Culture Of Anaerobic Bacteria Research Articles

Carvacrol and thymol reduce swine waste odor and pathogens: stability of oils.

An incomplete anoxic fermentation of livestock waste results in offensive odor emissions. Antimicrobial additives may be useful in controlling odor emissions and pathogens. Natural antimicrobial compounds, carvacrol or thymol at 16.75 mM (2.5 g/l) completely inhibited the production of the offensive odor compounds, isobutyrate, valerate, isovalerate, and cresol, and significantly reduced other short-chain volatile fatty acids and gas emissions from swine waste. Fecal coliforms were reduced from 6.3 x 10(6) to 1.0 x 10(3) cells per ml 2 days after treatment with carvacrol (13.3 mM) and were not detectable within 14 days. Total culturable anaerobic bacteria were reduced from 12.4 x 10(10) to 7.2 x 10(8) cells per ml after 2 days and were suppressed below this level for 28 days. Lactate production was not prevalent in untreated swine waste indicating that the microbial populations differ from those in cattle waste. Carvacrol and thymol were stable in swine waste under anoxic conditions for 62 days with 90 to 95% of the additive being recovered in the waste solids. In conclusion, carvacrol and thymol are not metabolized in anoxic swine waste and they are potentially useful in controlling odor emissions and pathogens in swine waste.

Interventional ERCP in patients with cholestasis. Degree of biliary bacterial colonization and antibiotic resistance

Interventional ERCP in patients with cholestasis. Degree of biliary bacterial colonization and antibiotic resistance. Biliary obstruction together with bacterial colonization of the bile duct may lead to development of acute cholangitis. The aim of our prospective study was to investigate the presence and degree of biliary bacterial colonization by means of bile aspiration during ERCP in patients with biliary obstruction. Furthermore, we evaluated antibiotic therapy regimens, which would cover the bacterial species obtained by ERCP and subsequent culture in each patient. In addition, analysis of risk factors was performed that would predispose to the development of cholangitis.80 patients with clinical and laboratory evidence of biliary obstruction underwent ERCP with initial aspiration of bile via the cannulation catheter. This material was used to culture aerobic and anaerobic bacteria and determine the colony count/ml bile, followed by identification of each species and antibiotic resistance testing. The minimal inhibitory concentration for Levofloxacin, Ciprofloxacin, Piperacillin, Ampicillin, Ceftriaxone, Imipenem, Gentamycin und Metronidazole was determined. Immediately after the ERCP or if the body temperature (after ERCP) rose to > 38 degrees C blood cultures were obtained. In 45 (56 %) patients biliary colonization with bacteria could be identified (56 %). In 20 patients a single isolate was cultured, in 25 cases mixed infection was present. A total of 83 species were isolated. The most common bacteria were E. coli, Enterococcus and Klebsiella. 9.6 % of all isolates were obligatory anaerobes. In 9 of 80 patients bloodcultures tested positive for bacterial growth (rate of bacteremia: 11.3 %). 10 patients had acute cholangitis clinically before ERCP, 13 patients developed signs of infection after ERCP. Statistically significant factors contributing to the risk of infection were age of the patient, the clinical condition of the patient before ERCP and the biliary colony count. Patients with development of infection after ERCP showed a significantly higher incidence of bacterial colonization of the biliary tree and a higher colony count. In all bacterial species Imipenem (4.5 %) or Levofloxacin (2.2 %) exhibited the lowest rate of in-vitro resistance. Based on these data, the implementation of Levofloxacin in combination with anaerobic coverage is advantageous as a calculated therapy for patients with acute cholangitis.

