Characterization of some anaerobic bacteria from the liquid phase of a mesophilic anaerobic digester fed with a prefermented cheese whey substrate

Bacterial counts on the contents of an anaerobic fixed-bed digester receiving a whey substrate were conducted using the modified roll tube technique. Average anaerobic counts after 48 hours incubation on lactate containing media were 3.12 × 10⁹ and 3.7 × 10⁹ ml^–1, respectively. These counts were between 140 and 190 times higher than aerobic counts on the same media. Seventy-four strains from both media were isolated and characterized, and the relationship between the organisms was calculated according to the similarity coefficient of Sokal and Michener [20]. The organisms were clustered using the unweighted pair group method and the results were presented in the form of a simplified dendrogram. The isolates clustered in three major groups (A, B, and C) at a similarity level of 76%. A small diffuse group of five organisms was also found. The organisms in two (A and B) of the major clusters were obligate anaerobes. Cluster A represented 34% of the isolates, cluster B represented 50%, and the facultative streptococci in cluster C, 11%. The isolates in clusters A and B could only tentatively be identified as members of the generaBacteroides andPeptostreptococcus, respectively. The isolates could not be positively identified.     

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 on two different media under aerobic and facultative conditions. Average counts of 16.6×10⁶ and 26.5×10⁶ ml^–1 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 of Sokal and Michener [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, mainlyEnterobacter, Klebsiella, andCitrobacter, withKlebsiella as the predominant genus; gram-positive bacteria, mainly enterococci; and gram-negative nonfermentive rods of the generaPseudomonas, Alcaligenes, andAcinetobacter. All the enterobacteria and enterococci were able to produce acetic acid, an intermediate in methanogenesis.     

Identification of the major anaerobic end products ofCellulomonas sp. (ATCC 21399)

Summary A novel approach of aerobic growth followed by anaerobic growth was used to identify the anaerobic end products of the facultative organismCellulomonas sp. (ATCC 21399) utilizing cellulose as the substrate. The organism was found to produce an equimolar mixture of ethanol and acetic acid as the two carbon end products.     

The isolation and characterization of the aerobic endospore-forming bacteria present in the liquid phase of an anaerobic fixed-bed digester,while treating a petrochemical effluent

Sixty-nine gram-positive endospore-forming rods were isolated from the liquid phase of an anaerobic digester, while treating a fatty acid-rich petrochemical effluent. These strains, including eight reference strains, were characterized and the similarities between the different strains were calculated using Sokal and Michener's simple matching coefficient. Phenotypic characteristics, determined by the API 20E and API 50CHB galleries, other biochemical tests, and morphological characteristics, were used for the numerical analysis. The strains were grouped into 12 (five major and seven minor) clusters. Nine of the clusters were positively identified asBacillus pumilus, B. subtilis, B. sphaericus, B. laterosporus, B. brevis, B. cereus, B. coagulans, B. megaterium, andB. circulans. Three clusters could not be identified using Gordon's classical system or the API identification system. Most of the aerobic endospore-forming rods (72%) utilized both acetic and propionic acid, and 17% utilized acetic acid as carbon source, but only under aerobic conditions. A small percentage of the strains studied (11%) was unable to utilize the fatty acids present in the petrochemical substrate, and no explanation could be given as to how they obtained their carbon source. Seventy-eight percent of the strains did not show growth in anaerobic agar. It was possible that sufficient oxygen, required for growth by these members of the genusBacillus, was introduced by the substrate. Since ample time had been allowed for population selection, their presence indicates that these aerobic strains can survive, grow, and compete in the digester environment but their relative importance and role in the primary digestion reactions is not clear.     

