Carbon nanotubes accelerate methane production in pure cultures of methanogens and in a syntrophic coculture

Carbon materials have been reported to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens improving methane production in anaerobic processes. In this work, the effect of increasing concentrations of carbon nanotubes (CNT) on the activity of pure cultures of methanogens and on typical fatty acid‐degrading syntrophic methanogenic coculture was evaluated. CNT affected methane production by methanogenic cultures, although acceleration was higher for hydrogenotrophic methanogens than for acetoclastic methanogens or syntrophic coculture. Interestingly, the initial methane production rate (IMPR) by Methanobacterium formicicum cultures increased 17 times with 5 g·L^?1 CNT. Butyrate conversion to methane by Syntrophomonas wolfei and Methanospirillum hungatei was enhanced (～1.5 times) in the presence of CNT (5 g·L^?1), but indications of DIET were not obtained. Increasing CNT concentrations resulted in more negative redox potentials in the anaerobic microcosms. Remarkably, without a reducing agent but in the presence of CNT, the IMPR was higher than in incubations with reducing agent. No growth was observed without reducing agent and without CNT. This finding is important to re‐frame discussions and re‐interpret data on the role of conductive materials as mediators of DIET in anaerobic communities. It also opens new challenges to improve methane production in engineered methanogenic processes.     

Characterization of marine isoprene‐degrading communities

Isoprene is a volatile and climate‐altering hydrocarbon with an atmospheric concentration similar to that of methane. It is well established that marine algae produce isoprene; however, until now there was no specific information about marine isoprene sinks. Here we demonstrate isoprene consumption in samples from temperate and tropical marine and coastal environments, and furthermore show that the most rapid degradation of isoprene coincides with the highest rates of isoprene production in estuarine sediments. Isoprene‐degrading enrichment cultures, analysed by denaturing gradient gel electrophoresis and 454 pyrosequencing of the 16S rRNA gene and by culturing, were generally dominated by Actinobacteria, but included other groups such as Alphaproteobacteria and Bacteroidetes, previously not known to degrade isoprene. In contrast to specialist methane‐oxidizing bacteria, cultivated isoprene degraders were nutritionally versatile, and nearly all of them were able to use n‐alkanes as a source of carbon and energy. We therefore tested and showed that the ubiquitous marine hydrocarbon‐degrader, Alcanivorax borkumensis, could also degrade isoprene. A mixture of the isolates consumed isoprene emitted from algal cultures, confirming that isoprene can be metabolized at low, environmentally relevant concentrations, and suggesting that, in the absence of spilled petroleum hydrocarbons, algal production of isoprene could maintain viable populations of hydrocarbon‐degrading microbes. This discovery of a missing marine sink for isoprene is the first step in obtaining more robust predictions of its flux, and suggests that algal‐derived isoprene provides an additional source of carbon for diverse microbes in the oceans.     

Contributions to the biotechnolgical production of sweeteners from stevia rebaudiana bertoni. III. Accumulation of secondary metabolites by means of a precursor and by elicitation of cell cultures

Gibberellin A₃ fed to cell suspension cultures of Stevia rebaudiana showed a fast conversion to stevioside. The product was detected within one day after gibberellin addition and achieved its maximum concentration after one week. However, using special production media (without precursor or elicitor), stevioside was produced only two to seven weeks after inoculation [1]. Elicitation of suspension cultures was performed with Stevia specific and non-specific fungi and with yeast extract. Although production of some secondary metabolites was induced, stevioside was not synthesized.     

The Potential of Bacterial Isolates for Emulsification with a Range of Hydrocarbons

