Characterization of 3-chlorobenzoate degrading aerobic bacteria isolated under various environmental conditions

The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O(2)) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low μ(max) values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations.     

Heat stable lipase activity of thermotolerant bacteria from hot springs at Orissa, India

Thermotolerant bacteria (35 in toto) isolated from three hot springs (Atri, Taptapani and Deuljhari, Orissa), were screened for lipase activities. Of these, nine strains of Bacillus spp. and three strains of Pseudomonas spp. showed heat stable lipase activity at 60 degrees C. The hydrolytic activity of these bacteria was tested using Tween-20 and Tween-80 as substrates at different temperatures using plate assay and titration techniques. The hydrolytic activity at different pH values and salt concentrations was investigated.     

Comparing Metabolic Functionalities,Community Structures,and Dynamics of Herbicide-Degrading Communities Cultivated with Different Substrate Concentrations

Two 4-chloro-2-methylphenoxyacetic acid (MCPA)-degrading enrichment cultures selected from an aquifer on low (0.1 mg liter⁻¹) or high (25 mg liter⁻¹) MCPA concentrations were compared in terms of metabolic activity, community composition, population growth, and single cell physiology. Different community compositions and major shifts in community structure following exposure to different MCPA concentrations were observed using both 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and pyrosequencing. The communities also differed in their MCPA-mineralizing activities. The enrichments selected on low concentrations mineralized MCPA with shorter lag phases than those selected on high concentrations. Flow cytometry measurements revealed that mineralization led to cell growth. The presence of low-nucleic acid-content bacteria (LNA bacteria) was correlated with mineralization activity in cultures selected on low herbicide concentrations. This suggests that LNA bacteria may play a role in degradation of low herbicide concentrations in aquifers impacted by agriculture. This study shows that subpopulations of herbicide-degrading bacteria that are adapted to different pesticide concentrations can coexist in the same environment and that using a low herbicide concentration enables enrichment of apparently oligotrophic subpopulations.     

Comparison of the WST-8 colorimetric method and the CLSI broth microdilution method for susceptibility testing against drug-resistant bacteria

The minimum inhibitory concentrations (MICs) obtained from the susceptibility testing of various bacteria to antibiotics were determined by a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone as an electron mediator and compared with those obtained by the broth microdilution methods approved by the Clinical and Laboratory Standard Institute (CLSI). Especially for drug-resistant bacteria, the CLSI method at an incubation time of 24h tended to give lower MICs. The extension of incubation time was necessary to obtain consistent MICs for drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococi (VRE) and multi-drug resistant Pseudomonas aeruginosa (MDRP) in the broth microdilution method. There was excellent agreement between the MICs determined after 24h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of consistent MICs for drug-resistant bacteria.     

Identification and characterization of humic substances-degrading bacterial isolates from an estuarine environment

Bacterial isolates were obtained from enrichment cultures containing humic substances extracted from estuarine water using an XAD-8 resin. Eighteen isolates were chosen for phylogenetic and physiological characterization based on numerical importance in serial dilutions of the enrichment culture and unique colony morphology. Partial sequences of the 16S rRNA genes indicated that six of the isolates were associated with the alpha subclass of Proteobacteria, three with the gamma-Proteobacteria, and nine with the Gram-positive bacteria. Ten isolates degraded at least one (and up to six) selected aromatic single-ring compounds. Six isolates showed ability to degrade [(14)C]humic substances derived from the dominant salt marsh grass in the estuary from which they were isolated (Spartina alterniflora), mineralizing 0.4-1.1% of the humic substances over 4 weeks. A mixture of all 18 isolates did not degrade humic substances significantly faster than any of the individual strains, however, and no isolate degraded humic substances to the same extent as the natural marine bacterial community (3.0%). Similar studies with a radiolabeled synthetic lignin ([beta-(14)C]dehydropolymerisate) showed measurable levels of degradation by all 18 bacteria (3.0-8.8% in 4 weeks), but mineralization levels were again lower than that observed for the natural marine bacterial community (28.2%). Metabolic capabilities of the 18 isolates were highly variable and generally did not map to phylogenetic affiliation.     

Bioenergetic aspects of halophilism.

