Biodegradation of cyclic nitramines by tropical marine sediment bacteria

Undersea deposition of unexploded ordnance (UXO) constitutes a potential source of contamination of marine environments by hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). The goal of the present study was to determine microbial degradation of RDX and HMX in a tropical marine sediment sampled from a coastal UXO field in the region of Oahu Island in Hawaii. Sediment mixed cultures growing in marine broth 2216 (21°C) anaerobically mineralized 69% or 57% (CO₂, 25 days) of the total carbon of [UL-¹⁴ C]-RDX (100 M) or [UL-¹⁴ C]-HMX (10 M), respectively. As detected by PCR-DGGE, members of -proteobacteria (Halomonas), sulfate-reducing -proteobacteria (Desulfovibrio), firmicutes (Clostridium), and fusobacterium appeared to be dominant in RDX-enrichment and/or HMX-enrichment cultures. Among 22 sediment bacterial isolates screened for RDX and HMX biodegradation activity under anaerobic conditions, 5 were positive for RDX and identified as Halomonas (HAW-OC4), Marinobacter (HAW-OC1), Pseudoalteromonas (HAW-OC2 and OC5) and Bacillus (HAW-OC6) by their 16S rRNA genes. Sediment bacteria degraded RDX to N₂O and HCHO via the intermediary formation of hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and methylenedinitramine. The present findings demonstrate that cyclic nitramine contaminants are likely to be degraded upon release from UXO into tropical marine sediment.     

Biodegradation and conversion of alkanes and crude oil by a marine Rhodococcus

A hydrocarbon degrader isolated from a chronically oil-polluted marine site was identified as Rhodococcus sp. on the basis of morphology, fatty acid methyl ester pattern, cell wall analysis, biochemical tests and G + C content of DNA. It degraded upto 50% of the aliphatic fraction of Assam crude oil, in seawater supplemented with 35 mM nitrogen as urea and 0.1 mM phosphorus as dipotassium hydrogen orthophosphate, after 72 h at 30 ° and 150 revolutions per minute. The relative percentage of intracellular fatty acid was higher in hydrocarbon-grown cells compared to fructose-grown cells. The fatty acids C₁₆ , C16_{16 :1} C₁₈ and C_{18 : 1} were constitutively present regardless of the growth substrate. In addition to these constitutive acids, other intracellular fatty acids varied in correlation to the hydrocarbon chain length supplied as a substrate. When grown on odd carbon number alkanes, the isolate released only monocarboxylic acids into the growth medium. On even carbon number alkanes only dicarboxylic acids were produced.     

Towards efficient crude oil degradation by a mixed bacterial consortium

A laboratory study was undertaken to assess the optimal conditions for biodegradation of Bombay High (BH) crude oil. Among 130 oil degrading bacterial cultures isolated from oil contaminated soil samples, Micrococcus sp. GS2-22, Corynebacterium sp. GS5-66, Flavobacterium sp. DS5-73, Bacillus sp. DS6-86 and Pseudomonas sp. DS10-129 were selected for the study based on the efficiency of crude oil utilisation. A mixed bacterial consortium prepared using the above strains was also used. Individual bacterial cultures showed less growth and degradation than did the mixed bacterial consortium. At 1% crude oil concentration, the mixed bacterial consortium degraded a maximum of 78% of BH crude oil. This was followed by 66% by Pseudomonas sp. DS10-129, 59% by Bacillus sp. DS6-86, 49% by Micrococcus sp. GS2-22, 43% by Corynebacterium sp. GS5-66 and 41% by Flavobacterium sp. DS5-73. The percentage of degradation by the mixed bacterial consortium decreased from 78% to 52% as the concentration of crude oil was increased from 1% to 10%. Temperature of 30 degrees C and pH 7.5 were found to be optima for maximum biodegradation.     