A study was undertaken to investigate the distribution of biosurfactant producing and crude oil degrading bacteria in the oil contaminated environment. This research revealed that hydrocarbon contaminated sites are the potent sources for oil degraders. Among 32 oil degrading bacteria isolated from ten different oil contaminated sites of gasoline and diesel fuel stations, 80% exhibited biosurfactant production. The quantity and emulsification activity of the biosurfactants varied. Pseudomonas sp. DS10‐129 produced a maximum of 7.5 ± 0.4 g/l of biosurfactant with a corresponding reduction in surface tension from 68 mN/m to 29.4 ± 0.7 mN/m at 84 h incubation. The isolates Micrococcus sp. GS2‐22, Bacillus sp. DS6‐86, Corynebacterium sp. GS5‐66, Flavobacterium sp. DS5‐73, Pseudomonas sp. DS10‐129, Pseudomonas sp. DS9‐119 and Acinetobacter sp. DS5‐74 emulsified xylene, benzene, n‐hexane, Bombay High crude oil, kerosene, gasoline, diesel fuel and olive oil. The first five of the above isolates had the highest emulsification activity and crude oil degradation ability and were selected for the preparation of a mixed bacterial consortium, which was also an efficient biosurfactant producing oil emulsifying and degrading culture. During this study, biosurfactant production and emulsification activity were detected in Moraxella sp., Flavobacterium sp. and in a mixed bacterial consortium, which have not been reported before.     

Isolation and Characterization of a New Benzene,Toluene, and Ethylbenzene Degrading Bacterium, <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. B113

A bacterium designated strain B113, able to degrade benzene, toluene, and ethylbenzene compounds (BTE), was isolated from gasoline-contaminated sediment at a gas station in Geoje, Korea. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belonged to the genus Acinetobacter. The biodegradation rates of benzene, toluene, and ethylbenzene were relatively low in MSB broth, but the addition of yeast extract had a substantial impact on the biodegradation of BTE compounds, which suggested that yeast extract might provide a factor that was necessary for its growth or BTE biodegradation activity. However, interestingly, the biodegradation of BTE compounds occurred very quickly in slurry systems amended with sterile soil. Moreover, if soil was combusted first to remove organic matters, the enhancement effect on BTE biodegradation was lost, indicating that some insoluble organic compounds were probably beneficial for BTE degradation in contaminated sediment. This study suggests that strain B113 may play an important role for biodegradation of BTE in the contaminated site.     

The quantitative significance of Syntrophaceae and syntrophic partnerships in methanogenic degradation of crude oil alkanes

Libraries of 16S rRNA genes cloned from methanogenic oil degrading microcosms amended with North Sea crude oil and inoculated with estuarine sediment indicated that bacteria from the genera Smithella (Deltaproteobacteria, Syntrophaceace) and Marinobacter sp. (Gammaproteobacteria) were enriched during degradation. Growth yields and doubling times (36 days for both Smithella and Marinobacter) were determined using qPCR and quantitative data on alkanes, which were the predominant hydrocarbons degraded. The growth yield of the Smithella sp. [0.020 g(cell-C)/g(alkane-C)], assuming it utilized all alkanes removed was consistent with yields of bacteria that degrade hydrocarbons and other organic compounds in methanogenic consortia. Over 450 days of incubation predominance and exponential growth of Smithella was coincident with alkane removal and exponential accumulation of methane. This growth is consistent with Smithella's occurrence in near surface anoxic hydrocarbon degrading systems and their complete oxidation of crude oil alkanes to acetate and/or hydrogen in syntrophic partnership with methanogens in such systems. The calculated growth yield of the Marinobacter sp., assuming it grew on alkanes, was [0.0005 g(cell-C)/g(alkane-C)] suggesting that it played a minor role in alkane degradation. The dominant methanogens were hydrogenotrophs (Methanocalculus spp. from the Methanomicrobiales). Enrichment of hydrogen-oxidizing methanogens relative to acetoclastic methanogens was consistent with syntrophic acetate oxidation measured in methanogenic crude oil degrading enrichment cultures. qPCR of the Methanomicrobiales indicated growth characteristics consistent with measured rates of methane production and growth in partnership with Smithella.     

