Microb Ecol (1986) 12:173-179

MICROBIAL ECOLOGY Springer-VerlagNew York Inc. 1986

Characterization of Aerobic and Facultative Anaerobic Bacteria from the Liquid Phase of an Anaerobic Fixed-Bed Digester Treating a Cheese Whey Substrate J. de Haast and T. J. Britz Departments of Dairy Scienceand Microbiology,Universityof the Orange Free State, P.O. Box 339, Bloemfontein9300, South Africa

Abstract.

Bacterial counts on the liquid phase o f an anaerobic, fixed-bed digester, treating a deproteinated, prefermented cheese whey substrate, were conducted on two different media under aerobic and facultative conditions. Average counts of 16.6 x 106 and 26.5 x 106 ml -~ were obtained on the two media, with the nutritionally poorer of the two media giving the highest average count. Seventy-five isolates from both media, incubated aerobically as well as in anaerobic jars, were obtained. These isolates as well as 13 reference strains were phenotypically characterized. The similarities between cultures were calculated using the similarity coefficient o f Sokal and Micbener [16]. The organisms were clustered using the unweighted pair group method, and the results presented as a simplified dendrogram. The isolates could be divided into 3 main groups: gram-negative fermentative rods, mainly Enterobacter, Klebsiella, and Citrobacter, with Klebsiella as the predominant genus; gram-positive bacteria, mainly enterococci; and gram-negative nonfermentive rods o f the genera Pseudomonas, Alcaligenes, and Acinetobacter. All the enterobacteria and enterococci were able to produce acetic acid, an intermediate in methanogenesis.

Introduction The complete process of anaerobic digestion involves a complex mixture o f interacting microbial species, most of which do not produce methane [ 1]. Studies on the acidogenic phase o f anaerobic digestion have been carried out and reviewed by various workers [5, 8, 10, 1 1, 18-20] who have established the presence o f large numbers of aerobic, facultative anaerobic, and obligate anaerobic genera in anaerobic digesters. Improvements in the isolation techniques of anaerobic bacteria have shown that most of the acidogenic bacteria are strict anaerobes, with counts higher than those of the facultative anaerobic and aerobic bacteria [5, 7, 18, 19]. In spite of these findings, the aerobic and facultative anaerobic bacteria still form a significant and constant portion of the total digester population [7, 8, 18]. Their main role is probably the rapid utilization o f oxygen, thereby restoring anaerobic conditions suitable for growth of strict anaerobes [7, 19] and the fermentation o f sugars [8]. The bacterial population

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of the acidogenic phase can therefore be divided into the functional bacteria, b a c t e r i a a c c i d e n t a l l y i n t r o d u c e d , a n d p o t e n t i a l l y h a r m f u l b a c t e r i a . F o r t h e first g r o u p , t w o f u n d a m e n t a l p r i n c i p l e s a p p l y . F i r s t , a f u n c t i o n a l o r g a n i s m is o n e t h a t p e r f o r m s a c h e m i c a l r e a c t i o n k n o w n t o o c c u r in t h e d i g e s t e r ; s e c o n d , it s h o u l d b e p r e s e n t in n u m b e r s sufficient t o p e r f o r m t h i s r e a c t i o n a t a r a t e c o m p a r a b l e w i t h t h a t o c c u r r i n g in t h e d i g e s t e r [ 12]. T h e p r e s e n t u n d e r s t a n d i n g o f b a c t e r i a l p o p u l a t i o n s in a n a e r o b i c d i g e s t e r s is r a t h e r l i m i t e d [22] a n d b a s e d m a i n l y o n a n a l y s e s o f b a c t e r i a i s o l a t e d f r o m sewage sludge, animal manure digesters, or the rumen. Studies have revealed that different substrates and carbon sources stimulated the development of t y p i c a l p o p u l a t i o n s [13, 17]. T h u s , a c h a r a c t e r i s t i c p o p u l a t i o n [21] is l i k e l y t o d e v e l o p in a d i g e s t e r b u t is d e p e n d e n t u p o n t h e s u b s t r a t e w i t h w h i c h it is fed. During the cheesemaking process, approximately 90% of the original volume o f m i l k is e x p e l l e d f r o m t h e c u r d in t h e f o r m o f w h e y [2]. L a c t o s e c o n s t i t u t e s about 70% (w/w) of the total solids of whey, while mainly whey proteins and inorganic matter are responsible for the balance. Therefore, whey should be a v e r y s u i t a b l e s u b s t r a t e f o r a n a e r o b i c d i g e s t i o n . S i n c e o p t i m u m d i g e s t i o n is d e p e n d e n t o n t h e m i c r o b i a l p o p u l a t i o n p r e s e n t in t h e s y s t e m i t is i m p o r t a n t to k n o w w h a t t h e m i c r o b i a l c o m p o s i t i o n is a n d w h i c h o r g a n i s m s a r e p r e s e n t . I n t h i s s t u d y , efforts w e r e m a d e to i s o l a t e , e n u m e r a t e , a n d p e r f o r m a p r e l i m i n a r y characterization of the predominant aerobic and facultative anaerobic bacteria from the liquid phase of a mesophilic anaerobic fixed-bed digester fed with a whey substrate.

