Mierob Ecol (1987) 13:159-168
MICROBIAL ECOLOGY | Springer-VerlagNew York Inc. 1987
Characterization of Aerobic, Facultative Anaerobic, and Anaerobic Bacteria in an Acidogenic Phase Reactor and Their Metabolite Formation W. A. Joubert and T. J. Britz Department of Microbiology, University of the Orange Free State, Bloemfontein, 9300, South Africa
Fifty-two aerobic and facultative anaerobic and 57 anaerobic bacterial isolates were obtained from an acidogenic phase digestion system. These isolates were characterized and the similarities between the different strains were calculated using Sokal and Michener's similarity coefficient. The aerobic and facultative anaerobic strains clustered in two major groups with the strains of the first main group being gram-negative fermentative rods, representing the genera Klebsiella, Enterobacter, Escherichia and Aeromonas. Isolates of the second group were gram-positive streptococci similar to Streptococcus lactis. The strict anaerobic isolates also clustered into two main groups with strains of cluster A being identified as members of the genus Fusobacterium while strains in cluster B were members of the genus Bacteroides. Hypothetical mean organisms were calculated for each cluster and used in further culture studies. The major products o f the continuously fed acidogenic phase reactor were ethanol and acetic, propionic, and butyric acids. In batch cultures, ethanol, acetic acid, diacetyl, and 2,3-butanediol were formed by the strains as major products both under aerobic and anaerobic conditions. The ability of the aerobic and facultative anaerobic strains to be metabolically active under anaerobic conditions indicates a prominent role in acidogenic reactors.
Introduction The anaerobic degradation of organic substrates to methane and carbon dioxide involves four major bacterial groups, of which the acidogenic group is the largest . This group comprises about 90% of the total population in digesters  and is able to degrade polysaccharides to ethanol, fatty acids, carbon dioxide, and hydrogen . Many workers have reported on a range of acidogenic bacteria which have been isolated from anaerobic digesters [2, 25]. The methanogens, which perform the ultimate steps of the anaerobic degradation process, are unable to utilize substrates such as sugars and are therefore dependent upon the metabolic activities of the fermentative bacteria. It has been suggested  that the separation of the acidogenic and methanogenic bacteria may be useful if hydrolysable carbohydrates are to be digested
w . A . Joubert and T. J. Britz
a n a e r o b i c a l l y . T h e b e h a v i o r o f t h e a c i d o g e n i c p h a s e s h a s b e e n s h o w n t o affect the p e r f o r m a n c e o f t h e m e t h a n o g e n i c p h a s e [ 1, 5] i n serial c o n n e c t e d r e a c t o r s . I t is t h e r e f o r e n e c e s s a r y t o o b t a i n m o r e i n f o r m a t i o n a b o u t t h e f a c t o r s t h a t affect the m a i n f e r m e n t a t i o n p r o d u c t s a n d p a t h w a y s o f t h e a c i d o g e n i c p o p u l a t i o n . T h i s c o u l d f u r t h e r m o r e assist in t h e p r e v e n t i o n o f u n w a n t e d m e t a b o l i c p r o d u c t f o r m a t i o n w h i c h c o u l d c a u s e r e a c t o r i n s t a b i l i t i e s [ 1]. S i n c e it h a s b e e n f o u n d t h a t t e m p e r a t u r e , in c o m p a r i s o n w i t h p H , h as a m o r e p r o n o u n c e d effect o n m e s o p h y l i c single stage r e a c t o r r e s p o n s e s , t h e effect o f t h i s p a r a m e t e r o n m e t a b o l i t e f o r m a t i o n w a s i n v e s t i g a t e d in t h i s s t u d y . T h u s t h e m a i n o b j e c t i v e s o f this s t u d y w e r e t o i s o l a t e a n d c h a r a c t e r i z e t h e aerobic, facultative anaerobic, and anaerobic bacteria present in an acidogenic phase reactor. T h e m e t a b o l i c p r o d u c t s o f the v a r i o u s isolates were also determined. T h e differences in f e r m e n t a t i o n p r o d u c t c o m p o s i t i o n occurring in batch c u l t u r e s a n d in t h e c o n t i n u o u s l y f e d r e a c t o r , as i n f l u e n c e d b y t e m p e r a t u r e , w e r e compared.
