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,facultative anaerobic,and anaerobic bacteria in an acidogenic phase reactor and their metabolite formation

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 generaKlebsiella, Enterobacter, Escherichia andAeromonas. Isolates of the second group were gram-positive streptococci similar toStreptococcus lactis. The strict anaerobic isolates also clustered into two main groups with strains of cluster A being identified as members of the genusFusobacterium while strains in cluster B were members of the genusBacteroides. Hypothetical mean organisms were calculated for each cluster and used in further culture studies. The major products of 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.     

Characterization of Subsurface Bacteria Associated with Two Shallow Aquifers in Oklahoma

The bacterial microflora of two shallow aquifers (saturated subsurface zones) in Oklahoma was characterized by direct observation with light and electron microscopy, by plating, and by examination of colony morphology and distribution. Isolated bacterial strains were also examined. Total cell counts varied only slightly (2.9 × 10⁶ to 9.8 × 10⁶ g [dry wt]⁻¹) from sample to sample, whereas colony counts varied widely (6.3 × 10² to 6.5 × 10⁶ CFU g [dry wt]⁻¹). Colony counts on nutritionally rich media were lower than on low-nutrient media, especially in samples from the saturated zone. The variety of colony types growing on nutritionally rich media decreased with increasing depth and saturation. Colony counts of anaerobic bacteria also decreased with depth but were at least 100-fold lower than aerobic counts on most media. Cell morphologies of bacteria grown aerobically on plates included short rods, cocci, and actinomycete-like forms. Direct light microscopic observation of sediments revealed short, rod-shaped, and coccoid bacterial cells; endospores, actinomycete spores, and eucaryotic forms were not observed by light microscopy. Electron microscopic observation of bacteria released from the samples revealed that 85 to 90% of them were coccoid, gram-positive, Arthrobacter-like organisms, some of which were dividing or contained completed division septa; other types of gram-positive and gram-negative bacteria were present in lower numbers. Isolated bacterial strains were able to grow on both nutritionally rich and low-nutrient media. A higher proportion of gram-negative organisms was isolated than gram-positive organisms. Most of the isolates were capable of storing polyphosphate, poly-β-hydroxybutyrate, or polysaccharide. The results of this study suggest that the microbial population of these two shallow aquifers is dominated by aerobic, nutritionally versatile bacteria that can subsist on low concentrations of organic compounds without forming specialized resting cells. Other types of microorganisms, such as facultatively anaerobic bacteria and microeucaryotes, may also be present, but they represent only a small fraction of the microflora.     

Colonization of the Botanical Environment by Klebsiella Isolates of Pathogenic Origin

Growth, survival, and pathogenicity of Klebsiella growing in and on environmental foci were examined. Total coliforms present in raw wastes from pulp mills were in excess of 10⁵/ml, and 60 to 80% were Klebsiella. Fecal coliform counts ranged from 10¹ to 10⁵/ml. Klebsiella isolates from industrial effluents and a variety of human and bovine mastitis origins multiplied in pulp waste and commonly exceeded 10⁶ cells per ml. Pathogenic isolates also multiplied in dilute aqueous extracts of sawdust to comparable levels. Klebsiella strains from vegetable surfaces and human infections grew rapidly on the surfaces of potatoes and lettuce and exceeded 10³ organisms per g of surface peel and leaf after a 24h incubation at room temperature. After 7 weeks on potatoes stored at 5°C, some 10 to 30% of the day 1 Klebsiella counts were recoverable. Three Klebsiella isolates of pathogenic origin were passed 45 times through sterile pulp effluent (270 generations), and mean lethal dose levels in mice were periodically monitored. In two instances, a significant decrease in virulence was noted after 15 to 26 passes (90 to 156 generations). The third culture, of bovine mastitis origin, retained its original mean lethal dose value. Botanical milieu provided suitable habitats for the multiplication and colonization of Klebsiella isolates of disease origins in the same manner as indigenous isolates. Aquatic environments polluted with botanical material served as potential reservoirs for perpetuating the growth and spread of opportunistic Klebsiella pathogens that may ultimately colonize animals, humans, and aquatic organisms.     

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.     

