The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, beta-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20-44.5 degrees C and at pH values 5.2-7.4 with optimal growth at 37-41.5 degrees C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5.0, the optimum temperature was 40 degrees C for the endoglucanase and 50 degrees C for the xylanase. Both enzyme activities were inhibited at 70 degrees C Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

Characterization of 10 mesophilic cellulolytic clostridia isolated from a municipal solid waste digestor

By hybridization experiments with three cloned fragments carrying cellulase genes ofClostridium cellulolyticum, we tried to differentiate 10 cellulolytic mesophilic clostridia, isolated from a municipal solid waste digestor. On the basis of hybridization experiments, three major groups were found among the 10 isolates. The two endoglucanase genes,cel CCA andcel CCB ofC. cellulolyticum, hybridized with nine strains of our isolates, suggesting homology and widespread distribution of these genes. Withcel CCA the strain A31 exhibited a different pattern. In contrast to these nine strains, the strain A11 was found to share no or very weak homology with these two probes, which indicated that this strain of cellulolytic clostridia possesses nonidentical cellulase complex. None of these new strains hybridized withnif genes, indicating that these clostridia did not appear to be nitrogen-fixing bacteria. With other biochemical characteristics, we found that these bacteria appeared to be different from the presently known mesophilic cellulolytic clostridia.     

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.     

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.

     

The ultrastructure ofAcetivibrio cellulolyticus,a recently isolated cellulolytic anaerobe

The surface and internal structure of air-dried, freeze-dried, or critical-point-dried cells ofAcetivibrio cellulolyticus were determined by negative staining, and by scanning and transmission electron microscopy. The cell wall has five layers, the outermost being the widest. This outermost layer is soft and amorphous, adsorbs cellulose microfibrils, and shows projections of tenuous appearance. Results of staining with heavy ions indicate that the outermost layer carries a net positive charge. The flagellum is a tubular, uniform extension of this outermost layer, about, 20 nm in diameter and approximately 6.4 m long, with no visible internal details or basal body. The inner layers of the wall show no fine structure, other than differences in electron opacity. The cytoplasm is composed of a mixture of densely staining spheres and smaller globules in a background of fine particles. These particles are not completely destroyed by fixation with KMnO₄, which indicates that they are low in ribonucleic acid. There is no evidence for membranes around or outside of any of these bodies.     

Clumping characteristics and hydrophobic behaviour of an isolated bacterial strain from sewage sludge

Summary A clump-forming bacterial strain was isolated from a sludge community derived from a waste water treatment plant. The Gram-negative bacterium is hydrophobic and forms an extensive capsule while clumping in dilute medium (0.1% bacto peptone). Emulsan, a capsule inhibitor, does not affect the clumping ability of this bacterium. Clumps are not dispersed with high and low pH, detergents or chelators. Bacterial clumping selected by the waste water treatment processes appears to be a complex set of interactions within and between the strains of bacteria. This study reveals some of the complexities.     

A new strain of Desulfovibrio gigas isolated from a sewage plant

Summary A new strain of Desulfovibrio gigas was isolated from sludge of a sewage plant. The medium contained ethanol, sulfate, minerals, some vitamins but no source of combined nitrogen. Several enrichment cultures of sulfate reducers could be obtained by employing the method to sludge samples from various sewage plants. With respect to their morphology, the dominating bacteria were of the Desulfovibrio gigas type.     

Adhesive properties of five mesophilic, cellulolytic Clostridia isolated from the same biotope

Abstract Adhesion to cellulose of five strains of mesophilic, cellulolytic clostridia , isolated from a municipal waste digestor, was found to be a reversible phenomenon. The type of attachment for the five strains conformed to a multilayer adhesion. In a first step, attachment to the adhesion site occurred by cell-cellulose interaction. In a second step, cell-cell interactions were identified. The five strains adhered slightly better to magazine paper and Whatman No. 1 filter paper than to newspaper and cardboard. Two strains, C401 and A22, were studied in more detail. The two strains, harvested in stationary phase, presented a heterogeneous population which could be separated: (i) as 'unbound' cells, corresponding to cells remaining in suspension from cellulose-grown cultures; and (ii) as 'bound' cells, coming from two successive washes with 50 mM Tris HCl, pH 7.0, which released 'bound' cells. In adhesion measurements, eluted cells ('bound' cells) adhered better to the cellulose than the 'unbound' cells. Strain C401 adhered better than strain A22 to the cellulose: 1.9-fold for the 'bound' cells and 3.6-fold for the 'unbound' cells. Adhesion of the two isolates was enhanced by the presence of calcium (10 mM). Cellobiose and glucose had no effect on strain A22 adhesion. Conversely, adhesion of strain C401 to cellulose was enhanced by cellobiose at a concentration of 1.5 g I⁻¹, but 85% inhibited by a concentration of 5.0 g I⁻¹. The two strains adhered to the same site on Whatman filter paper and unspecific interactions between the two strains occur.     