Bioremediation of Aroclor 1242 by a consortium culture in marine sediment microcosm

Plant terpenes have proven to be effective in stimulation of polychlorinated biphenyls (PCBs) biodegradation in soil systems. However, data on the application of plant terpenes in marine sediments contaminated with PCBs remains limited. The aim of this study was to ascertain the roles of a PCB degrading consortium and plant terpenes in stimulation of PCB biodegradation in marine sediments. The consortium culture 1-2Mix (strains 1-2M and 1-2T in commensalism), a utilizer of biphenyl and a natural substrate was enriched and isolated from marine sediments from the Busan coast, South Korea. PCB degradation by this culture was shown to be more effectively induced by tangerine peel extract than other known substrates (limonene, pinene, and cymene). Coastal sediment microcosms inoculated with 1-2Mix were set up to elucidate the effect of the consortium and plant terpenes on degradation of Aroclor 1242. After four weeks, the highest removal rates of PCBs, compared with the control (autoclaved sediment and no inoculation of 1-2Mix), were observed in order of the inducers tested; biphenyl (71.1%), tangerine peel extract (69.5%), surfactant (66.0%), and limonene (63.0%). Bioaugmentation effect was doubled in the presence of natural substrates such as tangerine peel extract and limonene, indicating effectiveness of these substrates in biostimulation. It was concluded that the tangerine peel extract could replace biphenyl as a feasible induction substrate for effective remediation of PCBs in the marine sediment.     

Biodegradation of lindane by a native bacterial consortium isolated from contaminated river sediment

The aerobic biodegradation of lindane (γ-hexachlorocyclohexane) by a consortium of acclimated bacteria from sediment at a polluted site on the Suquia River, Cordoba, Argentina, is reported. The bacteria were acclimated for 30 days under aerobic conditions, using a minimal culture medium containing lindane (0.034 mM) as sole carbon source. Growth of the bacterial consortium decreased at a lindane concentration of 1.03 mM and was totally inhibited at 2.41 mM. The consortium showed initial lindane degradation rates of 4.92×10⁻³, 11.0×10⁻³ and 34.8×10⁻³ mM h⁻¹ when exposed to lindane concentrations of 0.069, 0.137 and 0.412 mM, respectively. Chloride concentration increased during aerobic biodegradation, indicating lindane mineralization. A metabolite identified as γ-2,3,4,5,6-pentachlorocyclohexene appeared during the first 24 h of biodegradation. Four different bacteria, identified as Sphingobacterium spiritivorum, Ochrobactrum anthropi, Bosea thiooxidans and Sphingomonas paucimobilis, were isolated. Pure strains of B. thiooxidans and S. paucimobilis degraded lindane after 3 days of aerobic incubation. This is the first report of lindane biodegradation by B. thiooxidans.     

Degradation of crude oil by an arctic microbial consortium

The ability of a psychrotolerant microbial consortium to degrade crude oil at low temperatures was investigated. The enriched arctic microbial community was also tested for its ability to utilize various hydrocarbons, such as long-chain alkanes (n-C₂₄ to n-C₃₄), pristane, (methyl-)naphthalenes, and xylenes, as sole carbon and energy sources. Except for o-xylene and methylnaphthalenes, all tested compounds were metabolized under conditions that are typical for contaminated marine liquid sites, namely at pH 6–9 and at 4–27°C. By applying molecular biological techniques (16S rDNA sequencing, DGGE) nine strains could be identified in the consortium. Five of these strains could be isolated in pure cultures. The involved strains were closely related to the following genera: Pseudoalteromonas (two species), Pseudomonas (two species), Shewanella (two species), Marinobacter (one species), Psychrobacter (one species), and Agreia (one species). Interestingly, the five isolated strains in different combinations were unable to degrade crude oil or its components significantly, indicating the importance of the four unculturable microorganisms in the degradation of single or of complex mixtures of hydrocarbons. The obtained mixed culture showed obvious advantages including stability of the consortium, wide range adaptability for crude oil degradation, and strong degradation ability of crude oil.     

