RDX degrading microbial communities and the prediction of microorganisms responsible for RDX bioremediation

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has caused significant soil and groundwater contamination. To remediate these sites, there is a need to determine which microorganisms are responsible for in situ biodegradation of RDX to enable the appropriate planning of bioremediation efforts. Here, studies are examined that have reported on the microbial communities linked with RDX biodegradation. Dominant microorganisms across samples are discussed and summarized. This information is then compared to current knowledge on RDX degrading isolates to predict which organisms may be responsible for RDX degradation in soils and groundwater. From the phyla with known RDX degrading isolates, Firmicutes and Proteobacteria (particularly Gammaproteobacteria) were the most dominant organisms in many contaminated site derived samples. Organisms in the phyla Deltaproteobacteria, Alphaproteobacteria and Actinobacteria were dominant in these studies less frequently. Notably, organisms within the class Betaproteobacteria were dominant in many samples and yet this class does not appear to contain any known RDX degraders. This analysis is valuable for the future development of molecular techniques to track the occurrence and abundance of RDX degraders at contaminated sites.     

Anaerobic degradation of dehydrodiisoeugenol by rumen bacteria

The degradation of dehydrodiisoeugenol (DDIE) by cow rumen bacteria was studied under strictly anaerobic conditions. After two days of cultivation, about 23% of DDIE (1.2 mM) was degraded to volatile fatty acids (VFA) such as acetic acid, propionic acid and butyric acid. The aromatic intermediates were vanillic acid, 5-methylvanillin and 3-methyl-4-hydroxybenzaldehyde, which suggested that the coumaran ring in DDIE was cleaved during degradation. These results indicate that the rumen anaerobes can degrade this lignin-related dimer to monoaromatic compounds and VFA.     

Chitinolytic enzymes produced by ovine rumen bacteria

Two strains of clostridia, isolated from the rumen fluid of sheep as potential antagonists toward anaerobic fungi showed a complete array of chitinolytic enzymes. Enzyme tests in cultures demonstrated endochitinase, exochitinase,N-acetylglucosaminidase, chitosanase and chitin deacetylase activities mainly in the extracellular fractions. In all samples, the highest was the activity of exochitinase (600–1100 nmol mL⁻¹ h⁻¹); the activity of endochitinase (280–500 nmol mL⁻¹ h⁻¹) was also significant. Chitinases were stimulated in the presence of reducing compounds and no dependence on cations was observed. In both strains different isoforms of chitinases of molar mass 36–96 kDa were detected. The chitinases from our isolates lyzed cell walls of anaerobic fungiin vitro and inhibited the activity of fungal β-1,4-endoglucanases. Of the two bacteria examined, one was more effective in both antifungal effects.     

Lyophilization of rumen fluid for use in culture media

The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g.     

Anaerobic digestion of cattail with rumen culture in the presence of heavy metals

The anaerobic digestion of cattail by rumen cultures in the presence of Cu(II), Cd(II) or Cr(VI) was investigated in this study. Three cases were respectively observed for the different metal dosages: promoted cattail degradation and methane production at a low heavy metal concentration, e.g., Cu(II) 2.4 mg/l, Cd(II) 1.6 mg/l, Cr(VI) 4.0 mg/l; reduced cattail degradation efficiency and methane production at a middle metal level; a severe inhibition to the cattail degradation at a high heavy metal dosage. The inhibition kinetics of Cu(II) on the digestion of cattail by rumen cultures was also analyzed and a simplified Andrews equation was used to describe such an inhibition. The inhibition constants for Cu(II) on the degradation of cattail, production of volatile fatty acids and formation of methane were estimated as 7.4, 9.5 and 6.4 mg/l, respectively. Comparative experimental results suggest that the order of toxicity degree of the tested metals on the rumen cultures was: Cd(II) > Cu(II) > Cr(VI).     

Lyophilization of rumen fluid for use in culture media.

The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g.     

Anaerobic degradation of naphthalene by a pure culture of a novel type of marine sulphate-reducing bacterium

Incubation of marine sediment in anoxic, sulphate-rich medium in the presence of naphthalene resulted in the enrichment of sulphate-reducing bacteria. Pure cultures with short, oval cells (1.3 by 1.3–1.9 μm) were isolated that grew with naphthalene as the only organic carbon source and electron donor for sulphate reduction to sulphide. One strain, NaphS2, was characterized. It affiliated with completely oxidizing sulphate-reducing bacteria of the δ-subclass of the Proteobacteria, as revealed by 16S rRNA sequence analysis. 2-Naphthoate, benzoate, pyruvate and acetate were used in addition to naphthalene. Quantification of substrate consumption, sulphide formation and formed cell mass revealed that naphthalene was completely oxidized with sulphate as the electron acceptor.     

