Water Research, Pergamon Press 1967. Vol. 1, pp 397--404. Printed in Great Britain.

A METHOD FOR THE ENUMERATION AND CULTIVA- TION OF ANAEROBIC "ACID-FORMING" BACTERIA

PRESENT IN DIGESTING SLUDGE

D. F. TOERIEN and M. L. SIEBERT

National Institute for Water Research, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria, South Africa

(Received 24 April 1967)

Abstract--A method is described for the enumeration and cultivation of anaerobic "acid- forming" bacteria from digesting sludge. In this method use is made of pre-reduced media in roll tubes containing oxygen-free gas atmospheres. Digester supernatant liquor is included in the culture media to provide the growth factors normally present in anaerobic digesters.

INTRODUCTION

VARIOUS methods have been proposed for the cultivation of anaerobic bacteria. In rumen microbiology HUNGATE (1950) developed a method by which fastidious anaerobic rumen bacteria could be enumerated and cultivated, and his original method has been improved and extended in the methods currently being used in rumen bacteriology (BRYANT and BURKEY, 1953 ; SMITI-I and HtmGATE, 1958 ; KISTNER, 1960; BRYANT and ROBINSON, 1961 and BRYANT, 1963). The basic method of HUNGATE (1950) has recently also been extended to the cultivation of fastidious anaerobic bacteria other than those of the rumen (MOORE, 1966; MOORE et al., 1966).

Early attempts to isolate strains of rumen bacteria had little success due to failure to simulate rumen conditions (HtrNGAr~, 1966). Culture procedures borrowed from medical, dairy or soil microbiology, without modifications to fit the rumen habitat were largely unsuccessful (HtrNGATE, 1966). According to HtrNGArE (1966) the lack of success of early attempts to cultivate rumen cellulolytic bacteria stemmed in part from (1) failure to supply the important nutritional factors in rumen fluid, including carbon dioxide, n-valeric acid and C4 and Cs branched-chain fatty acids (BRYANT and DOETSCH (1954); (2) lack of sufficiently anaerobic conditions. Methods in which Petri plates and anaerobic jars were used were of no use for the isolation of the more fastidious anaerobic rumen bacteria (HtrNGATE, 1966).

The basic method developed by HtmGAXE (1950) consisted of the use of prereduced media in test tubes fitted with rubber stoppers and containing oxygen-free gas atmo- spheres (usually carbon dioxide). To the growth media used, rumen fluid was added to supply the nutritional factors usually present in the rumen. Samples or bacteria were never, or for only very brief periods, exposed to oxygen during the procedures devel- oped by HtrNGATE (1950), and his work has been of great importance in the rapid progress in the study of rumen bacteriology that has taken place in recent years.

When digestion in the tureen and digestion in anaerobic digesters are compared, there is a striking similarity between the two processes. Both systems usually receive a complex feed which is degraded in a microbe-rich environment under conditions of very low redox potentials. In both systems the organic matter introduced is broken down to fatty acids. In anaerobic digestion all the fatty acids are converted to methane and carbon dioxide, while in ruminants the fatty acids are absorbed into the blood

397

398 D . F . TOERIEN and M. L. SIEBERT

stream and a little amount of methane is produced from carbon dioxide and hydrogen. Because of the similarity between digestion in the rumen and digestion in anaerobic

digesters, an attempt to enumerate or cultivate the anaerobic bacteria of digesting sludge can be based on the principles established in the methodology of rumen bacteriology. In this way the conditions for anaerobic digestion will be simulated in culture vessels.

The method described here is based on the methods used in rumen microbiology, particularly those developed by KISTNER (1960). The conditions required for anaerobic digestion were simulated by using pre-reduced digester supernatant liquor-containing growth media in Astell roll tubes fitted with rubber stoppers. The bottles contained oxygen-free gas atmospheres consisting of carbon dioxide, nitrogen and hydrogen.

METHODS AND MATERIALS

Oxygen-free conditions Air was displaced from solutions, media and culture vessels by purging with a gas

mixture containing 66 per cent carbon dioxide, 33 per cent nitrogen and 1 per cent hydro- gen or with 99 per cent carbon dioxide and 1 per cent hydrogen. The commercial gases were mixed in the required proportions and then stripped of oxygen by passage through a Deoxo catalytic purifier (Engelhard Industries, London). With the aid of a manifold and flow meters six equal currents of oxygen-free gas were obtained. The individual gas streams were sterilized by passage through sterile cotton wool contained in the barrels of Luer-Lok syringes. These were connected to the flow meters by thick-walled Latex tubing. For gassing bottles, the syringe barrels were provided with 20 gauge hypodermic needles of sufficient length to reach to the bottoms of the bottles. Rubber stoppers were provided for the bottles and gassing was carried out by inserting the needle between the rubber stopper and the neck of the bottle. After gassing for approximately 1.5 to 3 min (10 volume displacements) at a rate of 250 ml/min, the needle was withdrawn with the one hand while the stopper was held down with the fingers of the other.