Examination of microbial diversity in environments of increasing salt concentrations indicates that certain types of dissimilatory metabolism do not occur at the highest salinities. Examples are methanogenesis for H2 + CO2 or from acetate, dissimilatory sulfate reduction with oxidation of acetate, and autotrophic nitrification. Occurrence of the different metabolic types is correlated with the free-energy change associated with the dissimilatory reactions. Life at high salt concentrations is energetically expensive. Most bacteria and also the methanogenic Archaea produce high intracellular concentrations of organic osmotic solutes at a high energetic cost. All halophilic microorganisms expend large amounts of energy to maintain steep gradients of NA+ and K+ concentrations across their cytoplasmic membrane. The energetic cost of salt adaptation probably dictates what types of metabolism can support life at the highest salt concentrations. Use of KCl as an intracellular solute, while requiring far-reaching adaptations of the intracellular machinery, is energetically more favorable than production of organic-compatible solutes. This may explain why the anaerobic halophilic fermentative bacteria (order Haloanaerobiales) use this strategy and also why halophilic homoacetogenic bacteria that produce acetate from H2 + CO2 exist whereas methanogens that use the same substrates in a reaction with a similar free-energy yield do not.     

Nitrification and occurrence of salt-tolerant nitrifying bacteria in the Negev desert soils

Ammonia oxidation potential, major ammonia oxidizers and occurrence of salt-tolerant nitrifying bacteria were studied in soil samples collected from diverse ecosystems along the northern Negev desert. Great diversity in ammonia oxidation potential was observed among the soil samples, and ammonia oxidizers were the rate-limiting step of nitrification. Denaturing gradient gel electrophoresis and partial 16S rRNA gene sequences indicate that members of the genus Nitrosospira are the major ammonia oxidizers in the natural desert soil samples. Upon enrichment with different salt concentrations, salt-tolerant nitrifying enrichments were established from several soil samples. In two enrichments, nitrification was not inhibited by 400 mM NaCl. Electrophoretic analysis and partial 16S rRNA gene sequences indicate that Nitrosomonas species were dominant in the 400 mM salt enrichment. The results point towards the potential of the desert ecosystem as a source of stress-tolerant nitrifying bacteria or other microorganisms with important properties.     

盐爪爪根部微生物分布特征及盐浓度对碳源代谢分析的影响

【背景】耐盐植物盐爪爪广泛分布于盐碱荒漠区域,其根际存在明显的盐离子富集,形成"盐岛效应",研究此过程中微生物如何演替和适应,对揭示盐爪爪根际微生物的分布及其耐盐特性、"盐岛"的形成具有重要科学意义。【目的】揭示盐爪爪根际、根区及其周围环境土壤中微生物分布特征,确定不同盐浓度处理对根际微生物Biolog Eco微平板碳源代谢多样性的影响。【方法】采用Biolog Eco微平板技术,对采自新疆和硕地区的盐爪爪根际、根区和环境土壤微生物碳源利用情况进行分析,并采用不同浓度NaCl溶液对盐爪爪根际土壤样品进行稀释加样,评估盐浓度对根际微生物活性及多样性分析的影响。【结果】样品微生物代谢活性总体呈现根际根区环境的趋势;随着与根部距离的增加,样品中利用氨基酸、胺类碳源微生物所占比例明显降低,而利用吐温和肝糖等多聚化合物类碳源微生物所占比例明显增加;多样性分析表明,随着与根部距离的增加,Shannon指数(H)随之降低且存在明显差异。不同浓度NaCl溶液对采用BiologEco微平板技术分析盐爪爪根际微生物的代谢活性和代谢多样性有着明显的影响,其中采用5%NaCl溶液作为稀释加样液的根际微生物活性最高。【结论】盐爪爪根部微生物组成和分布有着明显的演变规律,且采用Biolog Eco微平板技术分析微生物代谢多样性时需进行培养条件的优化。     

The effect of culture conditions on hydrophobic properties of Pseudomonas aeruginosa

The cell surface hydrophobicity (CSH) plays an important role in a adhesion of bacteria on solid surfaces. CSH of 62 Pseudomonas aeruginosa strains isolated from humans and different animals was assessed using the ammonium sulfate salt aggregation test. Bacteria were grown for 24 h and 48 h at a room temperature (22 degrees C) and 37 degrees C on enrichment broth and agar (Biomed) and tryptic soy agar (Difco). The hydrophobic properties of the Pseudomonas aeruginosa strains were depended on the temperature, time of the culture of bacteria and the kind of media. CSH properties were most frequently expressed when the analyzed strains were cultured in enrichment broth. In a such conditions Pseudomonas aeruginosa strains were more hydrophobic when grown at 22 degrees C (94% after 24 h and 87% after 48%) than those at 37 degrees C (72% after 24 h and 71% after 48 h). Among strains cultured in tryptic soy agar at 37 degrees C, 48% after 24 h and 75% after 48 h were autoaggregating, representing very strong hydrophobic properties.     