Phenotypical divergences between populations of soil bacteria isolated on different media

Bacterial strains were randomly isolated from soil using three different media with glucose (TG), Tryptone Soya Broth (TTS), and succinate (TS) as carbon sources. Plate counts obtained were 12.0×10⁷, 4.5 ×10⁷, and 1.5×10⁷ g^–1 soil dry weight, respectively. The strains were characterized phenotypically by the API 20B test system. A cluster analysis of all isolates revealed 40 biotypes at 80% similarity, 23 in TG, 29 in TTS, and 27 in TS. Each of the 10 most common biotypes contained 10 to 2.5% of the isolates, and 17 biotypes contained one or two isolates. The common biotypes were unevenly distributed among the isolates from the different media. About 20% of the isolates from TG and TTS were unique for the particular medium, whereas among the isolates from TS, about 60% were unique. Thirty percent of the isolates belonged to biotypes that were common to all three populations. All media gave approximately the same high diversity measured as Shannon index and Equitability, indicating no direct correlation between plate count and diversity.     

Effect of selective media on recovery of obligately anaerobic gram-negative rods from human faeces

Total numbers and gross composition of the anaerobic human faecal flora were compared using non-selective and selective media. Combinations of selective agents to suppress the gram-negative part of the flora such as vancomycin and neomycin, vancomycin and kanamycin, or kanamycin and bile were found to reduce total numbers of recovered obligately anaerobic gram-negattive rods by 50–75%. With reference to experiments with penicillin as selective agent, underlying mechanisms for this phenomenon are discussed. It is concluded that selective media should not be used for quantitative enumeration of anaerobic gram-negative rods from the faecal flora.     

Isolation and characterization of an anaerobic syntrophic benzoate-degrading bacterium from sewage sludge

An anaerobic, motile, gram-negative, rod-shaped bacterium is described which degrades benzoate in coculture with an H₂-utilizing organism and in the absence of exogenous electron acceptors such as O₂, SO ₄ ⁼ or NO ₃ ^- . The bacterium was isolated from a municipal primary, anaerobic sewage digestor using anaerobic roll-tube medium with benzoate as the main energy source and in syntrophic association with an H₂-utilizing sulfate-reducing Desulfovibrio sp. which cannot utilize benzoate or fatty acids apart from formate as energy source. The benzoate utilizer produced acetate (3 mol/mol of substrate degraded) and presumably CO₂ and H₂, or formate from benzoate. In media without sulfate and with Methanospirillum hungatei (a methanogen that utilizes only H₂–CO₂ or formate as the energy source) added, 3 mol of acetate and 0.7 mol of methane were produced per mol of benzoate and CO₂ was probably formed. Low numbers of Desulfovibrio sp. were present in the methanogenic coculture and a pure coculture of the benzoate utilizer with M. hungatei was not obtained. The generation times for growth of the sulfate-reducing and methanogenic cocultures were 132 and 166h, respectively. The benzoate utilizer did not utilize other common aromatic compounds, C ₃ ^- –C₇ monocarboxylic acids, or C₄-C₆ dicarboxylic acids for growth, nor did it appear to use SO ₄ ⁼ , NO ₃ ^- or fumarate as alternative electron acceptors. Addition of H₂ inhibited growth and benzoate degradation.     

Thermophilic anaerobic bacteria which ferment hemicellulose: characterization of organisms and identification of plasmids

Seven thermophilic anaerobic bacteria which ferment xylan were isolated from natural geothermal features in the western United States. Typically, these strains were Gram-negative non-sporeforming rods with an unusual double-layered cell wall which resembled that observed in Thermobacteroides acetoethylicus. The strains differed from known thermophilic anaerobes in their ability to utilize a very wide variety of carbohydrates and in their ability to grow in a chemically-defined medium and/or at pH 3.5. Four of the strains contained cryptic plasmids of 1.2 or 1.5×10⁶ daltons. The taxonomic characteristics of the strains are discussed in terms of their relatedness to those of Thermoanaerobium, Thermoanaerobacter, and Thermobacteroides species.Contribution No. 3222 of the Central Research and Development Department     

Microbiological characterization of a fuel-oil contaminated site including numerical identification of heterotrophic water and soil bacteria