Isolation of bacteria producing polyhydroxyalkanoates (PHA) from municipal sewage sludge

Bacterial isolates from sludge samples collected at a local municipal sewage treatment plant were screened for bacteria producing polyhydroxyalkanoates (PHA). Initially Sudan black B staining was performed to detect lipid cellular inclusions. Lipid-positive isolates were then grown in a nitrogen limitation E2 medium containing 2% (w/v) glucose to promote accumulation of PHA before the subsequent staining with Nile blue A. The positive isolates were quantified initially with a u.v. spectrophotometer, for a very large number of isolates (105) and among them high PHA-producing isolates (15) were selected and were confirmed by gas chromatographic analysis. The GC analysis showed the polymers produced by 13 of the selected isolates to be polyhydroxybutyrate (PHB), and the remaining two isolates produced polyhydroxybutyrate-co-hydroxyvalerate (PHB-co-HV) copolymer. The proportion of the PHA-positive bacterial isolates showed variability in the number of PHA accumulators during various months. The correlation of PHB production with the cell dry weight (CDW) was found to be statistically significant. The metabolism of PHB in these selected 15 isolates was studied using the Nile blue A staining, which showed an initial increase in the fluorescence followed by a decline, on further incubation. All the selected 15 isolates were classified to genus level by studying their morphological and biochemical characteristics. There were seven Bacillus species, three Pseudomonas species, two Alcaligenes species, two Aeromonas species, and one Chromobacterium species.     

Simultaneous production of 2,3-butanediol, ethanol and hydrogen with a Klebsiella sp. strain isolated from sewage sludge

A Klebsiella sp. HE1 strain isolated from hydrogen-producing sewage sludge was examined for its ability to produce H(2) and other valuable soluble metabolites (e.g., ethanol and 2,3-butanediol) from sucrose-based medium. The effect of pH and carbon substrate concentration on the production of soluble and gaseous products was investigated. The major soluble metabolite produced from Klebsiella sp. HE1 was 2,3-butanediol, accounting for over 42-58% of soluble microbial products (SMP) and its production efficiency enhanced after increasing the initial culture pH to 7.3 (without pH control). The HE1 strain also produced ethanol (contributing to 29-42% of total SMP) and a small amount of lactic acid and acetic acid. The gaseous products consisted of H(2) (25-36%) and CO(2) (64-75%). The optimal cumulative hydrogen production (2.7 l) and hydrogen yield (0.92molH(2)molsucrose(-1)) were obtained at an initial sucrose concentration of 30gCODl(-1) (i.e., 26.7gl(-1)), which also led to the highest production rate for H(2) (3.26mmolh(-1)l(-1)), ethanol (6.75mmolh(-1)l(-1)) and 2,3-butanediol (7.14mmolh(-1)l(-1)). The highest yield for H(2), ethanol and 2,3-butanediol was 0.92, 0.81 and 0.59molmol-sucrose(-1), respectively. As for the overall energy production performance, the highest energy generation rate was 27.7kJh(-1)l(-1) and the best energy yield was 2.45kJmolsucrose(-1), which was obtained at a sucrose concentration of 30 and 20gCODl(-1), respectively.     

Degradation of keratin substrates by fungi isolated from sewage sludge

Four fungal species including two dermatophytes and two saprophytes were isolated from sewage sludge samples at Basrah (Iraq) they were tested for their degradative ability towards three types of keratin substrates (human hair, chicken feathers and wool). The rate of keratin degradation was expressed as weight loss over three weeks of incubation using a liquid culture medium. Human hair had the highest degradation rate by colonization of Chrysosporium pannicola and Microsporum gypseum at a rate of 62% and 4% respectively. Chicken feathers were highly degraded by Aspergillus flavus (32%) while wool degradation was highest by C. pannicola (45.5%) and Trichophyton mentagrophytes var. erinacei (38%). There was a significant difference (p < 0.00l) in keratin substrate degradation rates by the examined fungi. Keratinase activity was highest for C. pannicola and M. gypseum in the culture medium baited with human hair. Aspergillus flavus revealed the highest activity of this enzyme in cultures amended with chicken feathers while T. mentagrophytes var. erinacei showed highest keratinase activity in cultures with wool substrate. The amount of protein released into the culture medium varied among the tested fungi. The medium's alkalinity increased over incubation time from 6.5 to 7.8. Microscopic examination showed maceration of the keratin substrates by the fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phylogenetic and physiological characterization of mesophilic and thermophilic bacteria from a sewage sludge composting process in Sapporo,Japan