Anaerobic decolorization of an azo dye by a mixed culture

Wool dyeing wastewater contains xenobiotic compounds that can be removed by biotechnological processes. Studies on various dyes showed that anaerobic processes are suitable to alter azo dyes as a first step of the biodegradation process. These compounds are reduced by anaerobic consortia to aromatic amines and its ultimate degradation can be achieved by a further aerobic treatment. Studies on degradation rate of an wool acid dye were performed in batch systems inoculated with anaerobic biomass. A commercial diazo dye, Acid Red 73, was added to the synthetic medium in which glucose was used as sole carbon source. Results indicated that the Acid Red 73 was partially degraded by a mixed culture of anaerobic bacteria and a decolorization of 90% was obtained. Kinetics studies on removal of the colour showed that the decolorization rate was several times faster than the degradation rate of glucose for a range of dye concentrations between 60 mg/L and 400 mg/L. A first order kinetic model was used for dye concentrations up to 200 mg/L. For higher concentrations a model similar to the Michaelis‐Menten equation was better fitted to the experimental data.

Neural network modelling of a depollution process

In this paper an artificial neural network is developed to model a new depollution process that uses sequential cultures of anaerobic bacteria and yeasts to efficiently remove both carbon and nitrogen from wastewaters. A set of batch experimental runs are used to train and test various neural network topologies. It is shown that the neural network accurately tracks the dynamics of the biological species of the yeast reactor in the process and account for the influence of butyric acid, ammonia and pH on the overall efficiency of purification.

Evaluation of cot mattress inner foam as a potential site for microbial generation of toxic gases.

Recent reports of biovolatilisation of phosphorus and antimony by anaerobic bacteria and of leaching of phosphorus and antimony fire-retardant additives from PVC cot mattress covers, indicate that the polyurethane inner-foam of cot mattresses could be a site for generation of toxic gases of group 15 elements. A toxic gas hypothesis for sudden infant death syndrome (SIDS) involving polyurethane foam of cot mattresses was proposed and tested experimentally. Levels of antimony, phosphorus, arsenic and bismuth were determined at four sites for 44 SIDS and 50 control (no death) cot mattress foams. There was no evidence to suggest that the levels of these elements in cot mattress foam have a causal relation to SIDS. Leaching of antimony trioxide from PVC mattress covers could account for detectable levels of this element in 52% of the cot mattress samples analysed. Volatile forms of antimony, phosphorus, arsenic and bismuth was not detected in the headspace of mixed or monoseptic cultures of anaerobic bacteria containing polyurethane foam. Past microbial activity had given rise to involatile methylated species of antimony in some of the cot mattress foams tested (61%, n = 24). Abiotic oxidation of biogenic trimethylantimony together with physical adsorption of methylantimony forms to the polyurethane foam matrix could account for the apparent absence of "escaped" volatile antimony species in culture headspaces of incubation vial. There was no evidence to suggest that levels of trimethylantimony or total methylantimony forms in cot mattress foams have a causal relation to SIDS.

Oxyrase Cell-Membrane Preparations Simplify Cultivation of Anaerobic Bacteria

Clinical microbiologists have sought to isolate, identify, and characterize anaerobic bacteria since the organisms and their role in wound infections were discovered. Methods to create an environment for anaerobic growth have not changed for 100 years. A new approach—the Oxyrase enzymatic method—promotes anaerobiosis in the solid culture medium itself, allowing colonies to form without barriers associated with jars, bags, and chambers. In response to space restraints, increased workload, and cost-cutting directives, we evaluated the Oxyrase method for the primary isolation of anaerobic bacteria from clinical wound specimens. By using plated media (containing the oxygen-reducing substance) to isolate and identify anaerobic bacteria, a single technologist could report culture results more rapidly because all cultures—aerobic and anaerobic—for a patient could be processed simultaneously.

Electricity generation in microbial fuel cells using neutral red as an electronophore.

Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells.