Isolation of hydrogen-producing bacteria from granular sludge of an upflow anaerobic sludge blanket reactor

H₂-producing bacteria were isolated from anaerobic granular sludge. Out of 72 colonies (36 grown under aerobic conditions and 36 under anaerobic conditions) arbitrarily chosen from the agar plate cultures of a suspended sludge, 34 colonies (15 under aerobic conditions and 19 under anaerobic conditions) produced H₂ under anaerobic conditions. Based on various biochemical tests and microscopic observations, they were classified into 13 groups and tentatively identified as follows: From aerobic isolates,Aeromonas spp. (7 strains),Pseudomonas spp. (3 strains), andVibrio spp. (5 strains); from anaerobic isolates,Actinomyces spp. (11 strains),Clostridium spp. (7 strains), andPorphyromonas sp. When glucose was used as the carbon substrate, all isolates showed a similar cell density and a H₂ production yield in the batch cultivations after 12h (2.24–2.74 OD at 600 nm and 1.02–1.22 mol H₂/mol glucose, respectively). The major fermentation by-products were ethanol and acetate for the aerobic isolates, and ethanol, acetate and propionate for the anaerobic isolates. This study demonstrated that several H₂ producers in an anaerobic granular sludge exist in large proportions and their performance in terms of H₂ production is quite similar.     

Isolation and characterization of xylose- and xylan-utilizing anaerobic bacteria

By enrichment with xylose, nine mesophilic strains of anaerobic bacteria were obtained from various sources. Two isolates appear to belong to the genus Eubacterium. Six other strains belong to the genus Clostridium. Three of the isolated strains utilized larch wood xylan. The percentage of utilization of xylose and xylan and the yield of fermentation end products — viz. acetic acid and butyric acid-are equivalent to that of Clostridium acetobutylicum (ATCC 824) and reported thermophilic strains.     

Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost

In a search for potential ethanologens, waste compost was screened for ethanol-tolerant thermophilic microorganisms. Two thermophilic bacterial strains, M5EXG and M10EXG, with tolerance of 5 and 10% (v/v) ethanol, respectively, were isolated. Both isolates are facultative anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negative, Gram-negative rods that are capable of utilizing a range of carbon sources including arabinose, galactose, mannose, glucose and xylose and produce low amounts of ethanol, acetate and lactate. Growth of both isolates was observed in fully defined minimal media within the temperature range 50–80°C and pH 6.0–8.0. Phylogenetic analysis of the 16S rDNA sequences revealed that both isolates clustered with members of subgroup 5 of the genus Bacillus. G+C contents and DNA–DNA relatedness of M5EXG and M10EXG revealed that they are strains belonging to Geobacillus thermoglucosidasius. However, physiological and biochemical differences were evident when isolates M5EXG and M10EXG were compared with G. thermoglucosidasius type strain (DSM 2542^T). The new thermophilic, ethanol-tolerant strains of G. thermoglucosidasius may be candidates for ethanol production at elevated temperatures.     

Ecology and taxonomy of chitinoclasticCytophaga and related chitin-degrading bacteria isolated from an estuary

A total of 103 strains of estuarine, Chitinoclastic bacteria isolated from water, and sediment samples collected from the upper Chesapeake Bay, including 17 freshwater and 11 seawater isolates, were subjected to numerical taxonomy analysis. The isolates included 44 yellow-orange pigmented strains classified asCytophaga-like bacteria (CLB) of theCytophagaceae. Salt requirement of the strains ranged from tolerance to 1% NaCl to an absolute requirement for NaCl, with 1% NaCl satisfying this requirement. The largest phenon consisted of facultatively anaerobic, oligo-nitrophilic, and flexirubin pigment-producing freshwater and estuarine isolates, and included reference strains of bothCytophaga johnsonae Stanier andCytophaga aquatilis Strohl and Tait. Other phena, containing a smaller number of strains, comprised marine and estuarine isolates which did not produce flexirubin pigments, and required organic nitrogen for growth and for production of chitinolytic enzymes. Salt-requiring, flexirubin pigment-producing, chitin-degrading strains were, on occasion, isolated from estuarine samples and represented phena found in estuaries. Most of theCytophaga isolates, as well as chitin-degrading species not of the genusCytophaga that were isolated from Chesapeake Bay, clustered in phena representing previously described species of aerobic, zymogenic, chitinoclastic bacteria. When the frequency of occurrence of features related to environmental parameters, viz., pH, salinity, temperature range of growth, and growth on media lacking organic nitrogen, was calculated, ecological groupings of strains in the 2 major phena of CLB could be distinguished among the estuarine, chitin-degrading bacteria.     

