enumeration and isolation of anaerobic

J. Gen. App!. Microbiol., 24, 317-332 (1978)

ENUMERATION AND ISOLATION OF ANAEROBIC

BACTERIA IN SEWAGE DIGESTOR FLUIDS

ATSUKO UEKI, EIICHI MIYAGAWA, HAJIME MINATO, RYOZO AZUMA, AND TSUNEJI SUTO

National Institute of Animal Health, Kodaira-shi, Tokyo 187

(Received June 20, 1978)

Examination of non-methanogenic anaerobic bacteria in sewage digestor fluid was attempted by the anaerobic roll tube method. For the enu-meration of anaerobic bacteria in digestor fluid, rumen fluid-glucose-cellobiose-agar (RGCA) gave as good results as previously reported in the rumen and other ecosystems like intestines and feces, under the 100% CO2 gas phase. However, under the mixed gas phase (95 % N2 and 5 % C02), higher colony counts were obtained with YLS agar, which contained the supernatant of autoclaved sewage digestor fluid, than with RGCA. Colony counts of anaerobic bacteria decreased with the

progress of fermentation of waste and the proportion of facultative anaerobes in the isolated anaerobes also decreased. Large variations in the distribution pattern of bacteria were usually observed between the results obtained from three digestors investigated. However, Strep-tococcus and Gram-negative curved rods were commonly isolated from the three digestors as predominant groups.

The anaerobic roll tube method devised by HUNGATE (1) facilitated isolation and cultivation of strictly anaerobic bacteria, and a significant progress has been made in the studies of microbial ecosystem in the rumen (2). This method was also applied to isolate anaerobic bacteria from intestines (3, 4) and feces (5, 6).

However, application of this method to the enumeration and isolation of anaerobic bacteria in other ecosystems, such as the soil and sewage digestors has been very scarce. Our knowledge about anaerobic bacteria in these ecosystems is restricted to only a few kinds of bacteria and increase in the information about other anaerobic bacteria in these ecosystems is being expected.

The anaerobic ecosystem of sewage digestor resembles that of rumen. The

process of fermentation in digestors involves two stages, non-methanogenic and methanogenic. At the non-methanogenic stage, organic matters poured into digestors are decomposed to lower fatty acids and other low-molecular compounds.

317

318 UEKI, MIYAGAWA, MINATO, AZUMA, and SUTo VOL. 24

Then these are finally converted to methane and carbon dioxide at the subsequent stage (7).

In this study, we applied the anaerobic roll tube method to the enumeration and isolation of non-methanogenic anaerobic bacteria in sewage digestors to know how many and what kind of anaerobic bacteria are taking part in the decomposition of waste of domestic animals. This report contains the results of comparison of several media and gas phase to examine the suitable culture con-ditions for non-methanogenic bacteria by the strict anaerobic technique. Presump-tive identification of isolates and their distribution in each fermentor in digestor systems are also presented.

MATERIALS AND METHODS

Anaerobic sewage digestors. Schematic diagrams of three digestors (A, B, C) investigated are shown in Fig. 1. All of them are for digestion of piggery waste. Almost all of solid materials in the waste are removed before it is poured into the digestor. In (A) digestor, the waste accumulated in the reservoir is transferred to a fermentor once a day and in the other two digestors it is directly poured into fermentors from piggeries. Waste in the fermentors flows to the next through

pits of partitions between them. Reservoir, and fermentors I, II, III, and IV are indicated below as R, F-I, F-II, F-III, and F-IV, respectively. Sampling of digestor fluid. Digestor fluid was obtained from the middle

part of each fermentor and poured into a container and filled up to the top, which was immediately closed tightly. After the containers were transported to the laboratory, a portion of each digestor fluid was diluted by the one-tenth serial dilution technique with the anaerobic dilution solution (8) under the stream of 02-free gas for the enumeration of anaerobic bacteria. The remainder was

Fig. 1. Shematic diagram of anaerobic sewage digestors.

Reservoir and fermentors are expressed in text as follows: Reservoir, R; Fermentor

I, F-I; Fermentor II, F-II; Fermentor III, F-III; and Fermentor IV, F-IV.