Reducing solution

In rumen microbiology reducing agents added to culture media to maintain low redox potentials, included cysteine (HOHTANEN et al., 1952), sodium dithionite (SMITH and HUNGATE, 1958), cysteine hydrochloride (KISTNER, 1960) and a mixture of cysteine hydrochloride and sulphide (BRYANT, 1965). A mixture of cysteine hydro- chloride and sulphide was used in the method described here. To prepare the reducing solution 80 ml of a 0.1 N NaOH solution was boiled for 1 min and cooled in cold water without stirring. Cysteine hydrochloride (1.25 g) was placed in a 100 ml measur- ing cylinder fitted with a ground glass stopper. When cool, the boiled NaOH solution was transferred to the measuring cylinder and immediately purged with a stream of 99 per cent nitrogen and 1 per cent hydrogen. When the cysteine hydrochloride was completely dissolved, the volume was made up to 100 ml with distilled water and then 1.25 g of Na2S.9H20 was added to the cylinder and purging with gas continued until dissolved. This solution was then distributed in appropriate volumes into 4 oz or 1 oz McCartney bottles fitted with rubber stoppers, and gassed as previously described with 99 per cent nitrogen and 1 per cent hydrogen. These bottles were then sterilized at 121 ° C for 15 min and stored in the dark at 2 ° C until used.

A Method of Enumeration and Cultivation of Anaerobic "Acid-forming" Bacteria 399

Redox potential indicator

Redox potential indicators added to the growth media in rumen microbiology included resazurine (BRYAICr, 1959), indigodisulphonate "indigocarmine" (BRYANT, 1959; KISTNER, 1960), benzylviologen (SMITH and HUNGATE, 1958) and phenosafranine (BLACKBURN and HOBSON, 1962). The American workers have made extensive use of resazurine but KISTNER (1960) considered indigocarmine a better redox indicator for the low redox potentials to be maintained. In the method described here indigocarmine was used, although it was found to be less satisfactory in media stored for a long period. In cases such as these, the use of resazurine should be satisfactory.

Anaerobic dilution solution

During the enumeration of the anaerobic bacteria of digesters, samples of the digesters must be diluted under anaerobic conditions. For this purpose a solution with the following composition (TABLE 1) was used. To a litre of this solution, 10 ml of a

TABLE 1. COMPOSITION OF ANAEROBIC DILLFFION SOLUTION

Solution components (g/l)

K2HPO4 17"4 KH2PO, 6"8 NH,C1 1 "0 MgC12.6H20 0"5 Cysteine.HC1 0.1 Sodium thioglycollate 0'1 Yeast extract (Difco) 0.01 MnSO4.4H20 0.005 COC12.6H20 0'005 Fe-citrate 0.005 CaCI2.2H20 0.005 NaMoOa.2H20 0.006

0.05 per cent (w/v) solution of indigocarmine was added as redox indicator. The solution was then boiled for 1 rain and rapidly cooled in ice water. When cool, 4.36 g of NaHCOa was added and the solution slowly stirred until dissolved. Of this solution, 44.5 ml quantities were distributed into 4 oz McCartney bottles and gassed as previously described. Into each bottle 0.5 ml of the cysteine hydrochloride sulphide reducing solution was injected through the rubber stopper by means of a syringe. The stoppers were screwed down with Bakelite screw caps and the bottles sterilized at 121 ° C for 20 min.

Digester liquor supplement

As a source of the growth stimulants normally present during anaerobic digestion, a d!gester liquor supplement was added to the growth media. To obtain this supple- ment digester liquor from an active methane-producing laboratory-scale digester was centrifuged at 32,000 g for 60 min. The clear supernatant liquor was stored under a nitrogen atmosphere at 2 ° C and appropriate volumes added to growth media.