Seven soil samples and seven groundwater samples from a site contaminated with fuel-oil were investigated using several chemical and microbiological techniques. In soil samples, 500 to 7,500 mg/kg of total hydrocarbons were found. These samples contained no n-alkanes but iso- and branched chain alkanes. No polychlorinated biphenyls could be detected. Microbiological investigations included estimations of total cell counts, viable cell counts on different media, and numbers of methylotrophic, denitrifying, sulphate reducing, anaerobic (with the exception of methanogenic organisms), and hydrocarbon degrading bacteria. Viable and hydrocarbon degrading bacteria were found in all samples. A total of 1,366 pure cultures was characterized morphologically and physiologically and identified by numerical identification using a data base of more than 4,000 reference strains. Groundwater samples were dominated by gram-negative bacteria of the generaPseudomonas, Comamonas, Alcaligenes, andAcinetobacter, which were also found in soil samples. In addition, more grampositive bacteria belonging to the generaArthrobacter, Nocardia, andBacillus could be isolated from soil samples.     

Xylanolytic anaerobic thermophiles from Icelandic hot-springs

Anaerobic enrichment cultures inoculated with neutral and alkaline (pH 7.0–9.0) sediment and biomat samples from hot-springs in Hveragerdi and Fluir, Iceland, were screened for growth on beech xylan from pH 8.0 to 10.0 at 68° C: no growth occured in cultures above pH 8.4. Five anaerobic xylanolytic bacteria were isolated from enrichment cultures at pH 8.4; all five microbes were Gram-positive rods with terminal spores, and produced CO₂, H₂, acetate, lactate and ethanol from xylan and xylose. One of the isolates, strain A2, grew from 50 to 75° C, with optimum growth near 68° C, and from pH 5.2 to 9.0 with an optimum between 6.8 and 7.4. Taxonomically, strain A2 was most similar to Clostridium thermohydrosulfuricum. At pH 7.0, the supernatant xylanases of strain A2 had a temperature range from 50 to 78° C with an optimum between 68 and 78° C. At 68° C, xylanase activity occurred from pH 4.9 to 9.1, with an optimum from pH 5.0 to 6.6. At pH 7.0 and 68° C, the K _m of the supernatant xylanases was 2.75 g xylan/l and the V _max was 2.65 × 10^–6 kat/l culture supernatant. When grown on xylose, xylanase production was as high as when grown on xylan. Correspondence to: B. K. Ahring     

Bacteria associated with deep,alkaline, anaerobic groundwaters in Southeast Washington

The microbial diversity in two deep, confined aquifers, the Grande Ronde (1270 m) and the Priest Rapids (316 m), Hanford Reservation, Washington, USA, was investigated by sampling from artesian wells. These basaltic aquifers were alkaline (pH 8.5 to 10.5) and anaerobic (Eh –200 to –450 mV). The wells were allowed to free-flow until pH and Eh stabilized, then the microflora was sampled with water filtration and flow-through sandtrap methods. Direct microscopic counts showed 7.6 × 10⁵ and 3.6 × 10³ bacteria ml^–1 in water from the Grande Ronde and Priest Rapids aquifers, respectively. The sand filter method yielded 5.7 × 10⁸ and 1.1 × 10⁵ cells g^–1 wet weight of sand. The numbers of bacteria did not decrease as increasing volumes of water were flushed out. The heterotrophic diversity of these bacterial populations was assessed using enrichments for 20 functional groups. These groups were defined by their ability to grow in a matrix of five different electron acceptors (O₂, Fe(III), NO₃ ^–, SO₄ ^2–, HCO₃ ^–) and four groups of electron donors (fermentation products, monomers, polymers, aromatics) in a mineral salts medium at pH 9.5. Growth was assessed by protein production. Culture media were subsequently analyzed to determine substrate utilization patterns. Substrate utilization patterns proved to be more reliable indicators of the presence of a particular physiological group than was protein production. The sand-trap method obtained a greater diversity of bacteria than did water filtration, presumably by enriching the proportion of normally sessile bacteria relative to planktonic bacteria. Substrate utilization patterns were different for microflora from the two aquifers and corresponded to their different geochemistries. Activities in the filtered water enrichments more closely matched those predicted by aquifer geochemistry than did the sand-trap enrichments. The greatest activities were found in Fe(III)-reducing enrichments from both wells, SO₄-reducing enrichments from the Grande Ronde aquifer, and methanogenic enrichments from the Priest Rapids aquifer. Organisms from these aquifers may be useful for high-pH bioremediation applications as well as production of biotechnological products. These organisms may also be useful for modeling potential reactions near buried concrete, as might be found in subsurface waste depositories. Offprint requests to: T. O. Stevens.     