Biodegradation of crude oil by soil microorganisms in the tropic

Five microorganisms, three bacteria and two yeasts, capable of degrading Tapis light crude oil were isolated from oil-contaminated soil in Bangkok, Thailand. Soil enrichment culture was done by inoculating the soil in mineral salt medium with 0.5% v/v Tapis crude oil as the sole carbon source. Crude oil biodegradation was measured by gas chromatography method. Five strains of pure microorganisms with petroleum degrading ability were isolated: three were bacteria and the other two were yeasts. Candida tropicalis strains 7Y and 15Y were identified as efficient oil degraders. Strain 15Y was more efficient, it was able to reduce 87.3% of the total petroleum or 99.6% of n-alkanes within the 7-day incubation period at room temperature of 25 ± 2 °C.     

Biodegradation of crude oil across a wide range of salinities by an extremely halotolerant bacterial consortium MPD-M,immobilized onto polypropylene fibers

The bacterial consortium MPD-M, isolated from sediment associated with Colombian mangrove roots, was effective in the treatment of hydrocarbons in water with salinities varying from 0 to 180 g L(-1). Where the salinity of the culture medium surpassed 20 g L(-1), its effectiveness increased when the cells were immobilized on polypropylene fibers. Over the range of salinity evaluated, the immobilized cells significantly enhanced the biodegradation rate of crude oil compared with free-living cells, especially with increasing salinity in the culture medium. Contrary to that observed in free cell systems, the bacterial consortium MPD-M was highly stable in immobilized systems and it was not greatly affected by increments in salinity. Biodegradation was evident even at the highest salinity evaluated (180 g L(-1)), where biodegradation was between 4 and 7 times higher with immobilized cells compared to free cells. The biodegradation of pristane (PR) and phytane (PH) and of the aromatic fraction was also increased using cells immobilized on polypropylene fibers.     

Biodegradation of naphthalene by enriched marine denitrifying bacteria

Numerous studies have been investigated on the PAHs biodegradation in aerobic and anaerobic environments; however, the biodegradation of PAHs under anoxic conditions, especially denitrifying conditions, has drawn less attention. In this study, four series of batch experiments were conducted to investigate the effect of temperature, pH, naphthalene concentration and nitrate concentration on the naphthalene degradation under denitrification condition. Our results showed that the degradation of naphthalene was most favorable at pH 7 and 25 °C. Results also indicated that 30 mg/l naphthalene inhibited the biodegradation and the removal efficiency was only 20.2%. Significant degradation (91.7% and 96.3%) of naphthalene occurred when nitrate concentrations were 1.0 and 5.0 mM. Moreover, the maximum degradation rates were 0.13 and 0.18 mg-NAP/(l h) depending on the concentration of nitrate. Based on 16S rDNA analysis, the denitrifying enriched culture was mainly composed of ??-Proteobacteria (19 clones out of a total of 23 clones) and Actinobacteria (4 clones). Using a primer set specific for naphthalene degrading functional gene nahAc, two operational taxonomy units were obtained in the clone library of nahAc. Both of them were closely related to nahAc genes of known species of Pseudomonas. Quantitative polymerase chain reaction (qPCR) was employed to quantify the change of naphthalene-degrading population during the degradation of naphthalene using nahAc gene as the biomarker. The maximum degradation rate and removal efficiency were strongly correlated with nahAc gene copy number, with R² of 0.69 and 0.79, respectively.     

Degradation of crude oil by marine cyanobacteria

The marine cyanobacteria Oscillatoria salina Biswas, Plectonema terebrans Bornet et Flahault and Aphanocapsa sp. degraded Bombay High crude oil when grown in artificial seawater nutrients as well as in plain natural seawater. Oil removal was measured by gravimetric and gas chromatographic methods. Around 45-55% of the total fractions of crude oil (containing 50% aliphatics, 31% waxes and bitumin, 14% aromatics and 5% polar compounds) were removed in the presence of these cultures within 10 days. Between 50% and 65% of pure hexadecane (model aliphatic compound) and 20% and 90% of aromatic compounds (anthracene and phenantherene) disappeared within 10 days. Mixed cultures of the three cyanobacterial species removed over 40% of the crude. Additionally, these cultures formed excellent cyanobacterial mats when grown in mixed cultures, and thus have the potential for use in mitigating oil pollution on seashores, either individually or in combination.     