Enhanced crude oil biodegradative potential of natural phytoplankton‐associated hydrocarbonoclastic bacteria

Phytoplankton have been shown to harbour a diversity of hydrocarbonoclastic bacteria (HCB), yet it is not understood how these phytoplankton‐associated HCB would respond in the event of an oil spill at sea. Here, we assess the diversity and dynamics of the bacterial community associated with a natural population of marine phytoplankton under oil spill‐simulated conditions, and compare it to that of the free‐living (non phytoplankton‐associated) bacterial community. While the crude oil severely impacted the phytoplankton population and was likely conducive to marine oil snow formation, analysis of the MiSeq‐derived 16S rRNA data revealed dramatic and differential shifts in the oil‐amended communities that included blooms of recognized HCB (e.g., Thalassospira, Cycloclasticus), including putative novel phyla, as well as other groups with previously unqualified oil‐degrading potential (Olleya, Winogradskyella, and members of the inconspicuous BD7‐3 phylum). Notably, the oil biodegradation potential of the phytoplankton‐associated community exceeded that of the free‐living community, and it showed a preference to degrade substituted and non‐substituted polycyclic aromatic hydrocarbons. Our study provides evidence of compartmentalization of hydrocarbon‐degrading capacity in the marine water column, wherein HCB associated with phytoplankton are better tuned to degrading crude oil hydrocarbons than that by the community of planktonic free‐living bacteria.     

Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 μmol CH₄ g⁻¹ oil d⁻¹, orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC–MS and FTICR–MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.     

Pyrene degradation and copper and zinc uptake by Fusarium solani and Hypocrea lixii isolated from petrol station soil

Aims: This study aimed to isolate and identify potential polycyclic aromatic hydrocarbon (PAH)‐degrading and/or metal‐tolerant fungi from PAH‐contaminated and metal‐contaminated soils. Methods and Results: Pyrene‐degrading fungi were isolated from contaminated soil and tested for metal (Cu, Zn and Pb) compound solubilization and metal accumulation. Three strains of Fusarium solani and one of Hypocrea lixii were able to degrade more than 60% of initial supplied pyrene (100 mg l^?1) after 2 weeks. The isolates were grown on toxic metal (Cu, Pb and Zn)‐containing media: all isolates accumulated Cu in their mycelia to values ranging from c. 5·9 to 10·4 mmol per kg dry weight biomass. The isolates were also able to accumulate Zn (c. 3·7–7·2 mmol per kg dry weight biomass) from zinc phosphate‐amended media. None of the isolates accumulated Pb. Conclusions: These fungal isolates appear to show promise for use in bioremediation of pyrene or related xenobiotics and removal of copper and zinc from wastes contaminated singly or in combination with these substances. Significance and Impact of the Study: Microbial responses to mixed organic and inorganic pollution are seldom considered: this research highlights the abilities of certain fungal strains to interact with both xenobiotics and toxic metals and is relevant to other studies on natural attenuation and bioremediation of polluted sites.     

Anaerobic Bioremediation of Groundwater Containing a Mixture of 1,1,2,2-Tetrachloroethane and Chloroethenes

This study investigated the biotransformation pathways of 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA) in the presence of chloroethenes (i.e. tetrachloroethene, PCE; trichloroethene, TCE) in anaerobic microcosms constructed with subsurface soil and groundwater from a contaminated site. When amended with yeast extract, lactate, butyrate, or H₂ and acetate, 1,1,2,2-TeCA was initially dechlorinated via both hydrogenolysis to 1,1,2-trichloroethane (1,1,2-TCA) (major pathway) and dichloroelimination to dichloroethenes (DCEs) (minor pathway), with both reactions occurring under sulfidogenic conditions. In the presence of only H₂, the hydrogenolysis of 1,1,2,2-TeCA to 1,1,2-TCA apparently required the presence of acetate to occur. Once formed, 1,1,2-TCA was degraded predominantly via dichloroelimination to vinyl chloride (VC). Ultimately, chloroethanes were converted to chloroethenes (mainly VC and DCEs) which persisted in the microcosms for very long periods along with PCE and TCE originally present in the groundwater. Hydrogenolysis of chloroethenes occurred only after highly reducing methanogenic conditions were established. However, substantial conversion to ethene (ETH) was observed only in microcosms amended with yeast extract (200 mg/l), suggesting that groundwater lacked some nutritional factors which were likely provided to dechlorinating microorganisms by this complex organic substrate. Bioaugmentation with an H₂-utilizing PCE-dechlorinating Dehalococcoides spp. -containing culture resulted in the conversion of 1,1,2,2-TeCA, PCE and TCE to ETH and VC. No chloroethanes accumulated during degradation suggesting that 1,1,2,2-TeCA was degraded through initial dichloroelimination into DCEs and then typical hydrogenolysis into ETH and VC.     