Materials and M e t h o d s
Reactor An upflow hybrid reactor, combining a fixed film and upflow sludge blanket reactor, was used as the acidogenic phase reactor. The reactor (working volume = 770 ml) had been in continuous operation for 8 months, treating synthetic sucrose substrate (chemical oxygen demand = 9,800 rag/liter) at a hydraulic retention time of 11.0 hours, prior to the first isolations. To determine the fermentation products formed, the reactor was operated at 29~ 31~ 33~ 35 ~ 37~ and 40~ These temperatures were changed successively from 29~ to 40~ and back to 29~ The reactor was operated at a given temperature for 15 reactor volume changes before the fermentation products were determined. Operational parameters were as given by Joubert et al. .
Isolation and Enumeration Aerobic and facultative anaerobic bacteria were isolated from the acidogenic phase reactor (operating temperature = 35"C) fluid with sterile syringes. Isolations were performed six times with two weeks between consecutive isolations. Samples were immediately serially diluted to a 1 x l0 -~ dilution and plated, using a medium consisting of: sucrose, l0 g liter-~; yeast extract, 4 g liter-~; KHzPO4, 1.6 g liter-~; K2HPO4, 3.2 g liter-~; NH4C1, 0.5 g liter-l; CaC12, 0.16 g liter-l; MgCl2, 0.2 g liter-~; and agar, 15 g liter -~. Ten colonies were randomly picked from each plate and purified. All media were set at pH 7.0. Gaspak jars were used for the incubation of facultative anaerobic bacteria. Plates were incubated at 37* for 48 hours. Anaerobic bacteria were isolated nine times from the digester with two weeks between consecutive isolations. Samples taken for the isolation of strict anaerobic acidogenic bacteria (reactor operating temperature = 35"C) were immediately transferred to an anaerobic cabinet and serially diluted to a 1 x l0 -8 dilution. The sucrose medium, to which 0.5 g liter-~ cysteine hydrochloride was added, was anaerobically prepared using the serum bottle modification  of the Hungate technique [11 ]. The vials were incubated anaerobically at 37"C for 48 hours. About seven colonies were anaerobically picked up from each vial, using syringes, and transferred to the sucrose broth. The purity of the isolates was checked after 48 hours by anaerobically streaking on agar plates which were then incubated in the anaerobic cabinet at 37"C for 36 hours.
Bacteria in an Acidogenic Phase Reactor
Characterization The cellular morphology of all isolates was determined by bright field microscopy of gram-stained preparations. Motility was observed on wet mounts of 24-hour-old broth cultures, and stained bacterial flagella [ 19] were observed with bright-field optics. Gram-negative aerobic and facultative anaerobic bacteria were identified using API 20E and 20 NE kits according to the manufacturer's instructions. The standard abbreviations used by the manufacturer of the API kits (API System S.A., La Balme Les Grottes, 38390 Montalieu Vercieu, France) are used throughout this paper. The following tests were also performed: oxidation and fermentation of glucose in Hugh and Leifson's medium, oxidase, catalase, pigment formation on nutrient agar, and endospore formation . Gram-positive bacteria were identified according to Harrigan and McCance . The phenotypic characteristics of anaerobic bacteria were determined using API 20A kits which were inoculated and incubated in an anaerobic cabinet. Anaerobic sucrose broth was inoculated with each isolate and incubated at 37~ for 48 hours. Following incubation and analysis of the medium by high pressure liquid chromatography (HPLC), the ability of the isolates to utilize sucrose was described as positive (strong) or negative (weak). Isolates were grown on PYG-broth to determine the various metabolic end products as described by Holdeman et al. . The production of volatile fatty acids (VFA), alcohols, 2,3-butanediol and diacetyl, were determined using a Hewlett Packard 5830A gas chromatograph equipped with a flame ionization detector (FID) and a glass column (1.8 m x 1.5 m m ID), packed with Porapak