 394

G 79

Treatments arranged in increasing order of magnitude of

* H-value determined according to KRUSKALand WALLIS (1952). ~"IsdR determined according to REINACH (1966). ~: These values are significant at P = 0.005 level. Treatments connected by continuous line do not differ significantly (P = 0-05).

111'9

lsdR~

Experiment 34.47+ No. 1

Hvalue*

TABLE 4. STATISTICAL ANALYSIS OF BOTH EXPERIMENTS

Z

r~

O r~

-]

~7

r-

O

Enumeration Studies on Methanogenic Bacteria

551

e n u m e r a t i o n s seems to be a gas a t m o s p h e r e o f 70-80 p e r cent h y d r o g e n a n d 20-30 p e r cent c a r b o n d i o x i d e (MYLROIE a n d HUNGATE, 1954; SMITa a n d HUNGATE, 1958; BRYANT, 1965; SMlxrI, 1965, 1966). I t m u s t however be k e p t in m i n d t h a t n o t o n l y m e t h a n o g e n i c b a c t e r i a b u t also b a c t e r i a o f the t y p e represented b y Clostridium aceticum (WIERINGA, 1940), will d e v e l o p if present in the ecosystem. The presence o f TABLE 5. PAIRS OF TREATMENTSWHICH DIFFEREDSIGNIFICANTLY (First named partner is significantly higher) EXPERIMENT NO. I Pair H,A H,C E,A E,C

Composition of pairs Atm+RF Atm+RF CO2+RF+Form CO2+RF+Form

> > > >

Pair

Atm+DF+Form Atm+Form Atm+DF+Form Atm+Form

Composition of pairs

D,C A t m + R F + F o r m > A t m + F o r m

EXPERIMENT NO. 2

Pair Q,C Q,A Q,U Q,J Q,B Q,M Q,P Q,V N,J N,B N,M N,P N,V O,M O,P O,V

Composition of pairs Atm+RF+acet Atm+RF+acet Atm+RF+acet Atm+RF+acet Atm+RF+acet Atm+RF+acet Atm+RF+acet Atm+RF+acet Atm+DF+acet Atm+DF+acet Atm+DF+acet Atm+DF+acet Atm+DF+acet CO2+DF+acet CO2+DF+acet CO2+DF+acet

> > > > > > > > > > > > > > > >

Atm+Form Atm+DF+Form Atm+Form+acet COz+DF+gluc CO2+DF+Form CO2+RF+gluc Atm+acet COz+acet+Form CO2+DF+gluc CO2+DF+Form CO2+RF+gluc Atm+acet CO2+acet+Form CO2+RF+gluc Atm+acet CO2+acet+Form

Pair R,M R,P R,V D,P D,V H,P H,V G,V

Composition of pairs CO2+RF+acet CO2+RF+acet CO2+RF+acet Atm+RF+Form Atm+RF+Form Atm+RF Atm+RF Atm

> > > > > > > >

CO2+RF+gluc Atm+acet CO2+acet+Form Atm+acet CO2+acet+Form Atm+acet CO2+acet+Form CO2 + acet + Form

All media were of the same composition, except for additions of rumen fluid (RF), digester fluid (DF), formate (Form), acetate (acet), glucose (glue) and the gas atmospheres in bottles. Carbon dioxide (99 per cent) and hydrogen (1 per cent) are abbreviated (CO2) and the atmosphere 20 per cent CO2 and 80 per cent H2 abbreviated (atm). c a r b o n d i o x i d e a n d h y d r o g e n in a n a e r o b i c digesters should p r o v e selective n o t o n l y for m e t h a n o g e n i c b a c t e r i a b u t also for these o t h e r c a r b o n d i o x i d e - h y d r o g e n utilizing bacteria, suggesting t h a t s o m e o f the colonies in c a r b o n d i o x i d e - h y d r o g e n c o n t a i n i n g e n u m e r a t i o n bottles m i g h t be o f a n o n - m e t h a n o g e n i c origin. SMITH a n d HUNGATE (1958), BRYANT (1965) a n d SMITH (1965, 1966) all a p p a r e n t l y included r u m e n fluid in their e n u m e r a t i o n m e d i a for m e t h a n o g e n i c bacteria. T h e r u m e n fluid served as source o f a d d i t i o n a l g r o w t h factors for the m e t h a n o g e n i c

