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 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.     

A comparison of two strains of the anaerobic ciliate Trimyema compressum

Two strains of the anaerobic ciliate Trimyema compressum, isolated from different habitats, were compared. The cytoplasm of the ciliates contained hydrogenosome-like microbodies and methanogenic bacteria; the latter were lost during continued cultivation. In addition both strains harbored a non-methanogenic endosymbiont, which was lost in strain K. The ciliates lacked cytochromes, cytochrome oxidase and catalase but contained superoxide dismutase. Hydrogenase activity could be demonstrated only in strain N. In monoxenic culture strain K needed sterols as growth factors. The cells of both strains reacted similarly with respect to oxygen tolerance (up to 0.5 mg O₂/l), inhibition of growth by cyanide and azide, and resistance to antimycin A. Only cells of strain N showed growth inhibition by chloramphenicol. It is concluded that Trimyema compressum is an anaerobic, microaerotolerant organism, its microbodies show more resemblance to hydrogenosomes than to mitochondria.     

The Survival of antibiotic resistant and sensitiveEscherichia coli strains during anaerobic digestion

Summary Total counts ofEscherichia coli were followed during anaerobic digestion of pig slurry laboratory scale digesters at 37° C. Counts decreased rapidly during anaerobic digestion. Antibiotic resistant strains in most cases appeared to be more persistent in anaerobic digesters than sensitiveE. coli strains as calculated from the decimal decay rates.     

Oxygen tolerance of anaerobic bacteria isolated from human feces

The large bowel intestinal flora of mammals is made up mostly of O₂-intolerant anaerobic microorganisms which are irreversibly damaged by brief exposure to air. The aim of our work was to investigate the effect of atmospheric O₂ on human intestinal anaerobic microorganisms. Thirty O₂-intolerant bacterial strains that reached 100% mortality after 120 min of air exposure were isolated. Ten of these strains were tested for their atmospheric O₂ sensitivity as a function of air exposure time; all tested microorganisms showed a similar mortality trend on exposure to air. In fact, 50% of cells survive, on the average, after 4–5 min of atmospheric O₂; this percentage decreases to 3–5% after only 20 min, and after 40 min only one cell in a thousand survives; all strains reached 100% mortality in a time range of 100–120 min. The strains examined were identified as belonging to the generaEubacterium, Peptostreptococcus, andCoprococcus.     

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.     

Bioaugmenting anaerobic digestion of biosolids with selected strains of Bacillus, Pseudomonas, and Actinomycetes species for increased methanogenesis and odor control

The objective of this study was to evaluate the effects of bioaugmenting anaerobic biosolids digestion with a commercial product containing selected strains of bacteria from genera Bacillus, Pseudomonas, and Actinomycetes, along with ancillary organic compounds containing various micronutrients. Specifically, the effects of the bioaugment in terms of volatile solids destruction and generation and fate of odor-causing compounds during anaerobic digestion and during storage of the digested biosolids were studied. Two bench-scale anaerobic digesters receiving primary and secondary clarifier biosolids from various full-scale biological wastewater treatment plants were operated. One of the digesters received the bioaugment developed by Organica Biotech, while the other was operated as control. The bioaugmented digester generated 29% more net CH₄ during the 8 weeks of operation. In addition, the average residual propionic acid concentration in the bioaugmented digester was 54% of that in the control. The monitoring of two organic sulfide compounds, methyl mercaptan (CH₃SH) and dimethyl sulfide (CH₃SCH₃), clearly demonstrated the beneficial effects of the bioaugmentation in terms of odor control. The biosolids digested in the bioaugmented digester generated a negligible amount of CH₃SH during 10 days of post-digestion storage, while CH₃SH concentration in the control reached nearly 300 ppm_v during the same period. Similarly, peak CH₃SCH₃ generated by stored biosolids from the bioaugmented digester was only 37% of that from the control.     