Biodegradation of fuel oil hydrocarbons by a mixed bacterial consortium in sandy and loamy soils

The aerobic degradation of light fuel oil in sandy and loamy soils by an environmental bacterial consortium was investigated. Soils were spiked with 1 or 0.1% of oil per dry weight of soil. Acetone extracts of dried soils were analyzed by GC and the overall degradation was calculated by comparison with hydrocarbon recovery from uninoculated soils. In sandy soils, the sum of alkanes n-C(12) to n-C(23) was degraded to about 45% within 6 days at 20 degrees C and to 27-31% within 28 days, provided that moisture and nutrients were replenished. Degradation in loamy soil was about 12% lower. The distribution of recovered alkanes suggested a preferential degradation of shorter chain molecules (n-C(12) to n-C(16)) by the bacterial consortium. Partial 16S rDNA sequences indicated the presence of strains of Pseudomonas aeruginosa, Pseudomonas citronellolis, and Stenotrophomonas maltophilia. Toxicity tests using commercial standard procedures showed a moderate inhibition of bacterial activity. The study showed the applicability of a natural microbial community for the degradation of oil spills into soils at ambient temperatures.     

Biodegradation of polyalcohol ethoxylate by a wastewater microbial consortium

Polyalcohol ethoxylate (PAE), an anionic surfactant, is the primary component in most laundry and dish wash detergents and is therefore highly loaded in domestic wastewater. Its biodegradation results in the formation of several metabolites and the fate of these metabolites through wastewater treatment plants, graywater recycling processes, and in the environment must be clearly understood. Biodegradation pathways for PAE were investigated in this project with a municipal wastewater microbial consortium. A microtiter-based oxygen sensor system was utilized to determine the preferential use of potential biodegradation products. Results show that while polyethylene glycols (PEGs) were readily degraded by PAE acclimated microorganisms, most of the carboxylic acids tested were not degraded. Biodegradation of PEGs suggests that hydrophobe–hydrophile scission was the dominant pathway for PAE biodegradation in this wastewater community. Ethylene glycol (EG) and diethylene glycol (DEG) were not utilized by microbial populations capable of degrading higher molecular weight EGs. It is possible that EG and DEG may accumulate. The microtiter-based oxygen sensor system was successfully utilized to elucidate information on PAE biodegradation pathways and could be applied to study biodegradation pathways for other important contaminants.     

Degradation of nonane by bacteria from Antarctic marine sediment

A microbial enrichment culture tolerant of petroleum hydrocarbons was developed from sediment collected near Casey Station, Antarctica. To select cold-adapted microbes that would degrade diesel, enrichments were cultured at 0°C during six successive transfers to fresh medium, with Special Antarctic Blend diesel (SAB) as the sole carbon source. Biodegradation of components of the SAB was then measured in microcosms inoculated with the enrichment culture. After 16 weeks, the amount of biodegradation was small, but nonane (a C9 alkane) had degraded significantly more in inoculated microcosms than in sterile controls. DNA was then extracted from the enrichment cultures and a fragment of the 16S rRNA gene was amplified for denaturing gradient gel electrophoresis. Bands were excised from the gel and, following sequencing, were found to belong to the genera Pseudomonas and Colwellia.     