1978 Anaerobic Bacteria in Sewage Digestors 319

used to measure the following chemical parameters. Organic acids were analyzed according to the same method as for analysis of fermentation products of bacteria described below and the amount of ammonia was measured by the Phenate-hypochlorite method (9). A part of the digestor fluid was also used for micro-scopic observation, which was performed according to the method described by MINATO and SUTO (10). Culture media and method. Strictly anaerobic technique was a modification of that of HUNGATE (1,11) as described by AzUMA as "gas jet method" (12). Four kinds of media were used throughout this examination (Table 1). Rumen fluid-

glucose-cellobiose-agar (RGCA) was a modification of the medium described by BRYANT and BURKEY (8) and VL agar (12) was previously described. Sewage digestor fluid-glucose-cellobiose-agar (SGCA) was a modification of RGCA sub-stituting the supernatant of sewage digestor fluid for the rumen fluid. Digestor fluid obtained from anaerobic digestors was autoclaved and centrifuged twice at 16,500 x g for 30 min. The supernatant was used as "the supernatant of sewage di-

gestor fluid" for media. VLS agar was also devised for this examination. It con-tains 40 % of the supernatant of sewage digestor fluid, supplemented with Trypticase,

yeast extract, and beef extract with half as much concentration as in VL agar. Three kinds of gas phase, 100 % C02, mixed gas (95 % N2 and 5 % CO2), and

Table 1. Composition of media (g or ml/liter).

320 UEKI, MIYAGAWA, MINATO, AZUMA, and SUTO VOL. 24

100 % N2, were used throughout this examination. All the gases were passed through a column of hot reduced copper to remove traces of oxygen.

For the colony counts, usually 0.3 ml of 10-5, 10-°, and 10_7 dilutions of each fluid was inoculated, under the stream of 02-free gas, and mixed with the roll tube media. Then the roll tubes were formed. The number of colonies was counted precisely after 7 days of incubation at 30°. Butyl-rubber stoppers were used to close the tubes. Trypticase soy agar (BBL) (TS agar) was used for aero-bic counts by the agar plate method.

Isolation and identification of bacteria. From the roll tubes, in which about 30-250 colonies were counted, about 40 colonies were picked up randomly for each sample and transferred to the slants of the same medium under the stream of the same gas as those of the roll tubes. All the isolates were cultured at 30° for 2 or 3 days and then the purity, Gram-stain reaction, and their morphology were examined microscopically. At the same time, aerotolerance of each isolate was tested by streaking on TS agar plate supplemented with defibrinated sheep blood (10% v/v). Purification of bacteria was also carried out with the roll tubes.

Analysis of fermentation products. Volatile (acetic, propionic, isobutyric, butyric, isovaleric, and valeric) and non-volatile (lactic and succinic) fatty acids formed in 0.5 % (w/v) glucose-containing liquid medium, which contained the same components as those of VLS agar except maltose, cellobiose, starch, and agar, were analyzed by gas chromatographic method according to the methods described by SUTO et al. (13) and by SUTO (14), respectively. The gas chromato-

graph (Nikon Chromato Co.) used was equipped with a hydrogen-flame ionization detector, with N2 as a carrier gas. Columns packed with Chromosorb AW (60/ 80 mesh) were used for the analysis of all the fatty acids. For volatile fatty acids, a column coated with 10% PEG (poly-ethylene glycol adipate) and 2 H3P04 was used and for non-volatile fatty acids, which were converted to methyl esters by borontrifluoride-methanol complex (Tokyo Chemical Industry Co.) and extracted with chloroform, a column coated with 10 % FFAP was used. The column temperatures were 140° and 130°, respectively. Inlet temperature was 220° and detector temperature was 180°.

Presumptive identification. Presumptive identification was performed accord-ing to the identification scheme of HOLDEMAN and MOORS (15). All of the isolates were classified into groups in accordance with the presumptive identification.

RESULTS

State of anaerobic digestor The pH value and concentrations of NH3-N and organic acids in the fluid of digesters are shown in Tables 2-4. The pH value became higher with the pro-

gress of fermentation and it was always above 7. The concentration of organic


acids decreased with the progress of fermentation and that of NH3-N usually increased. Among the detected organic acids, acetic acid was the highest in concentration and that of other volatile fatty acids was very low. The con-centration of acetic acid decreased with the progress of fermentation, and this fact was remarkable in (C) digestor. Non-volatile fatty acids such is lactic and succinic acids were not detected in any fluid.