400 D.F. TOERIEN and M. L. SIEBERT

Composition of growth media The medium used for the enumeration of the anaerobic acid-forming bacteria

consisted of 5 per cent (v/v) digester liquor supplement and 95 per cent (v]v) of the following basal medium (TABLE 2). The p H of this medium was adjusted to p H 7" 1 with

TABLE 2. COMPOSITION OF THE MEDIUM USED TO ENUMERATE THE

ANAEROBIC "ACID-FORMING" BACTERIA FROM DIGESTING SLUDGE

Medium components (g/l)

Dextrin 13"6 Sucrose 20'4 Nutrient broth (Difco) 4.6 Casamino acids (enzymatic hydrolyzate, Difco) 2.8 KH2PO4 1.I NH4HCO3 0.27 Acetic acid 0.169 Propionic acid 0.112 Butyric acid 0.029 Valeric acid 0.014 Lactic acid 0.420 Tartaric acid 0.210 Succinic acid 0.252 K2SO4.AI(SO4)a .24H20 0.005 CuSO4.SH20 0.0034 COC12.6H20 0.0001 MgSO4.7H20 0.1 Mg(CHaCOO)2.4H20 0.174 MnSO4.H20 0.0008 Na2SiO3.9H20 0.04 CaC12.2H20 0.084 (NH4)2SO4.Fe(SO4)a.24H20 0.097

5 N N a O H and 20 g Bacto-Agar (Difco) and 10 ml of a 0.05 per cent (w/v) solution of indigocarmine were added per litre of medium. To obtain a suitable redox potential in the medium, 10 ml of the reducing solution was added to 1 1. of medium.

The composition of a medium for streaking of isolates or growth of pure cultures on slants is presented in TABLE 3.

Preparation of growth media Bacto-Agar (20 g) was weighed into a 3 1. flask and the appropriate volumes of

digester liquor supplement, basal medium and indigocarmine solution added. The medium was then boiled until the agar had dissolved. The mouth of the flask was covered by a rubber stopper containing tubes as illustrated in FIG. 1. Line C was attached to the gas line and the flask purged with a gas mixture (usually 66 per cent carbon dioxide, 33 per cent nitrogen and 1 per cent hydrogen for enumeration tubes and 99 per cent carbon dioxide and 1 per cent hydrogen for streaks or s lants) for approximately 10 min at a rate of 500 ml/min. At the end of this period all the screw clamps were dosed and a line A attached to a 5 ml B-D Cornwall continuous pipetting

A Method of Enumeration and Cultivation of Anaerobic "Acid-forming" Bacteria 401

unit. To reduce the r edox po ten t ia l o f the med ium, the a p p r o p r i a t e a m o u n t o f reducing agent was injected into the flask th rough the serum cap. The flask conta in ing the m e d i u m and the a t t ached p ipe t t ing uni t was then steril ized at 121 ° C for 20 min. The screw c lamp on line B was opened jus t before s ter i l izat ion and then closed again immedia t e ly af ter steri l izat ion. Dur ing ster i l izat ion the 18 gauge stainless steel needle of the p ipe t t ing uni t was p ro tec ted by a small test tube and co t ton wool. Immedia t e ly after s ter i l izat ion the flask was p laced in a water ba th a t 80 ° C and line C connected to the gas line. The screw c lamp on line C was opened and short ly af terwards the screw

TABLE 3. COMPOSITION OF THE MEDIUM USED FOR STREAKS AND

SLANTS OF ANAEROBIC "ACID-FORMING" BACTERIA

Glucose 5 g Bacto-Peptone (Difco) 0"5 g Yeast extract (Difco) 0.5 g Lab-Lemco (Oxoid) 0.5 g Digester supernatant liquor 370 ml Indigocarmine (0.05 per cent, w/v solution) 10 ml Fatty acid mixture 3"1 ml Mineral solution A 37.5 ml Mineral solution B 37.5 ml Vitamin solution A 1 ml Vitamin solution B 1 ml Distilled water 455 ml

The above medium was sterilized at 121 ° C for 20 min and after sterilization 70 ml of a sterile 9"1 per cent (w/v) solution of NaHCO3 under CO2 atmosphere, and 20 ml of sterile reducing solution were added to the flask containing the medium. Final pH of medium 6.8.

Mineral solution A consisted of 3 g K2HOP4 ml distilled water.

Mineral solution B consisted of 6 g NaC1, 6 g (NH4)2SO4, 3 g KH2PO4, 0.6 g CaC12.2H20, 1.23 g MgSO4.TH20 dissolved in 400 ml of dist. H20 and made up to 500 ml with distilled water.

Fatty acid mixture consisted of acetic acid--17 ml, propionic acid--4 ml, butyric acid---4 ml, iso-butyric acid--lml, n-valeric acid---1 ml, iso-valeric acid--1 ml, 2-methyl-butryic acid--1 ml, iso-caproic acid--1 ml.