Microbial biomass and activities associated with subsurface environments contaminated with chlorinated hydrocarbons

Soil microcosms and enrichment cultures from subsurface sediments and groundwaters contaminated with trichloroethylene (TCE) were examined. Total lipids, [I‐‘⁴C]acetate incorporation into lipids, and [Me‐³H]thymidine incorporation into DNA were determined in these subsurface environments. In heavily TCE‐contam‐inated zones (greater than 500 mg/L) radioisotopes were not incorporated into lipids or DNA. Radioisotope incorporation occurred in sediments both above and below the TCE plume. Phospholipid fatty acids (PLFA) were not detected, i.e., less than 0.5 pmol/L in heavily contaminated groundwater samples. In less contaminated waters, extracted PLFA concentrations were greater than 100 pmollL and microbial isolates were readily obtained. Degradation of 30–100 mg/L TCE was observed when sediments were amended with a variety of energy sources. Microorganisms in these subsurface sediments have adapted to degrade TCE at concentrations greater than 50 mg/L.     

Culture‐dependent and culture‐independent characterization of potentially functional biphenyl‐degrading bacterial community in response to extracellular organic matter from Micrococcus luteus

Biphenyl (BP)‐degrading bacteria were identified to degrade various polychlorinated BP (PCB) congers in long‐term PCB‐contaminated sites. Exploring BP‐degrading capability of potentially useful bacteria was performed for enhancing PCB bioremediation. In the present study, the bacterial composition of the PCB‐contaminated sediment sample was first investigated. Then extracellular organic matter (EOM) from Micrococcus luteus was used to enhance BP biodegradation. The effect of the EOM on the composition of bacterial community was investigated by combining with culture‐dependent and culture‐independent methods. The obtained results indicate that Proteobacteria and Actinobacteria were predominant community in the PCB‐contaminated sediment. EOM from M. luteus could stimulate the activity of some potentially difficult‐to‐culture BP degraders, which contribute to significant enhancement of BP biodegradation. The potentially difficult‐to‐culture bacteria in response to EOM addition were mainly Rhodococcus and Pseudomonas belonging to Gammaproteobacteria and Actinobacteria respectively. This study provides new insights into exploration of functional difficult‐to‐culture bacteria with EOM addition and points out broader BP/PCB degrading, which could be employed for enhancing PCB‐bioremediation processes.     

Growth of Syntrophomonas wolfei in pure culture on crotonate

A Synthrophomonas wolfei-Methanospirillum hungatei coculture was adapted to catabolize crotonate. S. wolfei was then isolated in axenic culture using agar spread plates and roll tubes with crotonate as the sole energy source. S. wolfei catabolized crotonate via a disproportionation mechanism similar to that of some Clostridium species. Growth on crotonate was very slow (specific growth rate of 0.029 h^–1) but the conversion of energy into cell material was very efficient with cell yields of 14.6 g (dry wt.) per mol of crotonate. S. wolfei alone did not catabolize butyrate, but butyrate was stoichiometrically degraded to acetate and presumably methane when S. wolfei was reassociated with M. hungatei. S. wolfei-M. hungatei cocultures accumulated some butyrate during growth on crotonate indicating that protons were not the sole electron acceptors used for crotonate oxidation by the coculture.     

Comparative study of the degradation of flax shive by strains of Pleurotus

Summary Among the four strains of Pleurotus tested, hybrid strain A was found to degrade flax shive faster than the other three strains. Strain A produced more polygalacturonase, pectinlyase, cellulase and laccase enzymes than the other three strains of Pleurotus. Maximum activities of laccase and polysaccharide degrading enzymes were correlated with high weight loss, reduction in the yield of lignin and holocellulose and the degree of polymerisation of holocellulose. The addition of crude extract of flax shive on unextracted-shive increased the production of primordia of all the four strains, but it did not increase the degradation of flax shive. Flavonoid type compounds were detected in the crude extract of flax shive and they may be responsible for the increase in the production of mushroom primordia.     