Q. The inlet, FID, and column temperatures were set at 180~ 250"C, and 120~ respectively. Nitrogen (55 ml min -1) was used as carrier gas. Sample volumes of 2 #1 were used. The production of lactic, succinic, fumaric, and pyruvic acids were determined on a Hewlett Packard 5830A gas chromatograph, equipped with a DID and a glass column of 1.8 m x 1.5 m m ID. The column was packed with 10% DEGS on DMCS-treated Chromosorb W. The FID and inlet temperatures were 250* and 130~ respectively. The column temperature was programmed to increase from 1100 to 170~ at a rate of 3~ min-l. Nitrogen was used as carrier gas. Methylated  sample volumes (2 gl) were used. Sokal and Michener's  similarity coefficient (SsM) was used to separately calculate the similarities between the aerobic and facultative isolates and the anaerobic isolates. Clustering was Performed using the single linkage cluster analysis [ 18]. Aeromonas hydrophila ATCC 9702, Klebsiella pneumonia A T C C 9997, and Escherichia coil A T C C 11775 were included as reference strains in the clustering of the isolates. The hypothetical median organism (HMO) was calculated for each cluster [ 171. In selecting strains from each subcluster for further study, the primary consideration was to choose strains that were as closely related to the H M O as possible. These were considered centrotype strains.
Culture Studies Sucrose broth (250 ml), in Erlenmeyer sidearm flasks (250 ml), were separately inoculated with the centrotype strains o f the different aerobic and facultative anaerobic clusters. It was decided to perform the culture studies under both aerobic and anaerobic conditions, since it is very unlikely that strict anaerobic conditions will exist throughout any anaerobic reactor. Flasks were aerobically incubated, without shaking, at 37~ for 36 hours and the optical density in each flask was subsequently set at 0.4. Pure culture studies were performed using 20 ml sucrose broth, inoculated with 1 ml standardized inoculum, and stationary incubated at 29 ~ 31 ~ 33 ~ 35 ~ 37 ~ and 400C. Broth for multiculture studies was inoculated with a 1 ml standardized inoculum of each culture. Optical densities were read at two hour intervals until a stationary phase persisted. Sample Volumes (3 ml) ware taken in midlog and stationary phases. Metabolic products were determined with a gas chromatograph. Culture studies of the anaerobic isolates were performed under strict anaerobic conditions using the same method.
W.A. Joubert and T. J. Bdtz
Results and Discussion
Clustering and Identification The 52 aerobic, facultative anaerobic isolates and three reference strains were grouped into two major clusters (A and B) with a final similarity level of 77%. Strains o f cluster A were all found to be gram-negative, fermentative rods while those in cluster B were gram-positive streptococci. The strains in group AI constituted 43% of the aerobic and facultative anaerobic strains. This group was identified as Klebsiella oxytoca by means o f the API Profile Index. The reference strain, Klebsiella pneumonia ATCC 9997, was a single unclustered strain (A2) which had a 93% similarity level with the strains in group AI. The strains in group A3 were identified, by means of the API system, as Enterobacter cloacae. Since no reference strain was available, this identification was accepted. A single unclustered strain (A4) was also identified as Enterobacter cloacae but was lysine decarboxylase-negative and produced acid from rhamnose. Strains grouped in subcluster A5, constituting 13% of the aerobic and facultative anaerobic isolates, were identified as strains o f the genus Aeromonas and had a 93% similarity to the reference strain, Aeromonas hydrophila ATCC 9071. The strains o f subcluster A6, which constituted only 6% of the isolates, were identified as Escherichia coli by means of the API system. The reference strain, Escherichia coli ATCC 11775, was also grouped in this cluster and showed a similarity o f 90% to the other strains in this subcluster. The strains o f cluster B 1 were gram-positive, catalase-negative, nonmotile streptococci which comprised 13% of the isolated aerobic and facultative anaerobic strains. These strains grew well under facultative anaerobic conditions, but showed limited growth under aerobic conditions. Strains were characterized according to Harrigan and McCance  and were found to resemble Strepto-