Quantitative and qualitative transitions of methanogen community structure during the batch anaerobic digestion of cheese-processing wastewater

Qualitative and quantitative shifts in methanogen community structure, associated with process performance data, were investigated during the batch anaerobic digestion of a cheese-processing wastewater, whey permeate. Denaturing gradient gel electrophoresis (DGGE) and real-time PCR techniques were applied to obtain qualitative and quantitative microbial data sets, respectively, based on methanogen 16S rRNA genes. Throughout the operation, dynamic variations in both qualitative and quantitative community structures were observed, with repeated shifts in dominance between the aceticlastic Methanosarcinaceae (suggested mainly by the detection of a Methanosarcina-like population) and the hydrogenotrophic Methanomicrobiales (suggested mainly by the detection of a Methanofollis-like population). This trend corresponded well to the diauxic utilization of acetate and longer-chain fatty acids (C₃–C₆), mainly propionate. Joint-plot non-metric multidimensional scaling (NMS) analysis demonstrated that the qualitative and quantitative community shifts had significant correlations with the composition of residual organic acids and the methane production rate, respectively. This suggests the potential use of microbial community shift analysis as an indicative tool for diagnosing anaerobic digestion processes. The results suggest that more attention should be directed to quantitative, as well as qualitative, approaches for a better understanding of anaerobic digestion, particularly in terms of biogas production efficiency, under dynamic and transitional conditions.     

Physiological adaptations and tolerance towards higher concentration of selenite (Se+4) in Enterobacter sp. AR-4, Bacillus sp. AR-6 and Delftia tsuruhatensis AR-7

Environmental contamination with selenium is a major health concern. A few bacterial strains have been isolated that can transform toxic selenite to non-toxic elemental selenium only at low concentrations (0.001–150 mM) in recent past. We have previously reported isolation and characterization of few selenite-tolerant bacterial strains. These strains were found to be resistant to selenite at (300–600 mM) concentrations. In the present study we have characterized some physiological adaptations of strains Enterobacter sp. AR-4, Bacillus sp. AR-6 and Delftia tsuruhatensis AR-7 during exposure to higher concentration of selenite under aerobic and anaerobic environments. Adaptive responses are largely associated with alteration of cell morphology and change in total cellular fatty acid composition. Interestingly, electron microscopy studies revealed substantial decrease in cell size and intracellular deposition of Se⁰ crystals when reduction is carried out under aerobic conditions. On the other hand, cell size increased with adhesion of Se⁰ on cell surface during anaerobic reduction. Fatty acid composition analysis demonstrated selective increase in saturated and cyclic fatty acids and decrease in unsaturated ones during aerobic transformation. Changes observed during anaerobic transformation were in surprising contrast as indicated by total absence of saturated and cyclic fatty acids. Results presented here provide evidences for putative occurrence of two distinct mechanisms involved in tolerance towards higher concentrations of selenite utilization under aerobic and anaerobic conditions. Further, prior exposure to higher concentration of Se⁺⁴ enabled rapid adaptation indicating role of inducible system in adaptation.     

Methanethiol utilization and sulfur reduction by anaerobic halophilic saccharolytic bacteria

A number of sulfur compounds were tested as sulfur sources for the growth of three strains of anaerobic halophilic saccharolytic bacteria isolated from hypersaline water bodies of the eastern Crimea (USSR). Dithionite and sulfite at 1 mM concentration completely inhibited the growth of all strains. Methanethiol turned out to be the sole sulfur source for growth ofHalobacteroides strains in the defined medium with glucose and leucine. Methanethiol also stimulated growth of cultures in the complex medium with yeast extract. TheHaloincola saccharolytica Z-7787 appeared to be capable of methanethiol formation from methionine. All organisms studied were capable of heterotrophic sulfur reduction, producing up to 13 mM H₂S, but no evidence that they gain energy from the process has been obtained. The extremely halophilicHalobacteroides lacumaris may participate in sulfidogenesis at the high salinity (20–30% NaCl). The ecological position of haloanaerobes in halophilic community is discussed.     

An evaluation of potential hydrogen sulfide adsorbents for the protection of palladium catalyst in anaerobic culture systems

Hydrogen sulfide is produced by many anaerobic bacteria and can irreversibly damage the palladium catalyst used for oxygen removal in anaerobic jars and cabinets. The present study used direct measurements of oxygen concentration to quantify catalyst activity following exposure to H₂S and volatile fatty acids in the presence of eight potential H₂S adsorbents. Most adsorbents were unsatisfactory, but two compounds not tested previously afforded full protection against the effects of H₂S alone and provided protection in the presence of volatile fatty acids. The investigation demonstrated the importance both of selecting an adsorbent suitable for use in anaerobic conditions and of heating anaerobic jar catalysts in order to maintain activity.     