Change in colony counts of bacteria in anaerobic sewage digestor fluid Colony counts of anaerobic and aerobic bacteria in the fluid of (A) digestor were obtained using RGCA under the atmosphere of 100% CO2 and TS agar,

Table 2. pH value of the sewage digestor fluid.

Table 3. Concentration of NH3-N in the fluid of (A) digestor.

Table 4. Concentrations of organic acids in the sewage digestor fluid.


respectively, for 3 days (Fig. 2). Viable counts of aerobic bacteria were far less than those of anaerobic bacteria, and the counts of both of them decreased with the progress of waste decomposition. This order of counts (R> F-I> F-IV) did not change during the test for 3 days. Colony counts of anaerobic bacteria ranged from 107 to 108/ml digestor fluid and counts in each fermentor did not change significantly for 3 days. Similar results were obtained in (C) digestor in these respects. Direct microscopic counts obtained from three digestors ranged from 1.1 x 109 to 3.7>< 1010/ml and these counts also generally decreased with the progress of fermentation. The predominant morphotypes observed microscopically were Gram-negative curved rods, Gram-negative rods, Gram-negative small rods, and Gram-positive cocci.

Comparison of colony counts of anaerobic bacteria in digestor^ fluid with different culture conditions Colony counts of anaerobic bacteria in fermentors (I and IV) of (A) digestor were obtained with three kinds of media; RGCA, VL agar, and SGCA, under the

gas phase of 100% CO2 and compared with each other (Table 5). In all the fluids the highest counts were obtained with RGCA and the counts decreased in the order of RGCA, VL, agar, and SGCA. Although SGCA was devised as a "habitat-simulating medium" (2) like RGCA, colony counts with this medium were less than those with RGCA and VL agar. Thus Trypticase, yeast extract, and beef extract were supplemented to SGCA and used for the colony counts. This medium was referred to as VLS

Fig. 2. Change of colony counts of bacteria in the fluid of (A) digestor for 3 days. Anaerobic bacteria were counted with RGCA under 100% C02 gas phase and aerobic bacteria were counted with TS agar. o, Reservoir; 0, fermentor I; •, fer-mentor IV; -, anaerobic bacteria; ---, aerobic bacteria.


agar. Comparison of colony counts obtained with VLS agar with those obtained with three other media is presented in Table 6. Under 100 % CO2 gas phase, the colony counts with VLS agar ranged in general between those with RGCA and VL agar. It was considered noteworthy that the highest colony counts of the fluid in the reservoir were obtained with VL agar, while those in fermentors

(F-I and F-IV) were obtained with RGCA. These results suggest that bacteria in the reservoir differ in the nutritional requirements from those in fermentors. Table 6 also presents the effect of gas phase on the colony counts. Under the gas

phase of 100% N2, colony counts with VLS agar were higher than those with VL agar in all the digestor fluids and they also exceeded those obtained with the same medium under 100 % CO2 except one sample. On the contrary, the colony counts with VL agar decreased under 100% N2 as compared with those under 100 CO2 with 2 exceptions among 6 samples. It seemed that viability of anaerobic bacteria in the digestor fluid was affected not only by the composition of culture medium, but also by that of gas phase. In fact preliminary observations showed that the bacteria making colonies under the gas phase of N2 differed in morphology and Gram stain from those under CO2 gas. Based on these findings, we examined

Table 5. Comparison of colony counts

(A) digestor obtained with three different mediaof anaerobic bacteria in the fluid of

Table 6. Comparison of colony counts of anaerobic bacteria in the fluid of

(A) digestor obtained with VLS agar and three other media and effect of gas composi-tion on them.