coccus lactis. The 57 strict anaerobic isolates were grouped into two clusters (A and B) with a final similarity level of 64%. The isolates grouped in subcluster A1 comprised 63% o f the total strict anaerobic isolates. Strains in this group were found to be gram-negative, strict anaerobic rods which did not hydrolyze esculin or produce indole. Strains were anaerobically grown on PY G broth and the metabolic products analyzed gas chromatographically. Butyric and lactic acids were produced as major products, while only small amounts of acetic acid were produced. When using the API 20A Profile Index, no positive identification could be reached. These strains were eventually identified, according to keys of Holdeman et al. , as Fusobacterium plauti. However, when the keys in Bergey's Manual of Systematic Bacteriology  were used, the strains were found to correspond to the characteristics o f Fusobacterium russii. Two unclustered strains, which had an 80% similarity to group A1, could not be identified with the usual keys [10, 15]. A total o f 33% of the strict anaerobic isolates were found to be grouped in subcluster B 1. These strains were gram-negative rods which grew well in PYG
Bacteria in an Acidogenic Phase Reactor
200~ E "--,150q
Fig. 1. The effect of varying temperature on metabolite formation, from a sucrose substrate, using a constant substrate pH and loading rate. (x7 acetic acid;  butyrie acid; 9 caproic acid; O ethanol; 9 formic acid; v propionic acid.)
broth at temperatures between 30 ~ and 400C. Strains showed a weak acid production from both glucose and lactose and did not produce acid from maltose or starch. These strains p r o d u c e d succinic and acetic acids as m a j o r fermentation products when grown on P Y G broth. N o gas p r o d u c t i o n was detected. Strains o f this cluster were identified as Bacteroides succinogenes by means o f Bergey's Manual o f Systematic Bacteriology [ 15]. T h r e e unidentified gram-positive, strict anaerobic rods were also isolated, but could not be identified using the standard systems [10, 15].
Fermentation Product Composition The results given in Fig. 1 illustrate the variation in metabolic products f o r m e d in the acidogenic phase reactor at different operational temperatures. These results were obtained from the continuously fed reactor using a synthetic sucrose substrate. T e m p e r a t u r e had a m a r k e d effect on metabolite formation, and definite concentration o p t i m a were detected for the various metabolic products (Fig. 1). In the temperature range investigated, ethanol and acetic, propionic, and butyric acids were m a j o r products, whereas caproic and formic acids were formed as m i n o r products. The o p t i m u m concentrations o f acetic (2,015 mg l i t e r l ) , priopionic (1,790 mg liter-~), caproic (370 mg liter-l), and formic acid (195 mg liter -l) and ethanol (1,405 mg liter -t) were obtained at 35 ~ 32 ~ 33 ~ 35 a, and 340C respectively. It has been shown  that certain acidogenic products, in a two-phase reactor, can enhance m e t h a n e p r o d u c t i o n in the serially connected m e t h a n o genie phase reactor. F r o m Fig. 1 it can be concluded that the f o r m a t i o n o f energetically favorable acidogenic products can be achieved by manipulation o f the reactor temperature. This should be possible also i f the isolated strains can be manipulated individually or in combinations to yield the desired prodUcts. In preparation o f the inocula for the d e t e r m i n a t i o n o f pure culture metabolic products, it was found that, although the centrotype strains o f the Aeromonas and Streptococcus subclusters were metabolically active in the sucrose broth,
W.A. Joubert and T. J. Britz