M a t e r i a l s and M e t h o d s

Reactor A downflow fixed-bed reactor [4], operated at 35"C with no recycling or mixing facilities, was used for the anaerobic digestion of a deproteinated, prefermented whey substrate. The reactor consisted of a glass column containing an inert cylindrical polyethylene bacterial carrier. The reactor had a working volume of 3.5 liter and a surface area to liquid volume ratio of about one. Start-up and operational specifications are as described by de Haast et al. [3, 4]. The original inoculum of the reactor had been sewage sludge, but at no stage during the rest of the study was any added again. Before the start of this study, the reactor had been fed continuously for more than 6 months with prefermented cheese whey (COD = 13,000 mg liter-~), which contained large amounts of lactate [4]. A hydraulic retention time of 5 days was used throughout the study.

Isolation and Characterization Two different media were used for the enumeration and isolation of aerobic and facultative anaerobic bacteria. Medium PYL-A consisted of peptone, 5 g liter-'; trypticase, 5 g liter '; yeast extract, 10 g liter-'; salt solution [9], 4 ml liter-t; hemin, 5 mg liter-'; vitamin K, 0.001%; agar, 20 g liter-t; and sodium lactate, 10 g liter -~. Medium MI consisted of yeast extract, 4 g liter-'; sodium lactate, 10 g liter-'; KH2PO4, 1.6 g liter-'; K2HPO4, 3.2 g liter-'; NH4C1, 0.5 g liter-l; CaC12, 0.2 g liter-t; MgC12, 0.2 g liter-'; and agar, 20 g liter-'. All media were prepared aerobically and the pH set at 7.0. Digester fluid was sampled at the upper portion of the digester as well as at the outlet. The

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samples were serially diluted, plated on PYL-A and M1 media and incubated at 37"C for 48 hours, after which the colonies were counted. Plates were also incubated anaerobically, in a Gaspak jar, within 1 hour of pouring the media. Seventy-five colonies were randomly picked from the plates, checked for purity, and freeze-dried. Cellular morphology was determined by phase contrast microscopy of live cells and by bright field microscopy of gram's-stained preparations. Motility was determined by observing wet mounts prepared from 24 hour old PYL-A broth cultures incubated at 22~ and 37"C. API 20E and API 50CH microtube kits, and where necessary, the API 20 NE system, were used to determine Phenotypic characters according to the instructions of the manufacturer. Standard API abbreviations for tests are used in this paper. The following tests were also performed: oxidase test, nitrate reduction, growth on McConkey's agar, oxidation and fermentation of glucose in Hugh and Leifson's ~edium, catalase test, growth on Cristal Violet agar (2 ppm), and the ability to produce diffusible Pigment on King, Ward, and Raney's (KWR) medium [6]. The production of volatile fatty acids (VFA) in PYL-A medium was determined by gas chro9matographic analysis of 48-hour-old broth cultures incubated with and without shaking the flasks. VFA were determined using a Hewlett Packard chromatograph model 5830A equipped with a ttame ionization detector and a column (1.8 m x 1.5 mm ID) packed with Porapak Q (80--100 mesh). The column temperature was 195~ and detector temperature 250~ with an inlet temperature of 210"C. Nitrogen was used as the carrier gas [3]. The following reference strains were included in the study: Acinetobacter calcoaceticus ATCC 23055, Achromobacter zerosis ATCC 14780, Aeromonas h.ydrophila ATCC 9071, Alcaligenes faecalis ATCC 8750, Citrobacterfreundii ATCC 8090, Enterobacter aerogenes ATCC 13048, Enterobacter cloacae ATCC 13047, Escherichia coil ATCC 11775, Klebsiella pneumonia ATCC 9997, Pseudomonas aeruginosa ATCC 10145, Pseudomonas fluorescens ATCC 13525, Streptococcus faecalis ATCC 19433, and Streptococcus durans University of the Orange Free State culture collection (UOFSCC) 166. The similarities between the cultures were calculated using the similarity coefficient (SsM) of Sokal and Michener [16], and isolates were clustered by the unweighted pair group method.