Vitamin solution A consisted of: aneurine HC1--125 nag, L-ascorbic acid--125 mg, riboflavin--125 mg, biotin--500 mg, cobione--30 rag, pyridoxinum HC1--250 mg, folic acid--50 rag, p-aminobenzoic acid 250 mg, nicotinic acid--125 rag, choline-- 125 rag. These substances were dissolved in 250 ml double distilled water and were stored at 2 ° C.

Vitamin solution B consisted of 125 mg of a-tocopherol dis- solved in 250 ml absolute ethanol and stored at 2 ° C.

c lamps on line A and then line B. In this way a s teady s t ream of oxygen-free sterile gas f lowed th rough the flask at a ra te o f 0.5 1/min for the whole o f the tube filling per iod. Where necessay a p p r o p r i a t e amoun t s o f sterile N a H C O a solut ions were then a d d e d to the growth media . The med ium, in 4.5 ml quant i t ies was then injected via the au to-

402 D.F. TOERIEN and M. L. SIEBERT

matic pipette through the rubber stoppers of previously gassed and sterilized Astell roll tubes.

Procedure for enumeration of anaerobic bacteria Samples (5 ml) were withdrawn from each digester by means of a sterile 5 ml all-

glass syringe fitted with a 20 gauge Luer-Lok stainless steel needle. For every digester a serial dilution series was prepared by the transfer of 5 ml samples from dilution bottle to dilution bottle. All the transfers were made with sterile 5 ml syringes.

Four replicate portions of 0.5 ml each of three different dilutions of the contents of each digester were injected into Astell roll tubes containing growth medium. Sterile

Line C - - ' ~ ' ~ Line O

~ L i n e A -'-~ I~l H H I~-Rubb#r slopper

l T M

Fro. 1. Flask used during tubing of media.

all-glass 2 ml syringes were used for these inoculations. The roll tubes were spun in the modified Astell roll tube spinner (TOERIEN and SmBERT, 1967), and then incubated at 30 ° C for 8 days before the colonies of those dilutions which yielded between twenty and two hundred colonies per tube were counted. Whenever the redox indicator indicated the entrance of air into a tube, the count of that tube was disregarded in the calculation of the average number of bacteria for that digester.

Isolation of pure cultures of anaerobic bacteria

Pure cultures of anaerobic bacteria were isolated by the following procedure. Roll tubes containing the medium described in TABLE 3 were heated in a water-bath until the media melted and were then spun in the modified Astell roll tube spinner (TOERIEN and SIEBERT, 1967) until the agar had solidified. Growth of a colony from an enumera- tion roll tube was then anaerobically streaked onto the medium in the spinned bottle, using the device of Moom~ (1966). The streaked bottles were then incubated at 30 ° C until well-developed growth was observed. Single colonies from these streak bottles were then picked and anaerobically transferred to slants in AsteU roll tubes. During the transfers a procedure similar to that described by MOORE (1966) was followed. All transfers were made using a platinum wire loop since nichrome wire loops tend to oxidize the media and are therefore unsuitable (MooRE, 1966).

FIG. 2. A streak roll tube (left) and an enumeration roll tube (right).

(Facing p. 402)

FIG. 3. A slant culture.

A Method of Enumeration and Cultivation of Anaerobic "Acid-forming" Bacteria 403

RESULTS AND DISCUSSION

Using the principles of the methodology of rumen microbiology, a method for the enumeration and cultivation of anaerobic "acid-forming" bacteria from digesting sludge has been developed.

Using the described method thirty-six replicate bottles of one sample from an anaerobic digester yielded an average count of 133.67 colonies per bottle with a standard deviation of 33.74. The thirty-six replicas were divided in eighteen groups of two, twelve groups of three, nine groups of four, six groups of six, four groups of nine and three groups of twelve. An analysis of variance was carried out on each grouping and from this the coefficient of variation was determined for each grouping. The results are presented in TABLE 4. It can be seen that the coefficient of variation did not

TABLE 4. COEFFICIENT OF VARIATION FOR DIFFERENT GROUPINGS

OF THIRTY-SIX REPETITIVE ENUMERATIONS

Grouping Coefficient of variation

18 groups of 2 26.42 12 groups of 3 25.94 9 groups of 4 26.06 6 groups of 6 26.61 4 groups of 9 26.27 3 groups of 12 25.94

vary very greatly between the different groupings and it was decided to use four replicate bottles for each sample as a standard procedure.