Anaerobic degradation of o-phenylphenol by mixed and pure cultures

Summary An anaerobic enrichment culture that degraded 0.4 mmol/l per day of o-phenylphenol was selected from sediment of a waste water pond of a sugar factory. From the consortium an o-phenylphenol-degrading bacterium, strain B10, was isolated. Strain B10 could not degrade other aromatic substances, including phenylacetic acid, benzoate, o-hydroxybenzoate, p-hydroxybenzoate and phenol. Best growth was observed with glucose, pyruvate, lactate, methanol and H₂/CO₂ as substrates. o-Phenylphenol was slowly degraded if supplied as the only carbon source and was cometabolized in the presence of >5 mmol/l glucose. Strain B10 has not yet been assigned to a known species or family.     

Anaerobic arsenite oxidation by novel denitrifying isolates

Autotrophic microorganisms have been isolated that are able to derive energy from the oxidation of arsenite [As(III)] to arsenate [As(V)] under aerobic conditions. Based on chemical energetics, microbial oxidation of As(III) can occur in the absence of oxygen, and may be relevant in some environments. Enrichment cultures were established from an arsenic contaminated industrial soil amended with As(III) as the electron donor, inorganic C as the carbon source and nitrate as the electron acceptor. In the active enrichment cultures, oxidation of As(III) was stoichiometrically coupled to the reduction of NO(3) (-). Two autotrophic As(III)-oxidizing strains were isolated that completely oxidized 5 mM As(III) within 7 days under denitrifying conditions. Based on 16S rRNA gene sequencing results, strain DAO1 was 99% related to Azoarcus and strain DAO10 was most closely related to a Sinorhizobium. The nitrous oxide reductase (nosZ) and the RuBisCO Type II (cbbM) genes were successfully amplified from both isolates underscoring their ability to denitrify and fix CO(2) while coupled to As(III) oxidation. Although limited work has been done to examine the diversity of anaerobic autotrophic oxidizers of As(III), this process may be an important component in the biological cycling of arsenic within the environment.     

Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture.

A total of 10 strains of rumen bacteria, Selenomonas ruminantium HD4, Megasphaera elsdenii B159, Butyrivibrio fibrisolvens A38, Streptococcus bovis JB1, Lactobacillus vitulinus GA1, Bacteroides ruminicola B14, B. ruminicola GA33, Ruminococcus albus 7, Ruminococcus flavefaciens C94, and Bacteroides succinogenes S85, were grown in energy-limiteH of the medium reservoir was lowered approximately 0.3 pH units, and the energy source concentration remaining in the culture vessel, optical density, cell mass, and pH were determined. A low pH appeared to have a detrimental effect on cell yields. Large variations were seen among strains in both the magnitude of yield depressions at lower pH values and in the pH at which the culture washed out. Lactate analysis indicated ta are discussed in relation to the effect of pH on the efficiency of protein synthesis in the rumen and rumen microbial ecology.     

Proteinase isoenzyme patterns of Bacteroides nodosus: distinction between ovine virulent isolates, ovine benign isolates and bovine isolates

Bacteroides nodosus isolates from ovine virulent footrot and ovine benign footrot and bovine isolates of low virulence for sheep were distinguishable from each other by their proteinase isoenzyme patterns after polyacrylamide gel electrophoresis. Variants of low virulence were not always distinguishable from their virulent parent strains. The molecular weights of the isoenzymes ranged from 70000 to 129000. The relationship of isoenzyme patterns to virulence is discussed.     

Anaerobic degradation of phenylacetic acid by mixed and pure cultures

Summary A phenylacetic acid-degrading mixed culture was enriched from effluent of an anaerobic reactor for the treatment of waste water from cellulose bleaching. From this consortium a phenylacetic acid-degrading pure culture, strain DSU3, was isolated and, due to its typical morphology and substrate spectrum, tentatively classified as a Desulfosarcina sp. It could grow on and degrade phenylacetic acid, cyclohexane carboxylate, cyclohexylacetate, benzoate, fumaric acid and several volatile fatty acids, while phenol, o-hydroxybenzoate, p-hydroxybenzoate and glucose were not utilized. Production of mandelic acid from phenylacetic acid by the enrichment culture and utilization of benzoate, an intermediate of the mandelic acid pathway, by strain DSU3 may presumably indicate degradation of phenylacetic acid via the mandelic acid pathway.     

Anaerobic digestion of cellulose by pure and mixed bacterial cultures

Summary By enrichment technique, nine anaerobic mixed bacterial cultures were isolated, five of which showed stable cellulolysis. All cultures fermented cellulose and produced different fermentative products. Mixed culture BOC 25 yielded major levels of acetate and ethanol (39.6 and 12.0 mmol/l, respectively) and minor levels of propionate (2.5 mmol/l) and digested filter paper cellulose to the extent of 32.5% w/v. BOC 25 digested cellulosic and lignocellulosic substrates and produced filter paper cellulase, carboxymethyl cellulase, Avicelase and -glucosidase. Strain DC 25, a cellulolyticClostridium was purified from one of the mixed cultures. The fermentation products of DC 25 from cellulose, cellobiose or glucose were ethanol, acetate, formate, H₂ and CO₂.     