Isolation of Nitrogen Fixing Bacteria from Fungus Termites

Biological nitrogen fixation by the microorganisms in the gut of termites is one of the singularly important symbiotic processes, since termites invariably thrive on nitrogen poor diet. Two isolates of free living aerobic and facultative anaerobic N fixing bacteria were obtained from the guts of fungus cultivating termite, Macrotermes sp. Among the total bacterial isolates from termite gut, the per cents of N fixing aerobes viz., Azotobacter and Beijerinckia spp were 49% and 37% from the salivary gland while facultative N fixing anaerobe viz., Klebsiella and Clostridium contributed (51% and 93%). The free living aerobic bacteria were identified as Azotobacter spp (19 x 10⁴ CFU mL^‐1) and Beijerinckia (13.2 x 10⁴ CFU mL^‐1) from the salivary gland of the termite; interestingly, foregut, mid gut and hind gut registered a low population of these bacteria. The isolates of Azotobacter were smooth, glistening, vicid in nature, rods, gram negative and cyst forming. Isolates of Beijerinckia sp. produced copious slime, tenacious, rods, gram negative with no cyst formations. Both the isolates emitted green fluorescence and produced acid. Facultative N fixing anaerobes were harbored in the hind gut. The isolates were identified as Klebsiella (20 x 10⁴ CFU mL^‐1) and Clostridium pasteurianum 39.1 x 10⁴ CFU mL^‐1. Klebsiella were straight rods arranged singly or in pairs, non‐motile, gram negative, whereas Clostridium pasteurianum was viscoid, motile with terminal spores. A positive correlation was observed between the extractable polysaccharides of these isolates and soil aggregation. The aggregates formed by the isolates increased soil aeration, porosity, water holding capacity and helped in better plant growth. Thus, the gut microflora of termite, apart from harnessing nitrogen from the atmosphere, also helps improving soil fertility.     

Cytophaga xylanolytica sp. nov., a xylan-degrading,anaerobic gliding bacterium

Gliding bacteria attached in masses to, and dominated the fermentation of, xylan powder in methanogenic and sulfidogenic enrichments from various freshwater sediments. Isolates of such bacteria were all gram-negative, slender rods (0.4×4-24 m) that formed no endospores, microcysts or fruiting bodies. Representative strain XM3 was a mesophilic, aeroduric anaerobe that grew by fermentation of mono-, di-, and poly-saccharides (but not cellulose) in a mineral medium containing up to 3% NaCl. However, CO₂/HCO _inf3 ^sup- was required in media for consistent initiation of growth. Fermentation products included acetate, propionate, succinate, CO₂, and H₂. Xylan-grown cells had xylanase and various glycosidase activities that were mainly or almost entirely cell-associated, respectively. Strain XM3 was weakly catalase positive, but oxidase negative; it possessed sulphonolipids and carotenoid, but not flexirubin, pigments; and its total cellular fatty acids were dominated by C_15:0 anteiso (75%), n (13%) and iso (2%) isomers. Strain XM3 had 45.5 mol% G+C in its DNA, and partial sequencing of its 16S rRNA placed XM3 within the Bacteroides-Flavobacterium phylogenetic group. Similar strains were isolated from marine sediments. Strain XM3 is herewith proposed as the type strain of the new species, Cytophaga xylanolytica. Results, which are discussed in terms of our current concept of the genus Cytophaga, suggest that the importance of C. xylanolytica in anaerobic biopolymer decomposition has not been fully appreciated.Dedicated to Professor Norbert Pfennig, in whose laboratory this project was initiated and who recently retired after more than four decades of contributions to microbiology     

Fermentation of maleate by a gram-negative strictly anaerobic non-spore-former,Propionivibrio dicarboxylicus gen. nov., sp. nov.