Pyruvate fermentation in light-grown cells ofRhodospirillum rubrum during adaptation to anaerobic dark conditions

Pyruvate fermentation inRhodospirillum rubrum (strains F₁, S₁, and Ha) was investigated using cells precultured on different substrates anaerobically in the light and than transferred to anaerobic dark conditions. Pyruvate formate lyase was always the key enzyme in pyruvate fermentation but its activity was lower than in cells which have been precultured aerobically in darkness. The preculture substrate also had a clear influence on the pyruvate formate lyase activity. Strains F₁ and S₁ metabolized the produced formate further to H₂ and CO₂. A slight production of CO₂ from pyruvate, without additional H₂-production, could also be detected. It was concluded from this that under anaerobic dark conditions a pyruvate dehydrogenase was also functioning. On inhibition of pyruvate formate lyase the main part of pyruvate breakdown was taken over by pyruvate dehydrogenase.When enzyme synthesis was inhibited by chloramphenicol, propionate production in contrast to formate production was not affected. Protein synthesis was not significant during anaerobic dark culture. Bacteriochlorophyll. however, showed, after a lag phase, a clear rise.Abbreviations Bchl Bacteriochlorophyll - CoA Coenzyme A - DSM Deutsche Sammlung von Mikroorganismen (Göttingen) - OD optical density - PHBA poly--hydroxybutyric acid - R Rhodospirillum     

A strictly anaerobic nitrate-reducing bacterium growing with resorcinol and other aromatic compounds

With resorcinol as sole source of energy and organic carbon, two stains of gram-negative, nitrate-reducing bacteria were isolated under strictly anaerobic conditions. Strain LuBRes1 was facultatively anaerobic and catalase- and superoxide dismutase-positive. This strain was affiliated with Alcaligenes denitrificans on the basis of substrate utilization spectrum and peritrichous flagellation. Strain LuFRes1 could grow only under anaerobic conditions with oxidized nitrogen compounds as electron acceptor. Cells were catalase-negative but superoxide dismutase-positive. Since this strain was apparently an obligate nitrate reducer, it could not be grouped with any existing genus. Resorcinol was completely oxidized to CO₂ by both strains. Neither an enzyme activity reducing or hydrolyzing the resorcinol molecule, nor an acyl-CoA-synthetase activating resorcylic acids or benzoate was detected in cell-free extracts of cells grown with resorcinol. In dense cell suspensions, both strains produced a compound which was identified as 5-oxo-2-hexenoic acid by mass spectrometric analysis. This would indicate a direct, hydrolytic cleavage of the resorcinol nucleus without initial reduction.     

Photosynthetic electron transport and anaerobic metabolism in purple non-sulfur phototrophic bacteria

Purple non-sulfur phototrophic bacteria, exemplifed byRhodobacter capsulatus andRhodobacter sphaeroides, exhibit a remarkable versatility in their anaerobic metabolism. In these bacteria the photosynthetic apparatus, enzymes involved in CO₂ fixation and pathways of anaerobic respiration are all induced upon a reduction in oxygen tension. Recently, there have been significant advances in the understanding of molecular properties of the photosynthetic apparatus and the control of the expression of genes involved in photosynthesis and CO₂ fixation. In addition, anaerobic respiratory pathways have been characterised and their interaction with photosynthetic electron transport has been described. This review will survey these advances and will discuss the ways in which photosynthetic electron transport and oxidation-reduction processes are integrated during photoautotrophic and photoheterotrophic growth.     

Growth, natural relationships, cellular fatty acids and metabolic adaptation of sulfate-reducing bacteria that utilize long-chain alkanes under anoxic conditions