Molecular analysis of enrichment cultures of ammonia oxidizers from the Salar de Huasco,a high altitude saline wetland in northern Chile

We analyzed enrichment cultures of ammonia-oxidizing bacteria (AOB) collected from different areas of Salar de Huasco, a high altitude, saline, pH-neutral water body in the Chilean Altiplano. Samples were inoculated into mineral media with 10 mM NH₄ ⁺ at five different salt concentrations (10, 200, 400, 800 and 1,400 mM NaCl). Low diversity (up to three phylotypes per enrichment) of beta-AOB was detected using 16S rDNA and amoA clone libraries. Growth of beta-AOB was only recorded in a few enrichment cultures and varied according to site or media salinity. In total, five 16S rDNA and amoA phylotypes were found which were related to Nitrosomonas europaea/Nitrosococcus mobilis, N. marina and N. communis clusters. Phylotype 1-16S was 97% similar with N. halophila, previously isolated from Mongolian soda lakes, and phylotypes from amoA sequences were similar with yet uncultured beta-AOB from different biofilms. Sequences related to N. halophila were frequently found at all salinities. Neither gamma-AOB nor ammonia-oxidizing Archaea were recorded in these enrichment cultures.     

Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations

Solar salterns, based on a multi-pond system, give a discontinuous gradient of salt concentrations. The heterotrophic bacterial populations of ponds containing from 10% salt to saturation have been studied. Saltern samples were spread on agar plates containing different media for halophilic bacteria and one medium made with water of the pond plus nutrients. Replica plating was done to determine the salt range for growth of the colonies. We studied 150 strains to determine the salt spectra of growth, the morphology, and nutrient requirements. The following conclusions were reached: (a) In salt concentrations above 10% (total salts), most bacteria are halophilic and few are halotolerant; (b) the two types of halophilic bacteria, moderate and extreme, show different distributions; in these ponds a narrow overlap exists between 25% and 32% salts with moderate halophiles predominating below this interval and extreme halophiles above it; (c) the populations of moderate halophiles are highly heterogeneous, and the salt concentration of their habitat affects their taxonomic composition, salt range for growth, and nutrient requirements. The population composition of extreme halophiles is less affected by the salt concentrations at which these bacteria are found.     

Feasibility of hydrogen production in thermophilic mixed fermentation by natural anaerobes

The biological sludge from an animal wastewater treatment plant was treated to enrich hydrogen-producing mixed bacteria, and effects on hydrogen yield were investigated during anaerobic fermentation at 55 degrees C. Enrichment of hydrogen-producing bacteria was conducted at pH adjustment of inocula to 3 and 5 with and without additional heat treatment (NHT and HT). The enriched mixed bacteria were cultivated at initial pHs of 5, 6, and 7 with synthetic organic wastewater containing different levels of nitrogen (2.0 and 0.8 g/l as total nitrogen) under static batch conditions. The main effects of heat treatment and enrichment pH were significant on hydrogen production. There was no significant effect of different nitrogen concentrations on hydrogen production. The methane-free biogas contained hydrogen levels of up to 64% for a fermentative condition that showed maximum hydrogen evolution (at culture pH 5 after enrichment at pH 5 with HT). The dominating intermediate metabolites were acetate, n-butyrate, and ethanol. Yields of produced hydrogen were significantly dependent upon levels of n-butyrate.     