552

M.L. SIEBERT,D. F. TOERIENand W. H. J. HATHNGH

bacteria. Rumen fluid may however support the growth of non-methanogenic bacteria, causing a higher colony count, and this would rule out the use of rumen fluid additions. If the addition of rumen fluid to a medium with carbon dioxide and hydrogen does not cause a significantly higher bacterial count than the medium without rumen fluid in a statistically planned experiment, there is no need for the addition of rumen fluid to the growth media during enumerations. This was indeed observed during this investigation (no significant differences between media G and H in both experiments). It seems logical to suggest that during enumeration studies on methanogenic bacteria of microbial ecosystems, a reduced medium containing mineral salts, vitamins and small amounts of fatty acids in concordance with a carbon dioxide-hydrogen gas atmosphere may be used. It must however be remembered though that other hydrogettutilizing bacteria might also grow on this medium, thus overestimating the methanogenic bacterial numbers, while on the other hand methanogenic bacteria requiring growth factors only present in rumen fluid (or digester fluid) would not grow, thus underestimating the methanogenic bacterial numbers. The addition of rumen fluid to media in two cases (pairs C, D, first experiment and pairs Q, P, second experiment) caused significantly higher counts. In all other applicable cases (pairs C,D, G,H and K,L, experiment No. 2), the addition of rumen fluid resulted in a higher number of colonies, although not significant. It seems that rumen fluid thus served as a growth stimulant or as source of energy or both. SMITH (1965) found that rumen fluid could not be replaced by substances such as yeast extract, beef extract or trypticase soy broth for the growth of his methanogenic isolates. The addition of rumen fluid to media for the propagation of pure cultures of methanogenic bacteria should therefore be beneficial, but the inclusion of rumen fluid in growth media for the enumeration of methanogenic bacteria cannot be advocated since the rumen fluid addition may support growth of non-methanogenic bacteria. T h e stimialation of growth by the addition of digester fluid is not as clear-cut as that o f r u m e n fluid, although in one case (pair N, P; experiment No. 2) such an addition caused a significantly higher count. For the same reasons as in the case of rumen fluid, the inclusion of digester fluid into media for enumerations of methanogenic bacteria cannot be advocated. The inclusion of formate into media caused a signficantly lower count in one case (pair C, E; experiment No. 1) and in all other applicable cases except one (pairs A,F; E,C; C,G and D,H) the colony counts of media containing formate were less than in the absence of formate. It seems that formate suppresses the growth of the bacteria and should not be included in substrate amounts into media for the enumeration of methanogenic bacteria. This repression of formate on bacterial growth is strange since formate serves as an energy source for some methanogenic bacteria (BRYANTe t al., 1967). The addition of acetate to growth media showed a peculiar pattern although none of the differences was significant. In the presence of acetate, media which contained rumen fluid or digester fluid showed higher bacterial counts (pairs N,F and H,Q; experiment No. 2, TABLE 5), while in the absence of rumen fluid or digester fluid, acetate seemed to have repressed the bacterial counts (pairs C,U and G,P; experiment No. 2, TABLE 5). These tendencies should be investigated still further since acetate is apparently one of the major intermediate substrates between the non-methanogenic and methanogenic populations in anaerobic digesters (MCCARTY e t al., 1963).

Enumeration Studies on Methanogenic Bacteria

553

The addition of glucose to various growth media yielded lower bacterial counts in three of the four cases (pairs C,S; G , K and H , L ; TABLE 5), but these counts were not significantly lower. The addition of glucose to washed suspensions of cells from a synthetic substrate digester repressed endogenous methanogenesis (TrIiEL--personal communication). In view of this fact lower counts might indicate a toxic effect of glucose on methanogenic bacteria, but these results are not clear-cut and should be further investigated. The magnitudes of the colony counts in both experiments (TABLES2 and 3) being in the range of 1-0 × 108 to 486 x 10 s bacteria per ml indicated that a sizeable portion of the total bacterial population of the anaerobic digester must have been able to grow. It seems justifiable to advocate the use of medium G (TABLE 1) for the enumeration of methanogenic bacteria since this medium yielded counts of 1.5 x 108 and 303 x 10 s bacteria per ml in experiments I and 2 respectively (TABLES 2 and 3). These values compared favourably to the values of I05-10 s (MYLRO1E and HUNGATE, 1954), 1 X 106 (SMITH, 1965), 1 x 105-1 x 10 7 (SMITH, 1966) methanogenic bacteria per ml in sludge digestion and the values of 2 x l0 s (SMITH and HUNGATE, 1958) and 107-109 (BRYANT, 1965) methanogenic bacteria per ml in rumen contents.