Metagenomic analysis of microbial consortium from natural crude oil that seeps into the marine ecosystem offshore Southern California

Crude oils can be major contaminants of the marine ecosystem and microorganisms play a significant role in the degradation of its main constituents. To increase our understanding of the microbial hydrocarbon degradation process in the marine ecosystem, we collected crude oil from an active seep area located in the Santa Barbara Channel (SBC) and generated a total of about 52 Gb of raw metagenomic sequence data. The assembled data comprised ~500 Mb, representing ~1.1 million genes derived primarily from chemolithoautotrophic bacteria. Members of Oceanospirillales, a bacterial order belonging to the Deltaproteobacteria, recruited less than 2% of the assembled genes within the SBC metagenome. In contrast, the microbial community associated with the oil plume that developed in the aftermath of the Deepwater Horizon (DWH) blowout in 2010, was dominated by Oceanospirillales, which comprised more than 60% of the metagenomic data generated from the DWH oil plume. This suggests that Oceanospirillales might play a less significant role in the microbially mediated hydrocarbon conversion within the SBC seep oil compared to the DWH plume oil. We hypothesize that this difference results from the SBC oil seep being mostly anaerobic, while the DWH oil plume is aerobic. Within the Archaea, the phylum Euryarchaeota, recruited more than 95% of the assembled archaeal sequences from the SBC oil seep metagenome, with more than 50% of the sequences assigned to members of the orders Methanomicrobiales and Methanosarcinales. These orders contain organisms capable of anaerobic methanogenesis and methane oxidation (AOM) and we hypothesize that these orders – and their metabolic capabilities – may be fundamental to the ecology of the SBC oil seep.     

Biodegradation of crude oil saturated fraction supported on clays

The role of clay minerals in crude oil saturated hydrocarbon removal during biodegradation was investigated in aqueous clay/saturated hydrocarbon microcosm experiments with a hydrocarbon degrading microorganism community. The clay minerals used for this study were montmorillonite, palygorskite, saponite and kaolinite. The clay mineral samples were treated with hydrochloric acid and didecyldimethylammonium bromide to produce acid activated- and organoclays respectively which were used in this study. The production of organoclay was restricted to only montmorillonite and saponite because of their relative high CEC. The study indicated that acid activated clays, organoclays and unmodified kaolinite, were inhibitory to biodegradation of the hydrocarbon saturates. Unmodified saponite was neutral to biodegradation of the hydrocarbon saturates. However, unmodified palygorskite and montmorillonite were stimulatory to biodegradation of the hydrocarbon saturated fraction and appears to do so as a result of the clays’ ability to provide high surface area for the accumulation of microbes and nutrients such that the nutrients were within the ‘vicinity’ of the microbes. Adsorption of the saturated hydrocarbons was not significant during biodegradation.     

Biodegradation of 1,1,1-trichloroethane by a carbon tetrachloride-degrading denitrifying consortium

A denitrifying consortium capable of degrading carbon tetrachloride (CT) was shown to also degrade 1,1,1-trichloroethane (TCA). Fed-batch experiments demonstrated that the specific rate of TCA degradation by the consortium was comparable to the specific rate of CT degradation (approximately 0.01 L/gmol/min) and was independent of the limiting nutrient. Although previous work demonstrated that 4-50% of CT transformed by the consortium was converted to chloroform (CF), no reductive dechlorination products were detected during TCA degradation, regardless of the limiting nutrient. The lack of chlorinated TCA degradation products implies that the denitrifying consortium possesses an alternate pathway for the degradation of chlorinated solvents which does not involve reductive dechlorination. Copyright 1998 John Wiley & Sons, Inc.     

Biodegradation of a crude oil by three microbial consortia of different origins and metabolic capabilities