Anaerobic degradation of veratrylglycerol-beta-guaiacyl ether and guaiacoxyacetic acid by mixed rumen bacteria

Veratrylglycerol-beta-guaiacyl ether (0.2 g/liter), a lignin model compound, was found to be degraded by mixed rumen bacteria in a yeast extract medium under strictly anaerobic conditions to the extent of 19% within 24 h. Guaiacoxyacetic acid, 2-(o-methoxyphenoxy)ethanol, vanillic acid, and vanillin were detected as degradation products of veratrylglycerol-beta-guaiacyl ether by thin-layer chromatography, gas chromatography, and gas chromatography-mass spectrometry. Guaiacoxyacetic acid (0.25 g/liter), when added into the medium as a substrate, was entirely degraded within 36 h, resulting in the formation of phenoxyacetic acid, guaiacol, and phenol. These results suggest that the beta-arylether bond, an important intermonomer linkage in lignin, can be cleaved completely by these rumen anaerobes.     

Anaerobic degradation of cellulose by mixed culture

A mixed culture in which cellulose is capable of being converted to methane and carbon dioxide was obtained from an inoculum procured from a sewage-treatment plant and maintained in a synthetic medium containing tissue paper and an inorganic salt and vitamin mixture. The culture was tested for its ability to degrade 12 different paper and cotton products under batch conditions in 3-l anaerobic fermenters. This culture degraded 6-8 mmol/l per week of cellulose, expressed as glucose equivalents, with total gas yields of 0.3 m3/kg of cellulose degraded. The gas produced contained between 56 and 59% of methane. Maximum cellulose degradation occurred at chemical oxygen demand:nitrogen:phosphorus level of 80:5:1 and was adversely affected by high stirring rate. Also the presence of higher proportions of lignin in cellulose products adversely affected the ability of this culture to degrade cellulose.     

Anaerobic degradation of toluene by pure cultures of denitrifying bacteria

Several denitrifying Pseudomonas spp., isolated with various aromatic compounds, were tested for the ability to degrade toluene in the absence of molecular oxygen. Four out of seven strains were able to degrade toluene in the presence of N₂O. More than 50% of the ¹⁴C from ring-labelled toluene was released as CO₂, and up to 37% was assimilated into cell material. Furthermore it was demonstrated for two strains that they were able to grow on toluene as the sole carbon and energy source in the presence of N₂O. Suspensions of cells pre-grown on toluene degraded toluene, benzaldehyde or benzoate without a lag phase and without accumulation of intermediates. p-Cresol, p-hydroxybenzylalcohol, p-hydroxybenzaldehyde or p-hydroxybenzoate was degraded much slower or only after distinct lag times. In the presence of fluoroacetate [¹⁴C]toluene was transformed to [¹⁴C]benzoate, which suggests that anaerobic toluene degradation proceeds through oxidation of the methyl side chain to benzoate.     

Hydration of oleic acid by a Selenomonas strain from the ovine rumen

J.A. HUDSON, C.A.M. MACKENZIE AND K.N. JOBLIN. 1996. A Selenomonas sp., isolated from the ovine rumen, was characterized with regard to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydration occurred only in stationary phase in a medium containing 0.1%, 0.5% (w/v) galactose or 0.5% (w/v) glucose, but not in a medium containing 1% galactose. Growth under a hydrogen headspace did not result in the production of stearic acid, the biohydrogenated product of oleic acid. Linoleic and linolenic acids (0.1% v/v) were not hydrated. It is concluded that the growing bacterium is unlikely to contribute to oleic acid hydration in the rumen.     

In vitro culture and interferon-tau secretion by ovine blastocysts

Embryos were collected surgically from superovulated ewes on days 7, 8, 9 and 10 (oestrus=day 0) to evaluate the long-term culture and interferon-tau (IFN-τ) secretion of ovine blastocysts. Embryos were cultured in 2 ml Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 15 mg/ml BSA in 5% CO₂ in air or DMEM without BSA in 5% CO₂, 7% O₂, and 88% N₂ at 39 °C, examined daily for morphological features and diameter and each day placed into fresh culture medium to enable daily measurement of IFN-τ secretion. Nine day-7 and two day-9 embryos were cultured in DMEM with BSA and nine continued to develop. The day-7 embryos reached a mean maximum diameter of 370.0±50.25 μm after 4 days in culture. Nineteen day-7, 12 day-8 and five day-10 embryos were cultured in DMEM without BSA but only six of the day-7 and one day-8 embryos survived for at least 7 days with the former reaching a mean maximum diameter on day 7 of 357±43.75 μm whereas all five day-10 embryos survived for at least 7 days reaching a mean maximum diameter on day 6 of 1038±155.8 μm. An anti-viral assay and a ELISA for IFN-τ were developed. There was a considerable variation in the time of onset and amount of IFN-τ secreted that did not seem to be related to embryo morphology. Of 28 day-7 embryos cultured, 60.7% were secreting IFN-τ after 1 day of culture whereas 87.5% of day-8 embryos were secreting IFN-τ after 1 day in culture. The mean concentration of IFN-τ secreted by day-8 embryos after 1 day in culture (10.99±2.55 ng/ml) was not significantly different to day-7 embryos after 2 days in culture (8.8±1.75 ng/ml).