Investigations on cellulose biodegradation in activated sludge plants

Cellulose is the major carbon substrate entering treatment plants for municipal waste waters. In the present investigation an attempt was made to study its degradation in activated sludge. Cellulolytic micro-organisms were enumerated in different treatment plants and at one plant they were assessed after different steps over a period of about 1 year. The degradation of cellulose contained in Nylon bags suspended in the mixed liquor was also studied and the activities of cellulase components were assayed. Finally, the concentrations of cellulose and lignin in the suspended solids taken from different treatment steps were determined. The results showed that active cellulolysis occurred in activated sludge. The degradation was mainly bacterial, although no significant enrichment of such bacteria was found in the sludge floc. Cellulase activity, however, showed an increase. Experiments with the Nylon bag indicated that 60% of the weight of cotton wool was degraded in 4–5 weeks. It was concluded that about 60% (w/w) of the cellulose entering the system could be degraded by bacteria during aerobic treatment, while 50–60% of that present in the surplus activated sludge was degraded during anaerobic sludge digestion.     

Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production

Microbiological investigation of anaerobic ammonium oxidizing (anammox) bacteria has until now been restricted to wastewater species. The present study describes the enrichment and characterization of two marine Scalindua species, the anammox genus that dominates almost all natural habitats investigated so far. The species were enriched from a marine sediment in the Gullmar Fjord (Sweden) using a medium based on Red Sea salt. Anammox cells comprised about 90% of the enrichment culture after 10 months. The enriched Scalindua bacteria displayed all typical features known for anammox bacteria, including turnover of hydrazine, the presence of ladderane lipids, and a compartmentalized cellular ultrastructure. The Scalindua species also showed a nitrate-dependent use of formate, acetate and propionate, and performed a formate-dependent reduction of nitrate, Fe(III) and Mn(IV). This versatile metabolism may be the basis for the global distribution and substantial contribution of the marine Scalindua anammox bacteria to the nitrogen loss from oxygen-limited marine ecosystems.     

Anaerobic degradation of organic compounds at high salt concentrations

A number of obligately anaerobic fermentative bacteria are known to degrade a variety of organic substrates such as sugars, amino acids, and others, in the presence of high salt concentrations (up to 3–4 M) to products such as hydrogen, CO₂, acetate and higher fatty acids, and ethanol. Our understanding of the fate of these products in hypersaline environments is still extremely limited. The occurrence of bacterial sulfate reduction is well established at salt concentrations of up to 24%; however, the bacteria involved have not yet been isolated in pure culture, and the range of electron donors used is unknown. Halophilic or halotolerant methanogenic bacteria using hydrogen/CO₂ or acetate as energy source are notably absent; methanogenesis under hypersaline conditions is probably limited to such substrates as methanol and methylamines, which cannot be expected to be major products of anaerobic degradation of most organic compounds.     

Bioenergetic Aspects of Halophilism

Examinination of microbial diversity in environments of increasing salt concentrations indicates that certain types of dissimilatory metabolism do not occur at the highest salinities. Examples are methanogenesis for H₂ + CO₂ or from acetate, dissimilatory sulfate reduction with oxidation of acetate, and autotrophic nitrification. Occurrence of the different metabolic types is correlated with the free-energy change associated with the dissimilatory reactions. Life at high salt concentrations is energetically expensive. Most bacteria and also the methanogenic archaea produce high intracellular concentrations of organic osmotic solutes at a high energetic cost. All halophilic microorganisms expend large amounts of energy to maintain steep gradients of NA⁺ and K⁺ concentrations across their cytoplasmic membrane. The energetic cost of salt adaptation probably dictates what types of metabolism can support life at the highest salt concentrations. Use of KCl as an intracellular solute, while requiring far-reaching adaptations of the intracellular machinery, is energetically more favorable than production of organic-compatible solutes. This may explain why the anaerobic halophilic fermentative bacteria (order Haloanaerobiales) use this strategy and also why halophilic homoacetogenic bacteria that produce acetate from H₂ + CO₂ exist whereas methanogens that use the same substrates in a reaction with a similar free-energy yield do not.     

Novel green sulfur bacteria phylotypes detected in saline environments: ecophysiological characters versus phylogenetic taxonomy

The taxonomic significance of salt tolerance or requirements in green sulfur bacteria has been analyzed with environmental populations and enrichment cultures from several saline systems (inland and coastal water bodies) with different salinities (salt composition and concentration). Novel phylotypes of green sulfur bacteria have been found in hypersaline and brackish environments and 16S rRNA gene sequence analysis affiliated them into phylogenetic groups in which neither halotolerant nor halophilic species have been known to date. Therefore, salt tolerance does not seem to be restricted to members of any specific subgroup but is widespread among all the different phylogenetic branches of the green sulfur bacteria group, and closely-related phylotypes can have dissimilar salt tolerance capacities. Thus the phenotypic characteristics and phylogenetic structure of the green sulfur bacteria present some incongruities. Phenotypic traits should be studied further in order to determine the ecophysiological features of green sulfur bacteria phylotypes.     