the effect of mixed gas phase (95 % N2 and 5 % C02) (Table 7). Under this gas phase, colony counts with VLS agar increased, except in one sample (F-I, Exp. 2), by 1.3-2.0 times as high as those under 100 % C02 gas phase. However, colony counts with RGCA decreased under the mixed gas as compared with those under CO2. Similar results were obtained with the digestor fluid from (B) digestor (Table 8). Table 9 presents colony counts of anaerobic bacteria in the digestor fluid from (C) digestor with VLS agar and RGCA under the mixed gas. In all the digestor fluids, much higher colony counts were obtained with VLS agar. Finally, we concluded that VLS agar with the mixed gas phase was the most effective for the enumeration of anaerobic bacteria in the digestor fluid as a single medium and

Table 7. Effect of the fluid of (A) digestor.

mixed gas phase on colony counts of anaerobic bacteria in

Table 8. Effect of the fluid of (B) digestor.

mixed gas phase on colony counts of anaerobic bacteria in

Table 9. Colony counts of anaerob

with RGCA and VLS agar.

is bacteria in the fluid of (C) digestor obtained


gas phase. Thus, in subsequent examinations these conditions were employed for the enumeration and isolation.

Characterization of bacteria isolated Isolates from the three digestors were characterized according to the pro-

cedure described above and the presumptive identification of each isolates was carried out. Based on the presumptive identification, all of the isolates were classified into several groups. Characters of each group are shown in Table 10.

Composition of the bacterial flora Figs. 3-5 show compositions of the bacterial flora. (A) digestor was examined twice, in July and October, and the results are shown in Fig. 3. Concerning the flora obtained in July, Group 1 (Streptococci) was the most dominant in the bacterial flora in the reservoir (45 %) and the proportion of this group decreased in F-I (26 %). Although in F-I the proportion of Group 2 increased, that of all the facultative anaerobes (Group 1 and 2) decreased as compared with those in the reservoir (52 %-~44 %). As to the strict anaerobes (Group 3-7), Gram-negative rods (Group 4) were the most dominant group in both the reservoir and F-I. In contrast, in October, the proportion of facultative anaerobes increased in the later stage of decomposition (F-I) (20 %-45 %). Gram-negative curved rods

(Group 4) and Gram-positive cocci (Group 6) were the dominant groups in the strict anaerobes in both the reservoir and F-I at this sampling time. Thus, as for the strict anaerobes, rather similar florae were obtained from the reservoir and F-I at each sampling time. The similarity indices (S.I.) between these florae were calculated according to the following formula (16) :

xi-yi x 100 S.I. between x and y=(x • y)=

xi--yi i

where xi and yi indicate the number of a bacterial group in the two samples (x and y) to be compared. S.I. values obtained were as follows: (JR•JF-I)=35.6,

(OR • OF-I)=46.7, (JR • OR)=71.2, and (JF-I • OF-I)=60.9, where J and 0 indicate July and October, respectively. These values showed that similarity of florae in the reservoir at two sampling periods was very low, and so was F-I. The florae of (B) digestor, also examined in July and October, are given in Fig. 4. As shown in it, at both sampling time, a large difference in the composition of the bacterial flora was observed between two fermentors, including the strict anaerobes in contrast to the results of (A) digestor. In July, the proportion of Streptococci (Group 1) in F-I (62%) largely exceeded those of other groups and sharply decreased to 16% in F-III and the proportion of all the facultative anaerobes also decreased from 73 % to 33 %. As for the strictly anaerobic bacteria, Gram-positive cocci (Group 6) were the dominant group in both fermen-tors and in F-III, Gram-negative curved rods (Group 4) were also other dominant


group. In contrast, in October, Streptococci were not such a large group in both fermentors. At this time, the proportion of strict anaerobes was much higher than that of facultative anaerobes. The proportion of facultative anaerobes was slightly lower in F-III (17.5 %) than in F-I (22 %). Group 4

(Gram-negative curved rods) was the highest in F-I (52%) and largely exceeded other groups. In F-III this group became one of minor groups and on the one

hand Group 3 (Gram-negative rods) and Group 6 (Gram-positive cocci) were isolated as the dominant groups. S.I. values were as follows : (JF-I • JF-III) 63.3, (OF-IS OF-III)=75.4, (JF-I . OF-I)=70.7, and (JF-III . OF-III)=75.4. These values also showed that similarity between these florae was very low.