The <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> species is abundant among hydrogen-producing facultative anaerobic bacteria in Lake Averno sediment

Lake Averno sediment was used to isolate the facultative anaerobic bacteria having the potential for H₂ production. Twenty-five out of 35 isolates recovered from the sediment sample produced hydrogen under anaerobic conditions from glucose with yields ranging from 0.1 to 0.49 mol H₂/mol glucose. Identification based on 16S rRNA gene sequence analysis revealed that most of them belong to the Firmicutes group, with a prevalence of the Paenibacillus polymyxa species. Seven distinct genomic fingerprints among the 11 P. polymyxa isolates were obtained using the random amplified polymorphic DNA (RAPD) technique. Glucose fermentation by P. polymyxa isolates was investigated. Glucose was totally consumed after 3 days of fermentation. The fermentation products were hydrogen (0.18–0.47 mol H₂/mol glucose), ethanol (0.1–0.5 mol ethanol/mol glucose), and 2,3-butanediol (0.1 mol 2,3-butanediol/mol glucose). Lower amounts of acetic, butyric, formic, lactic, and propionic acids were detected. All metabolic data concerning P. polymyxa isolates were analyzed by cluster analysis to reveal similarities and/or differences with clustering based on RAPD profiles. Despite the high metabolic similarity among almost all P. paenibacillus isolates, results of cluster analyses of metabolic and genetic data do not match completely.     

Propionibacterium species diversity in anaerobic digesters

Phenotypic characteristics of 60 strains ofPropionibacterium isolated from anaerobic hybrid digesters treating landfill leachate and a baker's yeast factory effluent were analysed using numerical taxonomy. With the use of the S_SM similarity coefficient, 92% of the anaerobic digester strains were grouped in eight major clusters. The isolates were identified by relating them to specific type strains and comparison of phenotypic characteristics. These clusters were equated with the classical speciesP. acidipropionici, P. freudenreichii, P. jensenii andP. thoenii using the current classification system. Some of the digester isolates were identified to specifies level using the current identification system, but based on overall similarity they were clustered among members of another species. Furthermore, the data indicated that there was low similarity between the digester isolates and the type strains ofP. jensenii andP. thoeni. A hypothesis is presented as to the role of these propionic acid-producing bacteria during the granulation process found in anaerobic digesters.     

Isolation and identification of threeCellulomonas spp. from forest soils

Three stains of cellulose-degrading, aerobic, mesophilic bacteria were isolated from forest soils and, from their cultural, biochemical, and physiological characteristics, they were identified as members of the genusCellulomonas. Unusual biochemical characteristics, e.g. urea hydrolysis, were observed in two isolates. These characteristics have not previously been reported for cellulomonads and may prove to be significant for characterization ofCellulomonas spp. The isolates were able to use urea as a N source in cellulose fermentation. All three strains were motile, with one to four peritrichous flagella observed. Amino acid and polysaccharide composition of the cell walls of the three isolates were identical.     

Understanding aerobic/anaerobic metabolism in Caldibacillus debilis through a comparison with model organisms