The phylogenetic and physiological characteristics of mesophilic and thermophilic bacteria isolated from a field-scale sewage sludge composter were determined by 16S rDNA and phenotype analyses. Of the 34 mesophilic isolates, 5 (15%), 16 (47%), and 3 (9%) displayed amylase, protease, and lipase activities, respectively. Among these isolates, the following species were identified based on their 16S rRNA gene sequences: Aneurinibacillus aneurinilyticus, Bacillus fortis, Bacillus subtilis, Brachybacterium paraconglomeratum, Brevibacterium otitidis, Dietzia maris, Pseudomonas xiamenensis, Staphylococcus lentus, Thermobifida fusca, Ureibacillus thermosphaericus, and Vagococcus lutrae. However, 15 isolates could not be identified as known taxa, thus indicating new bacterial taxa. Of these new taxa, it is likely that NoID A plays an important role in organic matter decomposition during composting based on its physiological characteristics. Sapporo sewage sludge compost contains a microbial ecosystem with novel bacterial biodiversity, comprising a high percentage of previously unrecognized species. This study improves our knowledge of the unique bacteria in sewage sludge compost, providing a future resource for bacterial genetic information and bacterial species of agricultural benefit.     

Phytoextraction of heavy metals from municipal sewage sludge by Rosa multiflora and Sida hermaphrodita

The aim of the study was to evaluate the efficacy of the multiflora rose var. “Jatar” (Rosa multiflora Thunb. ex Murray) and the Virginia fanpetals (Sida hermaphrodita Rusby) to phytoextract heavy metals from municipal sewage sludge. The 6-year field experiment involved four levels of fertilization with sewage sludge at doses of 0, 10, 20, 40, and 60 Mg DM (Dry Mass) sludge ha^?1. The increasing doses of sewage sludge were found to significantly increase the yield of multiflora rose and Virginia fanpetals biomass. They also significantly increased the content of heavy metals in these plants. The highest uptake of heavy metals by the multiflora rose and Virginia fanpetals crops was recorded at the fertilization dose of 60 Mg DM ? ha^?1. Our investigations show that the Virginia fanpetals was more efficient in the phytoextraction of Cr, Ni, Cu, Zn, and Cd from the sewage sludge than the multiflora rose, due to the greater yields and higher heavy metal uptake by the former plant. In turn, the multiflora rose phytoextracted greater amounts of Pb from the sewage sludge. The analyses indicate that the Virginia fanpetals can be used for phytoremediation (phytoextraction) of heavy metals contained in sewage sludge.     

The elimination of Salmonella typhimurium in sewage sludge by aerobic mesophilic stabilization and lime hydrated stabilization

This study observed the effects of two methods, aerobic mesophilic stabilization and lime hydrated stabilization of sewage sludge upon the survival of Salmonella typhimurium. Raw (primary) sludges from the mechanical biological municipal sewage treatment plant were used. Aerobic stabilization and lime hydrated stabilization were carried out in a laboratory fermentor. Aerobic stabilization was carried out in the mesophilic temperature range (from 25.70+/-0.40 to 37.82+/-1.38 degrees C). Lime hydrated was used at an amount of 10 kg/m(3) for the stabilization. Sludge samples were inoculated with a broth culture of S. typhimurium. Quantitative and qualitative examinations of the presence of S. typhimurium were carried out. Aerobic mesophilic stabilization caused elimination S. typhimurium within 48 h. The T(90) value of S. typhimurium was 6.66+/-0.20 h. During the lime hydrated stabilization pH values significantly increased from 5.66+/-0.07 to 12.12+/-0.02 (P<0.01). S. typhimurium was inactivated within 1h and the T(90) value was 0.19+/-0.01 h. Our study confirmed that the treatment of sewage sludge with lime hydrated was significantly more effective than the aerobic mesophilic stabilization, (P<0.01).     