Natural relationships, improvement of anaerobic growth on hydrocarbons, and properties that may provide clues to an understanding of oxygen-independent alkane metabolism were studied with two mesophilic sulfate-reducing bacteria, strains Hxd3 and Pnd3. Strain Hxd3 had been formerly isolated from an oil tank; strain Pnd3 was isolated from marine sediment. Strains Hxd3 and Pnd3 grew under strictly anoxic conditions on n-alkanes in the range of C₁₂–C₂₀ and C₁₄–C₁₇, respectively, reducing sulfate to sulfide. Both strains shared 90% 16 S rRNA sequence similarity and clustered with classified species of completely oxidizing, sulfate-reducing bacteria within the δ-subclass of Proteobacteria. Anaerobic growth on alkanes was stimulated by α-cyclodextrin, which served as a non-degradable carrier for the hydrophobic substrate. Cells of strain Hxd3 grown on hydrocarbons and α-cyclodextrin were used to study the composition of cellular fatty acids and in vivo activities. When strain Hxd3 was grown on hexadecane (C₁₆H₃₄), cellular fatty acids with C-odd chains were dominant. Vice versa, cultures grown on heptadecane (C₁₇H₃₆) contained mainly fatty acids with C-even chains. In contrast, during growth on 1-alkenes or fatty acids, a C-even substrate yielded C-even fatty acids, and a C-odd substrate yielded C-odd fatty acids. These results suggest that anaerobic degradation of alkanes by strain Hxd3 does not occur via a desaturation to the corresponding 1-alkenes, a hypothetical reaction formerly discussed in the literature. Rather an alteration of the carbon chain by a C-odd carbon unit is likely to occur during activation; one hypothetical reaction is a terminal addition of a C₁ unit. In contrast, fatty acid analyses of strain Pnd3 after growth on alkanes did not indicate an alteration of the carbon chain by a C-odd carbon unit, suggesting that the initial reaction differed from that in strain Hxd3. When hexadecane-grown cells of strain Hxd3 were resuspended in medium with 1-hexadecene, an adaptation period of 2 days was observed. Also this result is not in favor of an anaerobic alkane degradation via the corresponding 1-alkene. Received: 25 June 1998 / Accepted: 29 July 1998     

Formation of dimethylsulfide and methanethiol from methoxylated aromatic compounds and inorganic sulfide by newly isolated anaerobic bacteria

Formation of gas and of methylated sulfur compounds was observed in anaerobic enrichment cultures with methoxylated aromatic compounds as substrates. Via direct dilution of mud samples in defined reduced media supplemented with trimethoxybenzoate or syringate two new strains of anaerobic homoacetogenic bacteria (strain TMBS4 and strain SA2) were obtained in pure culture. Both strains produced dimethylsulfide and methanethiol during growth on methoxylated aromatic compounds. Growth tests and determination of stoichiometries demonstrated that the volatile sulfur compounds were formed from the methyl group at the aromatic ring and the sulfide added as reducing agent to the medium (R = aromatic residue): 2 R - O - CH₃ + H₂ S 2 R - OH + (CH₃)₂SDimethylsulfide was the major organic sulfur compound formed, whereas methanethiol appeared only as intermediate in small quantities. The isolates grew also with trihydroxybenzenes such as gallate, phloroglucinol, or pyrogallol without formation of methylated sulfur compounds. The aromatic compounds were degraded to acetate. The freshwater strain TMBS4 also fermented pyruvate. Other aliphatic or aromatic compounds were not utilized. External electron acceptors (sulfate, nitrate, fumarate) were not reduced. Both strains were mesophilic and formed rod-shaped, non-motile, Gram-negative cells. Spore formation was not observed. Tentatively, both isolates can be affiliated to the genus Pelobacter.Abbreviations TMB 3,4,5-trimethoxybenzoate - MT methanethiol - DMS dimethylsulfide     

Prolonged cultivation of an anaerobic bacterial community producing hydrogen

This paper studies various methods of long-term maintenance of the process of hydrogen evolution during the growth of an anaerobic bacterial community on a starch-containing medium. Continuos fermentation with periodic feeding and effluent removal for 72 days, allow to obtain from 0.10 to 0.23 H₂/l of medium/day. The regime of regular transfer lasted more than 100 days, forming an average of 0.81 l H₂/l of medium/day. The advantages and disadvantages of different methods of microbial hydrogen production during a dark starch fermentation process are presented. From the obtained H₂-producing microbial community, we isolated an anaerobic spore-forming bacterium (strain BF). Phylogenetic analysis of the 16S PNA gene sequence of the new strain showed that according to its genotype it belongs to the Clostridium butyricum species.     

Anammox bacteria enrichment in upflow anaerobic sludge blanket (UASB) reactor

We investigated the anaerobic ammonium oxidation (anammox) reaction in a labscale upflow anaerobic sludge blanket (UASB) reactor. Our aim was to detect and enrich the organisms responsible for the anammox reaction using a synthetic medium that contained low concentrations of substrates (ammonium and nitrite). The reactor was inoculated with granular sludge collected from a full-scale anaerobic digestor used for treating brewery wastewater. The experiment was performed during 260 days under conditions of constant ammonium concentration (50 mg NH₄/⁺-N/L) and different nitrite concentrations (50∼150 mg NO₂-N/L). After 200 days, anammox activity was observed in the system. The microorganisms involved in this anammox reaction were identified as CandidatusB. Anammoxidans andK. Stuttgartiensis using fluorescencein situ hybridization (FISH) method.     

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.