Results and Discussion In t h i s s t u d y t h e a v e r a g e t o t a l a e r o b i c a n d f a c u l t a t i v e c o u n t s f r o m t h e l i q u i d phase of the fixed-bed anaerobic digester conducted on PYL-A and M I agar were 16.59 • 106 a n d 26.45 x 106 m l - J, r e s p e c t i v e l y . T h e a v e r a g e c o u n t s f r o m the u p p e r p o r t i o n o f t h e d i g e s t e r c o n t e n t s w e r e l o w e r (7% a n d 17% for P Y L - A a n d M 1, r e s p e c t i v e l y ) t h a n t h o s e f r o m t h e d i g e s t e r effluent. T h i s s m a l l difference, h o w e v e r , w a s n o t s i g n i f i c a n t a n d c o u l d b e a r e f l e c t i o n o f t h e g o o d m i x i n g c a u s e d b y e s c a p i n g gas b u b b l e s . D e s p i t e t h e fact t h a t t h e s e c o u n t s w e r e o b t a i n e d from a fixed-bed anaerobic digester, they correspond favorably with those o b t a i n e d b y o t h e r w o r k e r s [8, 19] o n c o n v e n t i o n a l t y p e d i g e s t e r s . I t is i n t e r e s t i n g to n o t e t h a t t h e n u t r i t i o n a l l y p o o r e r o f t h e t w o m e d i a g a v e t h e h i g h e s t a v e r a g e COunts. N o d i f f e r e n c e s i n t h e b a c t e r i a l t y p e i s o l a t e d f r o m e a c h m e d i u m c o u l d be d e t e c t e d . T h e 75 s t r a i n s i s o l a t e d f r o m t h e l i q u i d p h a s e o f t h e d i g e s t e r a n d t h e r e f e r e n c e s t r a i n s w e r e c l u s t e r e d , u s i n g t h e SSM coefficient, i n t o s e v e n m a j o r g r o u p s ( A G ) w i t h a final s i m i l a r i t y l e v e l o f 75%. T h e s i m i l a r i t i e s b e t w e e n t h e c l u s t e r s are i l l u s t r a t e d in a s i m p l i f i e d d e n d r o g r a m (Fig. 1). A l l t h e s t r a i n s in c l u s t e r s A, B, E, F , a n d G w e r e f o u n d to b e g r a m - n e g a t i v e a n d t h o s e in c l u s t e r s C a n d D gram-positive. C l u s t e r A , t h e l a r g e s t c l u s t e r , c o n t a i n e d 4 8 % o f t h e i s o l a t e s a n d five r e f e r e n c e

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SUBPERCENTAGE S I M I L A R I T Y CLUSTER 70 80 90 100 !

!