Fig. 3. Composition of bacterial flora in the fluid of (A) digestor. The examination was made with VLS agar under mixed gas (95% N2 and 5 % C02)

phase in July and October. Colony counts of these samples (x lO8 f ml) : July R, 0.62; July F-I, 0.42; October R; 3.68; and October F-I, 1.49. See Table 9 for difinition of each bacterial group. R, reservoir; F-I, fermentor I. ®, Facultative anaerobes; Eli, strict anaerobes.

Fig. 4. Composition of bacterial flora in the fluid of (B) digestor. Colony counts (x 108/ml): July F-I, 5.78; July F-III, 1.10; October F-I, 1.29; and October F-III, 0.22. F-I, fermentor I; F-III, fermentor III. See legend to Fig. 3 for cultural conditions and further detail.

328 UEKI, MIYACAWA, MINATO, AZUMA, and SUTO VOL. 24

As shown in Fig. 5, the bacterial composition in (C) digestor, which was examined in September, was rather simple compared with other two digestors. The composition of bacterial flora of four fermentors resembled each other. Streptococci (Group 1) and the strictly anaerobic Gram-positive rods (Group 5) were the two most dominant groups in this digestor. Especially about the strict anaerobes, Group 5, which was composed of mostly one species (Eubacteriurn sp.), exceeded other groups in all the fermentors. This group has never been dominant in other digestors and was peculiar to the (C) digestor. The proportion of facultative anaerobes decreased with the progress of fermentation (47 %-~ 39 %--~42 /-*23 %) and, in contrast, that of strictly anaerobic bacteria increased. This decreasing tendency of facultative anaerobes in later stage of fermentation was also seen in (B) digestor and (A) digestor in July. S.I. values between the florae of fermentors were: (F-I . F-II)=29.5, (F-II . F-III)=17.3, and (F-III . F-IV)

= 26.3. The high similarity of florae in four fermentors was also indicated by these values. As shown above, difference of bacterial flora among the three digestor systems is large. However, it may be pointed out that the proportion of facultative anaerobes decreased with the progress of fermentation except in one case ((A) digestor in October), and the proportion of strict anaerobes was higher than that of facultative anaerobes, especially in the later stage of fermentation. Judging from the S.I, values, variation of flora in different seasons was large in both of (A) and (B) digestors, while it seems likely that at some fixed time bacterial flora in a digestor system is rather similar through the course of fermentation, as seen in

(C) digestor, and also the case of strict anaerobes in (A) digestor. When the S.I. values between the reservoir and fermentors of (A) and (B) digestors were calculated all together, the values between F-I of (A) and F-III of (B) were : (AJF-I . BJF-III) = 39.3, (AJF-I.BOF-III)=35.1, (AOF-I.BJF-III)=20.9, and (AOF-I BOF-

Fig. 5. Composition of bacterial flora in the fluid of (C) digestor. Colony counts (x 108/ml): F-I, 2.90; F-II, 1.75; F-III, 1.25; and F-IV, This was examined in September. See legend to Fig. 3 for cultural methods further details. F-I, F-II, F-III, and F-IV indicate fermentor I, II, III, and respectively.

1.00.

and

IV,

1978 Anaerobic Bacteria in Sewage Dggestors 329

III)=72.6. The former three values were rather low compared with those between other samples. These values suggest that there is a possibility that even in different digestor systems, the composition of bacterial flora becomes similar after the fermentation proceeds to some extent.

Concerning the dominancy, for the strict anaerobes, Gram-negative curved rods (Group 4) and Gram-positive cocci (Group 6) were rather commonly isolated as dominant groups in (A) and (B) digestors and, although the dominant group in

(C) digestor was different from these digestors, Group 4 was also constantly isolated from all the four fermentors. Group 3 (Gram-negative rods) was also dominant in some samples.

DISCUSSION

It is often said that it is very difficult to understand microbial ecosystems only by cultural method, because microbes which we can catch on media are only a small part of them present in that microbial ecosystem. For sewage digestors, little is known about microbes in them and increase in the investigation is expected hereafter. Therefore, at present, it seems that analysis of bacterial flora by the cultural method and also improvement of the method are significant for the first understanding of this microbial ecosystem.