CONCLUSIONS 1. No significant difference was found between the colony counts of a medium which contained a gas atmosphere of 78 per cent H2 and 22 per cent CO 2 as substrates, mineral salts, small amounts of fatty acids and vitamins, and media of similar composition to which rumen fluid or digester fluid additions were made. The use of the former medium for the enumeration of methanogenic bacteria during ecological studies is advocated. 2. Rumen fluid addition to growth media caused higher colony counts, but rumen fluid might be able to stimulate growth of non-methanogenic bacteria. 3. Digester fluid addition apparently has the same, but not as marked effect as the rumen fluid addition. 4. The addition of formate in substrate amounts probably represses bacterial counts. 5. The addition of acetate probably gave rise to higher bacterial counts in the presence of digester or rumen fluids but repressed the bacterial counts in the absence of digester or rumen fluids. 6. Glucose might be toxic for the methanogenic bacteria. Acknowledgements--The helpful discussions and criticisms of Dr. S. G. REINACH,Dr. O. J. COETZEE

and Mr. P. G. TrtmL are gratefully acknowledged.

REFERENCES BRYANTM. P. (1965) Rumen methanogenic bacteria. In Physiology of Digestion in the Ruminant. (Edited by DOO~r~RTYR. W. et aL) Butterworths, Washington. BRYANTM. P. and ROBINSONI. M. (1961) An improved non-selective culture medium for ruminal bacteria and its use in determining diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44, 1446-1456.

554

M . L . SIEBERT, D. F. TOERIEN and W. H. J. HATrINGH

bRYANT M. P., WOLIN E. A., WOLIN M. J. and WOLr~ R. S. (1967) Methanobacillus omelianskii, A symbiotic association of two species of bacteria. Arch. MikrobioL 59, 20-31. GRANVILLE W. A., SMITH P. F. and LONGLEY W. R. (1957) Elements o f the Differential and Integral Calculus. New revised edition, Ginn and Co. HATTINGH W. H. J., KOTZI~ J. P., THIEL P. G., TOERIEN D. F. and SIEBERT M. L. (1967) Biological changes during the adaptation of an anaerobic digester to a synthetic substrate. Water Research 1, 255-277. HEUKELEKIAN H. and HEINEMAN B. (1939) Studies on the methane bacteria--I. Development of a method for enumeration. Sewage Wks. J. 11,426-435. KRUSKAL W. H. and WALL1S W. A. (1952) Use of ranks in one-criterion variance analysis. J. Am. statist. Ass. 47, 583-621. MCCARTY P. L., JERIS J. S. and MURDOCH W. (1963) Individual volatile acids in anaerobic treatmetit. J. War. Pollut. Control Fed. 35, 1501-1516. MYLROIE R. L. and HONGATE R. E. (1954) Experiments on the methane bacteria in sludge. Can. J. Microbiol. 1, 55-64. REINACH S. G. (1966) Distri3ution-free Methods in Experimental Design. D.Sc.(Agric.) thesis, University of Pretoria. SIEBERT M. L. and HATrlNGH W. H. J. (1967) Estimation of the methane-producing bacterial numbers by the most probable number (MPN) technique. Water Research 1, 13-19. SMITH P. H. (1965) Pure culture studies of methanogenic bacteria. Proc. 20th Purdue Waste Conference, pp. 583-588. SMrrH P. H. (1966) The microbial ecology of sludge methanogeneses. Devs ind. Microbiol. 7, 156-161. SMITH P. H. and HONGATE R. E. (1958) Isolation and characterization of Methanobacterium ruminatium n.sp. J. Bact. 75, 713-718. THIEL P. G., TOERIEN D. F., HATTINGH W. H. J., KOTZE J. P. and SIEBERT M. L. (1968) Interrelations between chemical and biological characteristics in anaerobic digestion. In press. TOERIEN D. F. and HATTINGH W. H. J. (1968) Anaerobic Digestion--I. Microbiology. Water Research, to be published. TOERIEN D. F. and SIBERT, i . L. (1967) A method for the enumeration and cultivation of anaerobic "acid-forming" bacteria present in digesting sludge. Water Research 1, 397-404. WIERINGA K. T. (1940) The formation of acetic acid from carbon dioxide and hydrogen by anaerobic spore-forming bacteria. Antonie van Leeuwenhoek 7, 121-127.