GC Analysis of Organic Acids and Phenols Using On-Line Methylation with Trimethylsulfonium Hydroxide and PTV Solvent Split Large Volume Injection

The combination of on-line methylation using trimethylsulfonium hydroxide with large volume injection of 100 μL was evaluated for the analysis of organic acids and phenols in water. Solvent split injection was applied with complete evaporation of the solvent before analytes were transferred onto the GC column. Despite complete solvent removal, losses were very low compared to conventional splitless injection even for volatile acidic compounds such as propionic acid and phenol. This is explained by intermediate formation of low volatility trimethylsulfonium salts of the analytes which were held in the injector for long evaporation times of up to 10 min, if the evaporation temperature was as low as 10°C. Using a simple liquid/liquid extraction procedure, volatile fatty acids, dicarboxylic acids, benzoic acids and phenols could be detected in 5 mL of water at concentrations of 0.04–0.1 μmol/L with GC/MS in full scan mode. Lactic, pyruvic, and also malonic acids could only be detected at higher levels because of their limited extractability from water as well as their poorer methylation yields. The method provides an easy way to sensitively detect acidic compounds of medium to high volatility in water. It was applied for screening of organic acids and phenols in batch cultures of anaerobic bacteria of which one example is shown.

Inhibition of methanogenesis by methyl fluoride: studies of pure and defined mixed cultures of anaerobic bacteria and archaea

Methyl fluoride (fluoromethane [CH(inf3)F]) has been used as a selective inhibitor of CH(inf4) oxidation by aerobic methanotrophic bacteria in studies of CH(inf4) emission from natural systems. In such studies, CH(inf3)F also diffuses into the anaerobic zones where CH(inf4) is produced. The effects of CH(inf3)F on pure and defined mixed cultures of anaerobic microorganisms were investigated. About 1 kPa of CH(inf3)F, similar to the amounts used in inhibition experiments, inhibited growth of and CH(inf4) production by pure cultures of aceticlastic methanogens (Methanosaeta spp. and Methanosarcina spp.) and by a methanogenic mixed culture of anaerobic microorganisms in which acetate was produced as an intermediate. With greater quantities of CH(inf3)F, hydrogenotrophic methanogens were also inhibited. At a partial pressure of CH(inf3)F of 1 kPa, homoacetogenic, sulfate-reducing, and fermentative bacteria and a methanogenic mixed culture of anaerobic microorganisms based on hydrogen syntrophy were not inhibited. The inhibition by CH(inf3)F of the growth and CH(inf4) production of Methanosarcina mazei growing on acetate was reversible. CH(inf3)F inhibited only acetate utilization by Methanosarcina barkeri, which is able to use acetate and hydrogen simultaneously, when both acetate and hydrogen were present. These findings suggest that the use of CH(inf3)F as a selective inhibitor of aerobic CH(inf4) oxidation in undefined systems must be interpreted with great care. However, by a careful choice of concentrations, CH(inf3)F may be useful for the rapid determination of the role of acetate as a CH(inf4) precursor.

The effect of timothy silage on the bacterial population in rumen fluid of reindeer ( Rangifer tarandus tarandus) from natural summer and winter pasture

The seasonal effect of first cut (FC) and regrowth (RG) timothy ( Phleum pratense) silage on the rumen fluid bacterial population and the ability of this microbial ecosystem to digest cellulose was compared to that of reindeer from natural pasture (NP). FC with 27% leaves contained 30.4% dry matter (DM) cellulose and 6.2% DM water soluble carbohydrates (WSC), while RG with 89% leaves contained 18.4% DM cellulose and 30.0% DM WSC. The number of culturable anaerobic bacteria per ml rumen fluid on a habitat simulating medium (M8V) ranged between 1.1×10 8 and 95.0×10 8 in the reindeer ( n=18) investigated, and no significant ( P>0.05) differences were found between animals from NP and animals from the experimental groups in summer or winter. Carboxymethyl cellulose hydrolysing bacterial strains ( n=138) isolated from the rumen fluid of the animals ( n=18) using an acid-swollen cellulose medium were characterised as Butyrivibrio spp. (69.6%), Eubacterium spp. (1.4%), Clostridium sp. (0.7%) and unknowns (28.3%). Cellulolytic strains of B. fibrisolvens were isolated from only one of the calves, which had been fed RG in summer. The rumen bacterial composition in reindeer on NP resembled that of the experimental groups. In vitro digestibility trials demonstrates that the ability of the rumen microbiota to digest cellulose is generally reduced in reindeer fed silage in summer compared to winter. Ruminal malfunction occurred in some of the animals, but no correlation between the composition of the bacterial population in the fluid fraction of the rumen and the ability of this microbiota to ferment cellulose was found.