Caldibacillus debilis GB1 is a facultative anaerobe isolated from a thermophilic aero-tolerant cellulolytic enrichment culture. There is a lack of representative proteomes of facultative anaerobic thermophilic Bacillaceae, exploring aerobic/anaerobic expression. The C. debilis GB1 genome was sequenced and annotated, and the proteome characterized under aerobic and anaerobic conditions while grown on cellobiose. The draft sequence of C. debilis GB1 contains a 3,340,752 bp chromosome and a 5,386 bp plasmid distributed over 49 contigs. Two-dimensional liquid chromatography mass spectrometry/mass spectrometry was used with Isobaric Tags for Relative and Absolute Quantification (iTRAQ) to compare protein expression profiles, focusing on energy production and conversion pathways. Under aerobic conditions, proteins in glycolysis and pyruvate fermentation pathways were down-regulated. Simultaneously, proteins within the tricarboxylic acid cycle, pyruvate dehydrogenase, the electron transport chain, and oxygen scavenging pathways showed increased amounts. Under anaerobic conditions, protein levels in fermentation pathways were consistent with the generated end-products: formate, acetate, ethanol, lactate, and CO₂. Under aerobic conditions CO₂ and acetate production was consistent with incomplete respiration. Through a direct comparison with gene expression profiles from Escherichia coli, we show that global regulation of core metabolism pathways is similar in thermophilic and mesophilic facultative anaerobes of the Phylum Proteobacteria and Firmicutes.     

Gram-negative respiratory bacteria which cause ropy milk constitute a distinct cluster within the genusAcinetobacter

Eight strains of Gram-negative respiratory bacteria that produce ropiness in milk were characterized with respect to morphology, physiology, and DNA base composition. All were found to belong to one of the major groups of the genusAcinetobacter. The results of tests for the utilization of 50 different compounds as sole source of carbon and energy were used for a numerical analysis in which 20 strains ofAcinetobacter characterized by other investigators were included. The eight ropy-milk strains, and one previously described, were found to cluster together. The characteristics differentiating the members of this cluster from other strains ofAcinetobacter were determined.     

Aerobic and anaerobic metabolism in a facultative anaerobe Ep01 lacking cytochrome, quinone and catalase

Abstract A facultative anaerobe, strain Ep01 produced a mixture of pyruvate, formate, acetate and ethanol from glucose anaerobically, and acetate and pyruvate aerobically. Cell extract of anaerobic-grown cells contained active pyruvate formatelyase, aldehyde dehydrogenase and alcohol dehydrogenase, while cell extract of aerobic grown cells contained an active pyruvate dehydrogenase system, NaDH oxidase and NADH peroxidase. Levels of acetate kinase and phosphate acetyltransferase activities were not significantly different in cells grown under either condition. Based on the metabolic products and the emzyme activities, we propose the presence of two metabolic pathways in strain Ep01, namely, a pathway to form formate, acetate and ethanol under anaerobic conditions, and a pathway to form under aerobic conditions. This explains why strain Ep01 can grow well both under strictly anaerobic conditions and well-aerated conditions.     

The aerobic and anaerobic microbiology of pustular acne lesions

Specimens from 32 pustular acne lesions that were inoculated on media supportive for the growth of aerobic and anaerobic bacteria showed bacterial growth. Only aerobic or facultative bacteria were recovered in 15 (47%) specimens, only anaerobic bacteria in 11 (34%) specimens, and mixed aerobic and anaerobic bacteria in 6 (18%) specimens. A total of 57 isolates, 31 anaerobes (1.0 per specimen) and 26 aerobes (0.8 per specimen) were recovered. The predominant isolates were Staphylococcus sp. (19 isolates), Peptostreptococcus sp. (15), and Propionibacterium sp. (10). Twelve (37.5%) of the comedones yielded only one organism. This retrospective study highlighted the polymicrobial nature of over two-thirds of culture positive pustular acne lesions and suggests the potential for pathogenic role of aerobic and anaerobic organisms other than P. acnes and Staphylococcus sp. in acne vulgaris.     