STRAIN

1

Enterobacter cloacae

ATCC 13047

A2

A3

A4

I

Klebsiella pneumonia ATCC 9997 A5 A6

A7 A8 B

Enterobacter aerogenes ATCC 13048 CitrobacterJ)'eundii ATCC 8090 Escherichia coli ATCC 11775 Aeromonas hydrophila ATCC 9071

C1 Streptococcus faecalis ATCC 19433

C2 D

E1

Streptocuccus durans UOFSCC 166 Pseudomonas aemginosa ATCC 10145

Alcaligettes faecalis ATCC 8750

E E3 E4

E6 F I

I

I

Pseudomonas fluorescens ATCC 13525 Achromobacter zerosis ATCC 14780 Acinetobacter calcoaceticus ATCC 23055

G

Fig. 1. A simplified d e n d r o g r a m s h o w i n g the p h e n o t y p i c similarities o f aerobic a n d facultative anaerobic bacteria isolated f r o m an anaerobic digester fed with cheese whey.

strains. All the strains in this cluster were gram-negative fermentative rods, catalase positive, and oxidase negative and reduced nitrate, indicating a relationship to the Enterobacteriaceae. Subcluster A 1 consisted o f five isolates and the type strain E. cloacae A T C C 13047. As a result o f the A P 1 2 0 E identification

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and the inclusion o f the type strain, this subcluster was thus taken as representative o f E. cloacae. Subclusters A2, A3, and A4 were identified with the API 20E system as Klebsiella spp. All isolates ofsubcluster A3 were, however, indole positive while those in subcluster A2 and A4 were indole negative. This character is used to distinguish between K. oxytoca and K. pneumoniae [14]. Both subclusters A2 and A4, which included the reference strain, were identified with the API system as K. pneumoniae. Strains o f A2 and A4 could probably be considered as biovars o f K. pneumoniae [14]. 1'4o reference strains o f K. oxytoca (A3) were available, and the identification of the API 20E system was accepted. Cluster A6 consisted o f two isolates and the type strain, Citrobacter freundii ATCC 8090. These stains were all motile and utilized citrate. The API profile also identified the cluster as C. freundii. Only two isolates were clustered 9in A8 with an API profile identification o f E. sakazakii. Both strains formed yellow pigmented colonies on agar media. Cluster B with the reference strain was found to represent A. hydrophila. Cluster C consisted o f 13 strains o f gram-positive streptococci which grouped with a single gram-positive nonsporing rod (D) at a similarity level of 82%. Ten of the streptococci as well as the lactobacillus were isolated from the plates incubated in the anaerobic Gaspak jars. They were, however, able to grow aerobically on PYL-A plates. The 10 isolates in C 1 clustered with the reference strain S. faecalis at a similarity level of 93%. The other 3 streptococci formed a more diffuse subcluster with S. durans UOFSCC 166. Further characterization, of the 13 streptococci, according to the classical scheme of Sherman [6], confirmed that these isolates belong to the enterococci. All the isolates in cluster E were aerobic, gram-negative, nonfermentative, and catalase positive. The isolates in E1 clustered with P. aeruginosa ATCC 10145 and were identified by the API 20 NE system as P. aeruginosa. Subcluster E5, which also contained the type strain, was identified as P. fluorescens. Isolate E4, which was A D H negative, was identified by the API 20 NE system as Pseudomonas acidovorans. All the strains, including the type strain in subcluster E2, produced an alkaline reaction in Hugh and Leifson tubes and were identified as A. faecalis by the API 20 NE system. The three isolates in subcluster E3 were identified by the API 20 NE system as Acinetobacter calcoaceticus vat. lwoffi. Morphologically they corresponded with the Acinetobacter genus description [14]. However, the similarity of subcluster E3 to the type strain A. calcoaceticus ATCC 23055 (cluster F) was not very good. It is known that a nUmber o f phenotypic groups exist in this genus [14]. The only strain in cluster G was identified, according to the API 20E system, as Enterobacter agglomerans. This was the only enterobacterium that did not cluster in Group A. This strain was found to have fastidious growth requirements and was thus not further characterized. In this study it was found that the streptococci were mainly isolated from Plates that were incubated anaerobically. The nonfermentative gram-negative rods (cluster E), on the other hand, were only isolated from plates that were incubated aerobically. Although none o f the isolates in cluster E were able to grow anaerobically, 45% of the strains were isolated from the digester effluent. This indicates that aerobic bacteria such as Pseudomonas, Alcaligenes, and "4cinetobacter are able to survive, over a long period, the conditions found in anaerobic digesters.