Microorganisms from deep, high temperature sandstones: constraints on microbial colonization

Cores were collected from Late Cretaceous and Early Tertiary rocks in the Piceance Basin of western Colorado, USA, to investigate the origins of subsurface microorganisms under geological conditions likely to constrain microbial transport and survival. The sampled strata from 856–862, 1996–1997 and 2091–2096 m recorded peak paleotemperatures of 120–145°C from 40–5 million years ago, while present temperatures range from 43 to 85°C. Cores were analyzed for culturable anaerobic bacteria (Fe(III)- and Mn(IV)-reducing bacteria, fermenters, sulfate reducers, nitrate reducers and methanogens), ester-linked phospholipid fatty acid and selected enzyme and physiological activities. Measurable but low biomass (total phospholipid fatty acid) and anaerobic bacteria, primarily Fe(III) reducers and fermenters, were present in samples from the 856–862 m core. Cores from greater depths yielded only a single positive enrichment and lower biomass values. Methanogens and sulfate reducers were not detected in any of the samples nor were bacteria that could grow with methane and any added electron acceptors. 16S rRNA genes cloned from products of PCR amplification of DNA extracted from an 858 m, 65°C, Fe(III)-reducing enrichment were most closely related to bacteria in the genus Desulfotomaculum, Gram-positive, spore-forming sulfate-reducing bacteria. Assuming the maximum temperatures would have eliminated any entrained bacteria, these anaerobic microorganisms likely migrated into the shallower Wasatch formation within the last 5 million years. However, the deepest stratum sampled was hydrologically isolated and lacked any indication of microbial colonization by all biological measures. Hydrologic connection to the surface, high maximum temperatures and the presence of fractures are probably the primary factors that control distribution of microorganisms in these deep rock environments.

Bacteria in the Prostate Tissue of Men With Idiopathic Prostatic Inflammation

Although antibiotics represent the first line of treatment for prostatitis syndromes, physicians can document infection in remarkably few cases. We examined the relationship of genitourinary infection to inflammatory prostatitis in 85 subjects without bacteriuria. Evaluation consisted of cultures of urethra, urine and transperineal prostate biopsies, specifically for commensal and fastidious organisms, and leukocyte counts of expressed prostatic secretions. Men with inflamed expressed prostatic secretions (25) were more likely to have any bacterial isolation (p = 0.01), positive cultures for anaerobic bacteria (p = 0.03), higher total bacterial counts (p = 0.02) and more bacteria, species isolated (p = 0.02) in prostate biopsy cultures than men without expressed prostatic secretion inflammation (60). Bacterial colonization/invasion of the prostate may be associated with inflammatory prostatitis in some cases.

Isolation of non-sporing anaerobic rods from infections in children.

From 1974 to 1994, 2033 microbiological specimens from children were submitted for cultures for anaerobic bacteria. Fifty-seven isolates of Bifidobacterium spp. were obtained from 55 (3%) children, 67 isolates of Eubacterium spp. from 65 (3%) children and 41 isolates of Lactobacillus spp. from 40 (2%) children. Most Bifidobacterium isolates were from chronic otitis media, abscesses, peritonitis, aspiration pneumonia and paronychia. Most Eubacterium isolates were from abscesses, peritonitis, decubitus ulcers and bites. Lactobacillus spp. were mainly isolated from abscesses, aspiration pneumonia, bacteraemia and conjunctivitis. Most (> 90%) infections from which these species were isolated were polymicrobial and yielded a mixture of aerobic and anaerobic bacteria. The organisms most commonly isolated with the non-sporing anaerobic gram-positive rods were Peptostreptococcus spp., Bacteroides spp., pigmented Prevotella and Porphyromonas spp., Fusobacterium spp., Staphylococcus aureus and Escherichia coli. Most Bacteroides spp. and E. coli were isolated from intra-abdominal infection and skin and soft tissue infection around the rectal area, whereas most Prevotella, Porphyromonas and Fusobacterium isolates were from oropharyngeal, pulmonary and head and neck sites. The predisposing conditions associated with the isolation of non-sporing anaerobic gram-positive rods were previous surgery, malignancy, steroid therapy and immunodeficiency. Antimicrobial therapy was given to 149 (83%) of the 160 patients, in conjunction with surgical drainage or correction of pathology in 89 (56%).