Isolation and characterization of two glycerol-fermenting clostridial strains from a pilot scale anaerobic digester treating high lipid-content slaughterhouse waste

Two obligately anaerobic bacterial strains were isolated from the contents of a pilot scale, anaerobic digester treating slaughterhouse waste with a high protein and lipid content. The isolates, LIP1 and MW8, were characterized as spore-forming, Gram-positive rods, capable of fermenting glycerol. Isolate LIP1 was also observed to be lipolytic and was able to hydrolyse tallow and olive oil. Both isolates grew optimally at 37 degrees C and formed either acetate and formate (LIP1), or acetate and butyrate (MW8), as major glycerol fermentation products. Both isolates produced ethanol as the major reduced fermentation end-product. Neither MW8 nor LIP1 had growth and metabolism inhibited by the addition of stearic acid at concentrations normally considered bactericidal. Analysis of the 16S rRNA gene sequences, in conjunction with the phenotypic data, confirmed that the isolates are members of the genus Clostridium (sensu lato), clustering with species of clostridial clusters I (MW8) and XIVa (LIP1).     

The role of encapsulation in the pathogenesis of anaerobic gram-positive cocci

The pathogenicity of 22 anaerobic and facultative Gram-positive cocci (AFGPC) was investigated by inoculating them into mice and determining their ability to cause subcutaneous abscesses. Only 11 heavily encapsulated isolates (greater than 50% of the cells were encapsulated) induced abscesses. However, when the other 11 isolates were injected with Bacteroides sp. or facultative and aerobic bacteria, abscesses were formed in 8 of the 11 combinations. The AFGPC recovered from the mixed infections contained many encapsulated cells. Encapsulation also occurred in cocci injected with capsular material or with Formalin-killed cells of Klebsiella pneumoniae or capsule-positive Bacteroides sp. After acquisition of capsules, these strains could induce abscesses on reinoculation in mice.     

Isolation of cellulolytic mesophilic clostridia from a municipal solid waste digestor

Ten obligately anaerobic, cellulolytic mesophilic bacteria were isolated from a municipal solid waste digestor used for biogas production. The isolates were rod-shaped, spore-forming bacteria in anaerobic conditions, and stained Gram-positive in young cultures, and hence were identified asClostridium. Small regular translucent and unpigmented colonies were observed on cellulose plates. The strains were gelatinase-negative, hydrolyzed esculin and starch, and fermented xylose and arabinose. The lecithinase, lipase, and indole tests were negative. The major fermentation products from cellulose included ethanol and acetate. The morphological and other biochemical characteristics indicated that these clostridia did not correspond to any previously described species. All the strains produced high activities of extracellular cellulases in cellulose media and degraded paper. Offprint requests to: L. Benoit.     

Aerobic and anaerobic metabolism of Listeria monocytogenes in defined glucose medium.

A defined medium with glucose as the carbon source was used to quantitatively determine the metabolic end products produced by Listeria monocytogenes under aerobic and anaerobic conditions. Of 10 strains tested, all produced acetoin under aerobic conditions but not anaerobic conditions. Percent carbon recoveries of end products, typified by strain F5069, were as follows: lactate, 28%; acetate, 23%; and acetoin, 26% for aerobic growth and lactate, 79%; acetate, 2%; formate, 5.4%; ethanol, 7.8%; and carbon dioxide, 2.3% for anaerobic growth. No attempt to determine carbon dioxide under aerobic growth conditions was made. The possibility of using acetoin production to assay for growth of L. monocytogenes under defined conditions should be considered.     

Isolation and characteristics ofTreponema zuelzerae nov. spec., an anaerobic,free-living spirochete

Summary An anaerobic, free-living spirochete was isolated from mud. The organism can be cultivated in ordinary nutrient media, e.g. yeast extract-glucose. End products of glucose fermentation are: lactic, acetic, and succinic acids, CO₂, and H₂. In cultures of this organism spheroid bodies are formed, especially during the stationary growth phase. Studies of slide cultures showed that these bodies, when inoculated in fresh medium, do not give rise to spiral cells whereas a rapid multiplication of normal cells, also present in the inoculum, was observed. Since the organism is serologically related toTreponema pallidum, it has been assigned to the genusTreponema, and is here described asTreponema zuelzerae nov. spec. Part of this work was carried out at the Hopkins Marine Station of Stanford University, Pacific Grove, U.S.A., under a Rockefeller Foundation fellowship. Present address: Laboratorium voor Microbiologie, Wageningen, the Netherlands.