Using the described method bacterial counts as high as 200 x 107 bacteria/ml (in some cases more than 100x 10 a bacteria/ml) for bench-scale anaerobic digesters receiving synthetic substrates, have been obtained (TOERIEN, et al., 1967). Anaerobic bacterial counts of 30 x 106 bacteria/ml 24 x 107 bacteria/ml, 29 x 106 bacteria/ml and 9.5 x 106 bacteria/ml respectively for bench-scale anaerobic digesters receiving raw sewage sludge, paper mill effluent, abattoir effluent and brewery effluent have been recorded. On average these counts compare closely with the counts reported by MCCARTY et al. (1962). Their counts varied from 2 x 106 to 350 x 106 and were carried out on laboratory-scale anaerobic digesters operating on fatty acids, ethyl alcohol, nutrient broth, amino acids, glucose, starch and cellulose.

Photographs of an enumeration roll tube, a streak roll tube and a slant are shown in FIRS. 2 and 3.

Pure cultures of anaerobic "acid-forming" bacteria from digesting sludge have been isolated using the method described here. This method should prove easier to use than that described by BUI~ANI< et aL (1966) and should considerably simplify the study of anaerobic digester bacteria.

CONCLUSIONS

1. A new method for the enumeration and cultivation of anaerobic "acid-forming" bacteria from digesting sludge has been described.

404 D . F . TOERIEN and M. L. SIEBERT

2. T h i s m e t h o d , w h i c h is b a s e d o n t h e m e t h o d o l o g y o f r u m e n b a c t e r i o l o g y , s h o u l d

s e r v e as a u s e f u l t o o l i n t h e s t u d y o f t h e " a c i d - f o r m i n g " b a c t e r i a o f d i g e s t i n g s ludge .

Acknowledgement--The valuable suggestions and advice of Dr. A. KISTNER of Onderstepoort are gratefully acknowledged.

R E F E R E N C E S

BLACKBURN T. H. and HOBSON P. N. (1962) Further studies on the isolation of proteolytic bacteria from the sheep rumen. J. gen. Microbio129, 69-81.

BRYANT M. P. (1959) Bacterial species of the rumen. Bacteriol. Rev. 23, 125-153. BRYANT M. P. 1963) Symposium on microbial digestion in ruminants: Identification of groups of

anaerobic bacteria active in the rumen. J. anita. Sci. 22, 801-814. BRYANT M. P. (1965) Rumen methanogenic bacteria. In Physiology o f Digestion in the Ruminant. (Ed.

DOUGHERTY R. W. et al.). Butterworths, Washington. BRYANT M. P. and BURKEY L. A. (1953) Cultural methods and some characteristics of some of the

more numerous groups of bacteria in the bovine rumen. J. Dairy Sci. 36, 205-217. BRYANT M. P. and DOETSCH R. N. (1954) Factors necessary for the growth of Bacteroides succinogenes

in the volatile acid fraction of rumen fluid. Science N. Y. 120, 944-945. BRYANT M. P. and ROBINSON I. 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.

BURBANK N. C., COOKSON J. T., GOEVVNER J and BROOMAN D. (1966) Isolation and identification of anaerobic and facultative bacteria present in the digestion process. Air & Wat. Pollut. Int. J. 10, 327-342.

HUHTANEN C. N., ROGERS M. R. and GALL L. S. (1952) Improved techniques for isolating and purifying rumen organisms. J. Bact. 64, 17-23.

HUNGATE R. E. (1950) The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14, 1-49. HtmGATE R. E. (1966) The Rumen and its Microbes. Academic Press, New York. KISTNER A. (1960) An improved method for the viable counts of bacteria of the ovine rumen which

ferment carbohydrates. J. gen. Microbiol. 23, 565-576. MCCARTY P. L., JERIS J. S., McKINNEY R. E., REED K. and VATH C. A. (1962) Microbiology o f Anaero-

bic Digestion. Report no. R62-29, Sedgwick Laboratories of Sanitary Science, Massachusetts Institute of Technology, Massachusetts.

MOORE W. E. C. (1966) Techniques for routine culture of fastidious anaerobes. Int. J. Syst. Bact. 16, 173-190.

MOORE W. E. C., CATO E. P. and HOLDEMAN L. V. (1966) Fermentation patterns of some Clostridium species. Int. J. Syst. Bact. 16, 383-415.

SMITH P. H. and HUNGATE R. E. (1958) Isolation and characterization of Methanobacterium rumin- antium n. sp. J. Bact. 75, 713-718.

TOERIEN D. F. and SmBERT M. L. (1967) Modification of the Astel roll tube apparatus for the enumera- tion and cultivation of anaerobic bacteria. Lab. Pract. 16, 320.

TOERIEN D. F., SIEBERT M. L. and HATTINGH W. H. J. (1967) The bacterial nature of the "acid- forming" phase of anaerobic digestion. Wat. Res. To be published.