Veillonella infections in children

From 1974 to 1994, 2,033 specimens from children were submitted for cultures for anaerobic bacteria. Eighty-three Veillonella spp. were recovered from 83 children (4%). Most Veillonella species were recovered from abscesses, aspiration pneumonias, burns, bites, and sinuses. The infections were polymicrobial in 79 (95%) patients, but in 4 (5%) patients, Veillonella species were recovered in pure culture. The predisposing conditions associated with the recovery of these organisms were previous surgery, malignancy, steroid therapy, foreign body, and immunodeficiency. These data illustrate that Veillonella spp. are found infrequently in children, mostly in association with mixed infections, and are recovered mixed with mouth and bowel flora.

Thermostable dextranases: screening, detection and preliminary characterization

Screening methods have been developed for detection of micro‐organisms producing thermostable dextranases. They utilize the incorporation of Blue Dextran into agar or liquid culture media for isolation of active dextranase producers growing at temperatures above 55°C. A variety of high temperature environments in sugar factories and naturally occurring thermal water samples were excellent sources of dextranase producers. A number of aerobic and anaerobic thermophilic bacteria, isolated from these sources, were found to produce thermostable dextranases. Dextranases with the greatest thermostability were found in cultures of anaerobic bacteria grown above 65°C. Temperature optima were determined for several crude enzyme preparations, four of which exhibited temperature optima in the range 65–85°C.

Rapid bacterial degradation of polysaccharides in anoxic marine systems

Extracellular hydrolysis of organic macromolecules is often assumed to be the slow step in remineralization of organic matter. We tested this assumption by comparing the degradation of four polysaccharides (pullulan, laminarin, and two polysaccharides isolated from the marine cyanobacterium Synechococcus WH7335) to determine whether size, linkage position, or anomeric linkage affected rates or mechanisms of carbohydrate degradation by mixed cultures of anaerobic bacteria enriched from marine sediments. Gel permeation chromatography and nuclear magnetic resonance spectroscopy (NMR) were used to follow the extracellular conversion of high molecular weight polysaccharides to lower molecular weight polysaccharides and oligosaccharides which were subsequently remineralized. In all cases, substrate degradation was rapid. NMR spectra showed that preferential hydrolysis occurred at specific chemical linkages, and extracellular enzymatic hydrolysis of polysaccharides occurred far more rapidly than bacterial uptake and remineralization of the lower molecular weight oligosaccharides produced through enzymatic hydrolysis. Substrate size was not a significant determinant of remineralization rate: high molecular weight does not always correlate with slow degradation rate. The hypothesis that extracellular enzymatic hydrolysis is a slow step in the degradation of macromolecular organic matter in marine systems needs to be critically re-examined.