Granulation of digested sewage sludge in mesophilic UASB reactors treating distillery waste waters from sugar cane molasses

Five 4.02-1 UASB (Upflow Anaerobic Sludge Blanket) reactors were continuously operated at 30°C under different hydrodynamic regimes for more than 120 days. The effect of the upflow liquid velocity (ULV) over the range of 0.25 to 2.0 m/h on the biological characteristics of the granules formed by treating vinasses (waste water of alcohol distilleries) from sugar cane molasses was investigated constantly maintaining the volumetric loading rate (VLR) (8 g COD/l · d). Granular sludge was found at all the ULV tested. The size, shape, etc., of the granules indicated that the ULV had a considerable effect on the sludge cultivated in this type of system, thereby acting as a selection process for the biomass. The best results in relation to the time of appearance, size, shape, consistence, stability, composition, and the accumulation were observed in the ULV range between 0.25 and 0.5 m/h. Microscopic studies of the granules using optical and epifluorescence microscopes and the scanning electron micrograph (SEM) showed a heterogeneous biomass and revealed the cell characteristics.     

Characteristics of a new cellulolytic Clostridium sp. isolated from pig intestinal tract.

Gram-positive, spore-forming, motile, cellulolytic rods were isolated from 10(7) dilutions of pig fecal samples. The pigs had previously been fed pure cultures of the ruminal cellulolytic organism Clostridium longisporum. Isolates formed terminal to subterminal spores, and a fermentable carbohydrate was required for growth. Besides cellulose, the isolates utilized cellobiose, glycogen, maltose, and starch. However, glucose, fructose, sucrose, pectin, and xylose were not used as energy sources. Major fermentation products included formate and butyrate. The isolates did not digest proteins from gelatin or milk. Unlike C. longisporum, which has limited ability to degrade cell wall components from grasses (switchgrass, bromegrass, and ryegrass), the swine isolates were equally effective in degrading these components from both alfalfa and grasses. The extent of degradation was equal to or better than that observed with the predominant ruminal cellulolytic organisms. On the basis of morphology, motility, spore formation, fermentation products, and the ability to hydrolyze cellulose, the isolates are considered to be a new species of the genus Clostridium. It is unclear whether C. longisporum played a role in the establishment or occurrence of this newly described cellulolytic species. This is the first report of a cellulolytic Clostridium sp. isolated from the pig intestinal tract.     

Tolerance to copper and zinc of Acidithiobacillus thiooxidans isolated from sewage sludge

The effect of copper and zinc ions on sulphur oxidation by Acidithiobacillus thiooxidans, strain SFR01, isolated from anaerobic sewage sludge was assessed, resulting in tolerance levels up to 20 and 200 mmol l^–1 for copper and zinc, respectively. The tolerance levels obtained were higher than the concentration of copper and zinc usually found in the collected sewage sludge. The tolerance levels obtained indicate no constraints for sludge bioleaching of those metals due to their toxicities to the indigenous A. thiooxidans.     

Characteristics of a new cellulolytic Clostridium sp. isolated from pig intestinal tract.

Gram-positive, spore-forming, motile, cellulolytic rods were isolated from 10(7) dilutions of pig fecal samples. The pigs had previously been fed pure cultures of the ruminal cellulolytic organism Clostridium longisporum. Isolates formed terminal to subterminal spores, and a fermentable carbohydrate was required for growth. Besides cellulose, the isolates utilized cellobiose, glycogen, maltose, and starch. However, glucose, fructose, sucrose, pectin, and xylose were not used as energy sources. Major fermentation products included formate and butyrate. The isolates did not digest proteins from gelatin or milk. Unlike C. longisporum, which has limited ability to degrade cell wall components from grasses (switchgrass, bromegrass, and ryegrass), the swine isolates were equally effective in degrading these components from both alfalfa and grasses. The extent of degradation was equal to or better than that observed with the predominant ruminal cellulolytic organisms. On the basis of morphology, motility, spore formation, fermentation products, and the ability to hydrolyze cellulose, the isolates are considered to be a new species of the genus Clostridium. It is unclear whether C. longisporum played a role in the establishment or occurrence of this newly described cellulolytic species. This is the first report of a cellulolytic Clostridium sp. isolated from the pig intestinal tract.     

The effect of organic loading rate on foam initiation during mesophilic anaerobic digestion of municipal wastewater sludge

The impact of increasing organic load on anaerobic digestion foaming was studied at both full and bench scale. Organic loadings of 1.25, 2.5 and 5 kg VS m⁻³ were applied to bench-scale digesters. Foaming was monitored at a full scale digester operated in a comparable organic loading range over 15 months. The bench scale batch studies identified 2.5 kg VS m⁻³ as a critical threshold for foam initiation while 5 kg VS m⁻³ resulted in persistent foaming. Investigation of a full scale foaming event corroborated the laboratory observation that foaming may be initiated at a loading rate of ?2.5 kg VS m⁻³. Experimental findings on foam composition and differences in the quality characteristics between foaming and non-foaming sludges indicated that foam initiation derived from the combined effect of the liquid and gas phases inside a digester and that the solids/biomass ultimately stabilized foaming.