Three strains of maleate-fermenting anaerobic curved rods were isolated in pure culture from anaerobic freshwater mud samples. Among the isolates, strain CreMal1 was studied in detail. It was a mesophilic non-sporing gram-negative strict anaerobe, and grew not only on maleate but also on fumarate and l-malate, producing propionate and acetate stoichiometrically as end products. Succinate was an intermediate in the degradation of maleate. Nitrate, sulfate, and other sulfur compounds were not utilized as electron acceptors. It had 61 mol% guanine-plus-cytosine content, but possessed a single polar flagellum and did not utilize carbohydrates and lactate, unlike the genus Selenomonas. Therefore, strain CreMal1 is described as a member of Propionivibrio dicarboxylicus gen. nov., sp. nov., in the family Bacteroidaceae. Strain CreMal1 was deposited as type strain in the Japan Collection of Microorganisms and in Deutsche Sammlung von Mikroorganismen.Dedicated to Professor Dr. Norbert Pfennig on the occasion of his 65th birthday     

Characterization of anaerobic non-spore-forming bacteria in municipal sewage sludge and integrated paper-mill waste water

Summary Forty-three isolates of Gram-negative, mesophilic, non-spore-forming anaerobic cellulolytic bacteria were obtained from (i) an anaerobic reactor treating waste water from an integrated paper mill and (ii) an anaerobic sewage-sludge digestor. These isolates were studied for carbohydrate fermentation and fermentation products. By numerical techniques, 22 isolates could be placed in two groups: group A (10 isolates) and group B (12 isolates). The isolates belonging to group A showed degradation of filter paper in 2–7 days. They were slightly-curved long rods and similar toBacteroides cellulosolvens andAcetivibrio cellulolyticus. Acetic acid was produced as major product. The bacteria also produced ethanol, isobutanol, pyruvic and lactic acids. Group B strains degraded filter paper in 4–5 weeks. They were short rods and produced propionic, lactic, succinic and acetic acids as fermentation products. The remaining 21 isolates could disintegrate filter paper in 2–5 weeks. They showed variable fermentation patterns, both as to fermentable carbohydrates and end products. Except for one isolate, which showed obvious similarity toButyrivibrio fibrisolvens, the isolate differed distinctly from reference strains of ruminai origin.

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Caractérisation des bactéries anaérobies non-sporulantes dans la boue résiduaire de station d'épuration des eaux domestiques (boues d'égout) et dans l'eau résiduaire d'une usine intégrale de pâte à papier

Résumé 43 souches de bactéries cellulolytiques Gram-négatives, mésophiles, non-sporulantes, et anaérobies ont été isolées à partir (A) d'un réacteur anaérobie traitant l'eau résiduaire d'une usine intégrale de pâte à papier et (B) de digesteurs anaérobies de boues d'égout. Ces souches ont été étudiées quant à leurs propriétés de fermenter les hydrates de carbone et quant à leurs produits de fermentation. Des techniques numériques ont permis de classer 22 de ces souches dans deux groupes: le groupe A (10 souches) et le groupe B (12 souches). Les souches appartenant au groupe A dégradaient le papier filtre en 2 à 7 jours. Elles se présentaient sous la forme de longs bâtonnets curvilignes et ressemblaient àBacteroides cellulosolvens et àAcetivibrio cellulolyticus. Le metabolite majeur était l'acide acétique. Elles produisaient aussi de l'éthanol, de l'iso-butanol, et des acides pyruvique et lactique. Les souches appartenant au groupe B dégradaient le papier filtre en 4 à 5 semaines. Elles se présentaient sous la forme de bâtonnets courts et produisaient, comme métabolites, les acides propionique, lactique, succinique et acétique. Les 21 souches restantes dégradaient le papier filtre en 2 à 5 semaines. Elles présentaient des profils de fermentation variables tant en ce qui concernait les hydrates de carbone fermentés que les produits finaux. A part une souche qui présentait une similitude évidente avecButyrivibrio fibrisolvens, ces souches différaient de façon marquée d'avec les souches de référence du rumen.

     

Physicochemical and microbiological characteristics of groundwater from observation wells of a deep radioactive liquid waste repository

A radioactive liquid waste repository was found to be the habitat of a rich microbial community with a high catabolic potential. Groundwater from a depth of 162–189 m contained aerobic saprotrophic and anaerobic fermentative, sulfate-reducing, and denitrifying bacteria. Nitrate-reducing bacteria residing in this groundwater were isolated in pure cultures. Based on the results of their physiological studies, 16S rRNA sequencing, and phylogenetic analysis, the microorganisms isolated were ascribed to one phylogenetic branch, the γ-subclass of gram-negative bacteria. Among six isolates, four belonged to the genusAcinetobacter, whereas two others belonged to the generaComamonas andAeromonas. The data obtained indicate that the microflora of the repository can exert a certain effect on the chemical composition of the formation fluids and bearing rocks, as well as on the migration of radionuclides     

Characterization of anaerobic heterotrophic bacteria isolated from freshwater lake sediments.