Effect of dietary fiber on microbial activity and microbial gas production in various regions of the gastrointestinal tract of pigs

The microbial activity, composition of the gas phase, and gas production rates in the gastrointestinal tract of pigs fed either a low- or a high-fiber diet were investigated. Dense populations of culturable anaerobic bacteria, high ATP concentrations, and high adenylate energy charges were found for the last third of the small intestine, indicating that substantial microbial activity takes place in that portion of the gut. The highest microbial activity (highest bacterium counts, highest ATP concentration, high adenylate energy charge, and low pH) was found in the cecum and proximal colon. Greater microbial activity was found in the stomach and all segments of the hindgut in the pigs fed the high-fiber diet than in the pigs fed the low-fiber diet. Considerable amounts of O2 were found in the stomach (around 5%), while the content of O2 in gas samples taken from all other parts of the gastrointestinal tract was < 1%. The highest concentrations and highest production rates for H2 were found in the last third of the small intestine. No methane could be detected in the stomach or the small intestine. The rate of production and concentration of methane in the cecum and the proximal colon were low, followed by a steady increase in the successive segments of the hindgut. A very good correlation between in vivo and in vitro measurements of methane production was found. The amount of CH4 produced by pigs fed the low-fiber diet was 1.4 liters/day per animal. Substantially larger amounts of CH4 were produced by pigs fed the high-fiber diet (12.5 liters/day)(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of the presence of protozoa on the rumen microbial population of cattle.

Three unfaunated cattle, fitted with a rumen fistula at the age of 9 months, were fed with hay wafer and pelleted concentrate. After two unfaunated cows were inoculated with a mixed population of protozoal species, changes of the bacterial populations as well as volatile fatty acids (VFA) and NH3 production in the rumen were followed for one month. The protozoal inoculum was prepared by using the rumen fluid obtained from a fistulated cattle given hay and concentrate ration. The third cow was kept protozoa-free as a control animal. A mixed population of protozoa established in the rumen of two cows within one month after inoculation. The number of total culturable anaerobic bacteria, amylolytic bacteria and cellulolytic bacteria decreased after the inoculation of protozoa. In contrast, the numbers of methanogenic bacteria, sulfate-reducing bacteria and anaerobic fungi increased after the inoculation of protozoa. The number of lactate utilizers decreased within 9 days after the inoculation of protozoa, and thereafter increased to the original level. Neither the number of xylanolytic bacteria nor pectinolytic bacteria changed after the inoculation of protozoa. The proportion of Ruminobacter amylophilus of total culturable bacterial population was reduced most significantly by the presence of protozoa, while that of Butyrivibrio spp., Selenomonas spp. and Clostridium spp. was increased. The NH3 and VFA production increased along with the proportion of butyrate after the inoculation of protozoa. The presence of protozoa affected markedly not only the number and constitution of bacterial populations, but also the NH3 and VFA production in the rumen.

Methanogenesis in granular sludge exposed to oxygen

Substrate competition between methanogenic and facultative bacteria under highly aerobic conditions was investigated in batch experiments. Natural mixed cultures of anaerobic bacteria immobilized in granular sludge were able to concurrently utilize oxygen and produce methane when supplied with ethanol as substrate. The most oxygen tolerant sludge converted 3 to 25% of substrate chemical oxygen demand to methane after 3 days while 23 to 2 mg 1−1 of dissolved oxygen were present in the media. The tolerance of methanogens to oxygen and their coexistence with facultative bacteria were evident even after long periods of oxygen exposure. Eventually, methane oxidizing bacteria developed in the co-culture. The consumption of oxygen by facultative bacteria, creating anaerobic microniches inside the granules, is hypothesized to protect the methanogens.

Methanogenesis in granular sludge exposed to oxygen

Substrate competition between methanogenic and facultative bacteria under highly aerobic conditions was investigated in batch experiments. Natural mixed cultures of anaerobic bacteria immobilized in granular sludge were able to concurrently utilize oxygen and produce methane when supplied with ethanol as substrate. The most oxygen tolerant sludge converted 3 to 25% of substrate chemical oxygen demand to methane after 3 days while 23 to 2 mg 1−1 of dissolved oxygen were present in the media. The tolerance of methanogens to oxygen and their coexistence with facultative bacteria were evident even after long periods of oxygen exposure. Eventually, methane oxidizing bacteria developed in the co-culture. The consumption of oxygen by facultative bacteria, creating anaerobic microniches inside the granules, is hypothesized to protect the methanogens.