Improved growth medium for Campylobacter species.

Campylobacter species were grown in a base containing proteose peptone no. 3, yeast extract, K2HPO4, (NH4)2SO4, NA2SO3, soluble starch, and agar. Concentrations and sources of organic nitrogen and growth factors were critical, and the optimal pH range was 7.0 to 7.5. Cultures tolerated 0.7% NaCl in addition to the salt present in the organic constituents and were sensitive to surface-active agents at concentrations recommended for enrichment of other gram-negative bacteria. Cultures were maintained on the proposed medium for 1 year with transfer every 2 weeks.     

RAPID DETECTION OF MICROBIAL CONTAMINATION IN TRICLOSAN AND HIGH FLUORIDE DENTIFRICES USING AN ATP BIOLUMINESCENCE ASSAY

Abstract The Celsis ATP Bioluminescence method was optimized and validated to detect the presence of microbial contamination in High Fluoride and Triclosan dentifrice formulations. Several enrichment broths were evaluated by using a 24–27 h incubation period. The ATP concentrations of the enrichment broths were found to a range from 0.012 to 0.040 nM. None of the tested enrichment broths were found to exhibit any sample inhibition/enhancement effects on the ATP Bioluminescence reaction. Dentifrice suspensions were inoculated with bacteria, yeast, and mold. All test microorganisms (ca. 1–15 CFU/g) were detected within a 24–27 h incubation period by using TAT Broth Base enrichment broths containing different concentrations of the following ingredients: Tween 20, Neopeptone, Dextrose, Triton X-100, Thiosulfate, Sodium Dibasic Phosphate, and Glycine. Negative ATP response after 24–27 h of incubation at 35C indicates the absence of contamination from these products.     

Composition of free and adherent ruminal bacteria: inaccuracy of the microbial nutrient supply estimates obtained using free bacteria as reference samples and (15)N as the marker

Previous studies have indicated that (15)N enrichment of solid-associated bacteria (SAB) may be predicted from the same value in liquid-associated bacteria (LAB). The aims of this study were to confirm this and to measure the error in the nutrient supply from SAB, when LAB are used as the reference sample. For this purpose, the chemical and amino acid (AA) compositions of both the bacterial populations were studied in four experiments carried out on different groups of three rumen cannulated wethers. Diets (one in Experiments 1 and 4 and three in Experiments 2 and 3) had forage-to-concentrate ratios (dry matter (DM) basis) between 2 : 1 and 40 : 60, and were consumed at intake levels between 40 and 75 g DM/kg (BW)(0.75). The bacteria samples were isolated after continuous infusion of ((15)NH(4))(2)SO(4) (40, 18, 30 and 25 mg (15)N/day, in Experiments 1 to 4, respectively) for at least 14 days. In all experiments, SAB had consistently higher concentrations of organic matter (826 v. 716 g/kg DM, as average) and total lipids (192 v. 95 g/kg DM, as average) than LAB. Similar CP concentrations of both populations were observed, except a higher concentration in SAB than in LAB in Experiment 3. A consistent (in Experiment 4 only as tendency) higher AA-N/total N ratio (on average 17.5%) was observed in SAB than in LAB. The (15)N enrichment in SAB was systematically lower than in LAB. On the basis of the results of all studies a close relationship was found between the (15)N enrichment in SAB and LAB, which was shown irrespective of experiments. This relationship was established from Experiments 1 and 2 and the above cited previous results (n = 20; P < 0.001; R(2) = 0.996), and then confirmed from the results of Experiments 3 and 4. These relationships between SAB and LAB demonstrate that CP supply from SAB is underevaluated by, on average, 21.2% when LAB are used as the reference. This underevaluation was higher for true protein and even higher for the lipid supply (32.5% and 59.6%, respectively, as an average of the four experiments). Large differences in AA profile were observed between SAB and LAB. The prediction equation obtained using (15)N as the marker may be used to correct the errors associated with the traditional use of LAB as the reference sample, and therefore to obtain more accurate estimates of the microbial nutrient supply to the ruminants.