Strict anaerobic culture techniques were used to quantitatively and qualitatively evaluate the anaerobic heterotrophic bacteria present at the sediment-water interface of hyperutrophic Wintergreen Lake (Augusta, Mich.). Anaerobic plate counts remained constant from March through December, 1973, ranging from 2.4 X 10(6) to 5.7 X 10(6) organisms/g (dry weight) of sediment. The isolatable bacteria represented a small percentage of the total microbial community, which was shown by direct microscopic counts to be 2.0 X 10' organisms/g (dry weight) of sediment during June and July. Bacteria of the genus Clostridium dominated the isolates obtained, accounting for 71.8% of the 960 isolates examined. A single species, Clostridium bifermentens, comprised 47.7% of the total. Additional bacterial groups and the percentage in which they were isolated included: Streptococcus sp. (10.8%), unidentified curved rods (9.5%y, gram-positive nonsporing rods (5.6%), and motile gram-negative rods (1.9%). Temperature growth studies demonstrated the ability of all the isolates to grow at in situ sediment temperatures. Gas-liqid radiochromatography was used to determine the soluble metabolic end products from [U-14C]glucose and a U-14C-labeled amino acid mixture by representative sedimentary clostridial isolates and by natural sediment microbial communities. At in situ temperatures the natural sediment microflora produced soluble fermentative end products characteristic of those elaborated by the clostridial isolates tested. These results are considered strong presumptive evidence that clostridia are actively metabolizing in the sediments of Wintergreen Lake.     

Microbiological quality assessment of Moroccan camel's milk and identification of predominating lactic acid bacteria

Samples of camel's milk collected from different zones of Morocco were analysed to evaluate their microbiological quality and to identify predominating lactic acid bacteria (LAB). The following average colony-forming units (c.f.u.s) of aerobic total count, enterococci, faecal and total coliforms, LAB, yeasts,Staphylococcus aureus and spores of sulphite-reducing clostridia were recorded: 6.2 × 10⁷, 2.9 × 10⁴, 1.6 × 10⁴, 7.0 × 10⁶, 1.0 × 10⁷, 3.8 × 10⁴, 1.3 × 10⁵ and 6.0 c.f.u./ml, respectively. The enumeration results were markedly variable and coliforms were not detected in 1 ml of some samples. Bacteriological identification revealed a definite dominance of enterococci with Enterococcus faecalis as the main representative species. Besides Enterococcus, other genera including Pediococcus (28.2%), Streptococcus (4%), Lactococcus (8%) and Leuconostoc(1%) were isolated on de Man, Rogosa and Sharp (MRS) agar.     

Air-side ammonia stripping coupled to anaerobic digestion indirectly impacts anaerobic microbiome

Air-side stripping without a prior solid–liquid phase separation step is a feasible and promising process to control ammonia concentration in thermophilic digesters. During the process, part of the anaerobic biomass is exposed to high temperature, high pH and aerobic conditions. However, there are no studies assessing the effects of those harsh conditions on the microbial communities of thermophilic digesters. To fill this knowledge gap, the microbiomes of two thermophilic digesters (55°C), fed with a mixture of pig manure and nitrogen-rich co-substrates, were investigated under different organic loading rates (OLR: 1.1–5.2 g COD l⁻¹ day⁻¹), ammonia concentrations (0.2–1.5 g free ammonia nitrogen l⁻¹) and stripping frequencies (3–5 times per week). The bacterial communities were dominated by Firmicutes and Bacteroidetes phyla, while the predominant methanogens were Methanosarcina sp archaea. Increasing co-substrate fraction, OLR and free ammonia nitrogen (FAN) favoured the presence of genera Ruminiclostridium, Clostridium and Tepidimicrobium and of hydrogenotrophic methanogens, mainly Methanoculleus archaea. The data indicated that the use of air-side stripping did not adversely affect thermophilic microbial communities, but indirectly modulated them by controlling FAN concentrations in the digester. These results demonstrate the viability at microbial community level of air side-stream stripping process as an adequate technology for the ammonia control during anaerobic co-digestion of nitrogen-rich substrates.