Microbial consortia were obtained three by sequential enrichment using different oil products. Consortium F1AA was obtained on a heavily saturated fraction of a degraded crude oil; consortium TD, by enrichment on diesel and consortium AM, on a mixture of five polycyclic aromatic hydrocarbons [PAHs]. The three consortia were incubated with a crude oil in order to elucidate their metabolic capabilities and to investigate possible differences in the biodegradation of these complex hydrocarbon mixtures in relation to their origin. The efficiency of the three consortia in removing the saturated fraction was 60% (F1AA), 48% (TD) and 34% (AM), depending on the carbon sources used in the enrichment procedures. Consortia F1AA and TD removed 100% of n-alkanes and branched alkanes, whereas with consortium AM, 91% of branched alkanes remained. Efficiency on the polyaromatic fraction was 19% (AM), 11% (TD) and 7% (F1AA). The increase in aromaticity of the polyaromatic fraction during degradation of the crude oil by consortium F1AA suggested that this consortium metabolized the aromatic compounds primarily by oxidation of the alkylic chains. The 500-fold amplification of the inocula from the consortia by subculturing in rich media, necessary for use of the consortia in bioremediation experiments, showed no significant decrease in their degradation capability. Journal of Industrial Microbiology & Biotechnology (2002) 28, 252–260 DOI: 10.1038/sj/jim/7000236 Received 12 July 2001/ Accepted in revised form 11 November 2001     

Thermophilic sulfate-reducing bacteria in cold marine sediment

Abstract Sulfate reduction was measured with the ³⁵SO₄²⁻ -tracer technique in slurries of sediment from Aarhus Bay, Denmark, where seasonal temperatures range from 0° to 15°C. The incubations were made at temperatures from 0°C to 80°C in temperature increments of 2°C to search for presence of psychrophilic, mesophilic and thermophilic sulfate-reducing bacteria. Detectable activity was initially only in the mesophilic range, but after a lag phase sulfate reduction by thermophilic sulfate-reducing bacteria were observed. No distinct activity of psychrophilic sulfate-reducing bacteria was detected. Time course experiments showed constant sulfate reduction rates at 4°C and 30°C, whereas the activity at 60°C increased exponentially after a lag period of one day. Thermophilic, endospore-forming sulfate-reducing bacteria, designated strain P60, were isolated and characterized as D esulfotomaculum kuznetsovii . The temperature response of growth and respiration of strain P60 agreed well with the measured sulfate reduction at 50°–70°C. Bacteria similar to strain P60 could thus be responsible for the measured thermophilic activity. The viable population of thermophilic sulfate-reducing bacteria and the density of their spores was determined in most probable number (MPN) dilutions. The density was 2.8·10⁴ cells·.g⁻¹ fresh sediment, and the enumerations suggested that they were all present as spores. This result agrees well with the observed lag period in sulfate reduction above 50°C. No environment with temperatures supporting the growth of these thermophiles is known in the region around Aarhus Bay.     

Biodegradation of propanol and isopropanol by a mixed microbial consortium

The aerobic biodegradation of high concentrations of 1-propanol and 2-propanol (IPA) by a mixed microbial consortium was investigated. Solvent concentrations were one order of magnitude greater than any previously reported in the literature. The consortium utilized these solvents as their sole carbon source to a maximum cell density of 2.4 × 10⁹ cells ml⁻¹. Enrichment experiments with propanol or IPA as carbon sources were carried out in batch culture and maximum specific growth rates (μ_max) calculated. At 20 °C, μ _max values were calculated to be 0.0305 h⁻¹ and 0.1093 h⁻¹ on 1% (v/v) IPA and 1-propanol, respectively. Growth on propanol and IPA was carried out between temperatures of 10 °C and 45 °C. Temperature shock responses by the microbial consortium at temperatures above 45 °C were demonstrated by considerable cell flocculation. An increase in propanol substrate concentration from 1% (v/v) to 2% (v/v) decreased the μ _max from 0.1093 h⁻¹ to 0.0715 h⁻¹. Maximum achievable biodegradation rates of propanol and IPA were 6.11 × 10⁻³% (v/v) h⁻¹ and 2.72 × 10⁻³% (v/v) h⁻¹, respectively. Generation of acetone during IPA biodegradation commenced at 264 h and reached a maximum concentration of 0.4% (v/v). The results demonstrate the potential of mixed microbial consortia in the bioremediation of solvent-containing waste streams. Received: 14 December 1999 / Received revision: 3 April 2000 / Accepted: 7 April 2000