Although RGCA was developed to cultivate anaerobic bacteria in the rumen, it has been proved that this medium is also very effective for cultivation of anaerobic bacteria in other samples such as feces and intestines. We also used this medium as one of the most promising media for the enumeration and isola-tion of anaerobic bacteria from sewage digestors. RGCA also showed its superior-ity for the enumeration of anaerobic bacteria in the digestor fluid in three media used, under the atmosphere of 100 % CO2. On the contrary, we could not obtain high counts with SGCA. The supernatant of sewage digestor fluid which was added to SGCA contained urine and extracts of feces and soil. As the number of bacteria in digestor fluid is less than that in the rumen fluid, factors in the super-natant of sewage digestor fluid supplied from bacterial cell materials are less than those in the rumen fluid. Thus we can easily assume that this supernatant is largely different from the rumen fluid.

Since the major part of gas in the rumen is C02, it seems reasonable to use 100% CO2 gas phase for the cultivation of anaerobic bacteria in the rumen but, as the composition of gas in ecosystems like sewage digestor is different from that of rumen, it may be important to take into consideration the gas other than CO2 for the culture of anaerobic bacteria in these ecosystems. In our preliminary ex-

periments, we realized that bacteria making colonies under N2 gas phase were different from those under the CO2 gas phase in their morphology and Gram stain. It is well known that many kinds of bacteria such as Bacteroides and Streptococcus require CO2 essentially for theirr growth (2,17). Conversely, it has


not been known that there exist some bacteria which are affected by the gaseous N2 other than N2-fixing bacteria. However, colony counts obtained in this examination under N2 gas phase were not always lower than those under C02 gas

phase. Furthermore, in some cases, these counts were rather higher than those under C02 gas phase. These results indicate that N2 gas itself or absence of C02

gas enhances the growth of bacteria, but how these conditions of gas phase affect the growth of bacteria is not clear. Bacteria which essentially require C02 for their growth do not necessarily require 100 % C02 (17). Therefore, from these points of view, it seems that more kinds of bacteria can grow under a mixed gas phase of C02 and N2 than under the gas phase of only one of them. The colony counts and the number of isolated bacterial species indicated that the

combination of VLS agar and the mixed gas was the most effective as a single culture condition among the conditions used in this study for the enumeration and isolation of anaerobic bacteria. The reasons why VLS agar gave good results might be as follows : (i) The supernatant of digestor fluid supplied some growth factors for bacteria, (ii) because this medium was not so poor as SGCA by the addition of Trypticase and others, bacteria which were not able to grow in only the supernatant of digestor fluid could grow, but (iii) VLS agar was not so rich as VL agar that large colonies scarcely suppressed the growth of slow growers.

Colony counts of anaerobic bacteria changed with digestors and sampling time. It is not clear whether these variations indicate seasonal changes in bacterial flora or only the difference of input of waste into the digestors but, in most cases, colony counts in the digestor fluid were in the order of x 10$/ml of fluid. The colony counts decreased generally with the progress of fermentation, and the results of all the three digestors coincided well in this respect. The

proportion of facultative anaerobes in the isolated bacteria also usually decreased, and the concentration of acetic acid decreased with the progress of fermentation as well. SMITH and MAH (18) reported that the acetic acid-utilizing methanogenic

process proceeded in the sewage digestor. Above-mentioned results suggested that with the progress of waste fermentation Eh value of the digestor fluid became lower and the acetic acid-utilizing methanogenic process was proceeding in the fluid.

Aerobic bacterial counts were about 20 ° o to 30 % of the anaerobic counts in almost all the cases. In our preliminary examination, it seemed that the dominant bacteria isolated with the aerobic culture technique were facultative anaerobes and were able to grow under the condition of anaerobic technique. Thus it may be

possible to count them by the anaerobic technique. It could be concluded that the members of bacteria captured by the anaerobic method represented the

bacterial flora in the anaerobic digestor fluid, at least the predominant members that could be caught in test tubes. However, the recovery of bacteria caught in test tubes was so low, as indicated by the microscopic observations, that an improve-ment of cultural conditions was necessary.