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All the isolates in clusters A, B, a n d C were able to produce acetic acid f r o m lactate in P Y L - A b r o t h after a 48 h o u r incubation. Acetic acid concentrations ranged f r o m 134 m g liter- J to 2,272 m g liter- 1. T h e 13 streptococci p r o d u c e d 428 m g liter - j on average, while no acetic acid was p r o d u c e d by the single culture o f cluster D. N o acetic acid or only trace a m o u n t s were o b s e r v e d in b r o t h cultures o f the n o n f e r m e n t a t i v e bacteria o f cluster E. Only small concentrations o f p r o p i o n i c acid a n d other fatty acids (100 m g liter - j ) were observed. Considering the substrate used, the f e r m e n t a t i v e bacteria, except for clusters D a n d G, could possibly be considered functional bacteria in view o f the fact that a b o u t 70% o f the m e t h a n e p r o d u c e d f r o m organic m a t t e r is produced via acetic acid [ 15, 21 ]. T h e aerobic a n d facultative anaerobic m i c r o b i a l species present in the anaerobic digester used in this study were similar to those reported by other workers [5, 18, 20]. Thus, even w h e n using a relatively bacteria-free s u b s t r a t e - c o m p a r e d with fecal w a s t e s - - t h e r e are still aerobic and facultative bacteria present in the digester which f o r m a part o f the bacterial population. It has been generally a s s u m e d [8] that the aerobic a n d facultative bacteria are " p a s sengers" a n d o f doubtful i m p o r t a n c e in the functioning o f the digester. It has been shown [7, 8] that in fecal waste digesters the types o f p r e d o m i n a n t facultative bacteria in the digesters reflect the p r e d o m i n a n t types present in the fecal waste. Thus, it can be concluded that s o m e o f the facultative bacteria are continuously being a d d e d to these digesters as part o f the feedstock. In this study, the whey used as substrate did not at a n y stage contain the bacterial groups found in the fixed bed anaerobic reactor. T h e original source o f these bacteria was p r o b a b l y the sewage sludge used as i n o c u l u m at the start-up o f the digester. Since no further sludge was a d d e d a n d the aerobic a n d facultative bacteria were still present in relatively large n u m b e r s , after m o r e than 6 m o n t h s c o n t i n u o u s operation at an H R T o f 5 days, it is difficult to explain their presence as " p a s s e n g e r s . " A n o t h e r reason why the aerobic and facultative bacteria h a v e been considered "'passengers" is that they h a v e no cellulolytic or other hydrolase activities [7, 8] that would contribute to the degradation o f the energy sources in digesters treating d o m e s t i c and agricultural wastes. H o w e v e r , in this study, where the digester was fed with deproteinated, p r e f e r m e n t e d whey o f which the p r e d o m inant energy sources are lactate a n d lactose, the f e r m e n t a t i v e bacteria do not need cellulolytic or hydrolase activities for the b r e a k d o w n o f the p r i m a r y substrate. F u r t h e r m o r e , it was found that the m e t a b o l i c p r o d u c t s o f the facultative bacteria, w h e n grown anaerobically with lactate as carbon source, could be used by the m e t h a n o g e n s . D u e to the nature o f the substrate used and the m e t a b o l i c products f o r m e d , they m a y h a v e a functional role in the p r i m a r y digester reactions. Future research on the acidogenic p o p u l a t i o n should, therefore, be focused on their functional role, as well as their practical aspects such as synchronization with the terminal phases o f anaerobic digestion.

Acknowledgments. The financial assistance of the Central Research Fund of the University of the Orange Free State and the South African Dairy Foundation is appreciated. The technical assistance of E. W. Verwey and the friendly collaboration of P. J. Jooste is acknowledged.

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Characterization of aerobic and facultative anaerobic bacteria from the liquid phase of an anaerobic fixed-bed digester treating a cheese whey substrate.

Bacterial counts on the liquid phase of an anaerobic, fixed-bed digester, treating a deproteinated, prefermented cheese whey substrate, were conducted...
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