In the isolated facultative anaerobes, Streptococcus was the most dominant

genus, especially in the early stages of fermentation. Streptococcus existed at least in the order of 10~ to 108/ml. Thus, it was thought that lactic acid, the main fermentation product of this genus, may be produced and accumulated in the digestor fluid at a rather high concentration but, as described above, lactic acid could not be detected in any case and only volatile fatty acids were detected

(Table 3). From these results it seems that in the digestor fluid there exist some bacteria that utilize lactic acid. HoBSON and SHAW reported that the predominant bacteria isolated from several kinds of sewage digestor fluid belonged to the genera such as Streptococcus, Clostridium, and Bacteroides (19). We also isolated Streptococcus as a predomi-nant genus, but neither Clostridium nor Bacteroides was isolated as such a predom-inant group, if any. On the other hand, the predominant groups isolated from anaerobic digestors by TOERIEN (20) were Gram-positive branched or straight rods such as Bifidobacterium and Eubacterium. Gram-positive rods also occupied a predominant position in some samples in our examination, but it could not be generalized that this group was a predominant group in these ecosystems, and moreover, Gram-positive branched rods were not isolated in such a high proportion as his report indicated. As to the bacterial composition in anaerobic digestor fluid, further examinations should be attempted and then the

general aspects would be made clear.

REFERENCES

1) R. E. HUNGATE, Bacteriol. Rev., 14, 1 (1950). 2) R. E. HUNGATE, In The Rumen and Its Microbes, Academic Press, New York, (1966),

p. 20. 3) J. P. SALANITRO, I. G. FAIRCHILDS, and Y. D. ZG0R0NICKI, App!. Microbio!., 27, 678 (1974). 4) A. MARTHA, C. HARRIS, A. REDDY, and G. R. CARTNER, App!. Environ. Microbio!., 31, 907 (1976).

5) W. E. C. MOORE and L. V. HOLDEMAN, App!. Microbio!., 27, 961 (1974). 6) J. P. SALANITRO, I. G. BLAKE, and P. A. MUIRHEAD, App!. Environ. Microbio!., 33, 79

(1977). 7) R. A. MAH, D. M. WARD, L. BARESI, and T. L. GLASS, Annu. Rev. Microbio!., 31, 309

(1977). 8) M. P. BRYANT and L. A. BURKEY, J. Dairy Sci., 36, 205 (1953). 9) A. KAPLAN, In Methods of Biochemical Analysis, 17, ed. by D. CLICK, Interscience Publishers, New York (1969), p. 311.

10) H. MINATO and T. SuTo, J. Gen. App!. Microbiol., 22, 259 (1976). 11) R. E. HUNGATE, In Methods in Microbiology, 3B, ed. by J. R. NoRRIS and D. W. RIBBONS,

Academic Press, New York (1969), p. 117. 12) R. AzuMA and T. SUTO, Proc.1st Int. Conf. Culture Collection, p. 493 (1970). 13) T. SUTO, H. MINATO, S. ISHIBASHI, and K. OGIM0T0, Proc.1st Int. Conf Culture Collection,

p. 387 (1970). 14) T. SUTO, In Ushi no Rinshokensaho, ed. by R. NAKAMURA, T. YoNEMURA, and T. SuTo,

Nosangyoson Bunka Kyokai, Tokyo (1973), pp. 6-42, in Japanese.

332 UEKI, MIYAGAWA, M[NATO, AZUMA, and SUTO VOL. 24

15) L. V. HOLDEMAN and W. E. C. MOORE (ed.), Anaerobe Laboratory Manual, 3rd ed., Virginia Polytechnic Institute and State Univ. Anaerobe Laboratory, Blacksburg, Virginia

(1974). 16) C. FURUSAKA (ed.), In Dojo Biseibutsu Nyumon, Kyoritsu Shuppan, Tokyo (1969), p. 155,

in Japanese. 17) B. A. DEHORITY, J. Bacteriol., 105, 70 (1971). 18) P. H. SMITH and R. A. MAH, App!. Microbiol., :14, 368 (1966). 19) P. N. HoBSON and B. G. SHAW, Water Res., 8, 507 (1974). 20) D. F. TOERIEN, Water Res., 4, 129 (1970).

enumeration and isolation of anaerobic

Documents