Ilyobacter delafieldii sp. nov., a metabolically restricted anaerobic bacterium fermenting PHB

Enrichments from an estuarine sediment with crotonate as substrate resulted in the isolation of a motile, gram-negative, obligately anaerobic rod with pointed ends, designated strain 10cr1. The organism was asporogenous, did not reduce sulfur, sulfate, thiosulfate, nitrate, oxygen or fumarate, and had a mol %G+C ratio of 29. Strain 10cr1 was able to ferment crotonate, 3-hydroxybutyrate, lactate, pyruvate, and poly--hydroxybutyric acid (PHB). Acetate, propionate, butyrate, CO₂ and H₂ were the fermentation products. When grown on PHB there was accumulation of 3-hydroxybutyrate once growth had ceased, indicating degradation of PHB to the monomer. The 3-hydroxybutyrate formed during growth of the culture was fermented to acetate, butyrate and H₂. Experimental evidence suggested the production of an extracellular PHB depolymerase. The cells were not attached to the PHB granules. This is the first isolation of an anaerobic bacterium capable of degrading exogenous PHB. This strain is described as a new species, Ilyobacter delafieldii sp. nov., and strain 10cr1 (=DSM 5704) is designated as the type (and at present, only) strain.Abbreviations G+C guanine plus cytosine - OD optical density - PHB poly--hydroxybutyric acid - specific growth rate - HPLC high-performance liquid chromatography - YE yeast extract     

Utilization of (E)-2-butenoate (Crotonate) by Clostridium kluyveri and some other Clostridium species

Clostridium La 1 obtained from a Clostridium kluyveri culture was compared with a typical C. kluyvery strain (DSM 555). The former grows on crotonate and is unable to use ethanol-acetate as carbon sources. The latter grows on crotonate only after long adaptation periods. Resting cells of both strains show also pronounced differences in the fermentation of crotonate. This holds even for C. kluyveri grown on crotonate. Besides several other differences the most striking is that there is no hybridization between the DNA of both strains.Crotonate seems not to be a very special carbon source since C. butyricum and C. pasteurianum grow on crotonate medium supplemented by peptone and yeast extract.Non Standard Abbreviations EA-medium ethanol and acetate as carbon source - C-medium crotonate as carbon source - DSM Deutsche Sammlung von Mikroorganismen     

Syntrophus aciditrophicus uses the same enzymes in a reversible manner to degrade and synthesize aromatic and alicyclic acids

Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane-1-carboxylate and catalyses the novel synthesis of benzoate and cyclohexane-1-carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane-1-carboxylate synthesis. Enzymes involved in the metabolism of cyclohex-1,5-diene carboxyl-CoA to acetyl-CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane-1-carboxylate. Incorporation of ¹³C-atoms from 1-[¹³C]-acetate into crotonate, benzoate and cyclohexane-1-carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl-CoA intermediates to formate was detected. Ligases and membrane-bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl-CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.     

Effect of yeast extract on fluoranthene degradation and aromatic ring dioxygenase expressing bacterial community structure of a fluoranthene degrading bacterial consortium

Fluoranthene (Fla) is a high molecular weight polycyclic aromatic hydrocarbon that exerts hazardous effects on living organisms. An efficient Fla degrading bacterial consortium LP was enriched from an oil contaminated soil sample, with and without yeast extract as a supplement. Objective of the present study was to see if there was any differential effect of yeast extract addition on Fla degradation potential and aromatic ring dioxygenase expressing bacteria (ARDB) of the enrichments. Primary enrichment of the soil sample was carried out in minimal salt medium (MSM) added with 500 mg l⁻¹ Fla and 0.05% yeast extract (YMSM). Secondary, tertiary and subsequent enrichments were prepared in YMSM and MSM after every sixteen days of incubation. Fla was efficiently degraded by YMSM enriched culture than MSM enriched culture. However, when MSM enrichment was incubated longer instead of further subculturings, it also degraded Fla efficiently. All three enrichments exhibited growth of bacterial colonies on Fla sprayed minimal agar plates however only YMSM enrichment showed clear zone forming bacterial colonies. A positive effect was observed of yeast extract on ARDB population of LP consortium. To our limited knowledge this is first time that effect of yeast extract on ARDB population was studied.     

Analysis of the Anaerobic Microbial Community Capable of Degrading p-Toluene Sulfonate

Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp. SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate. Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene. p-Toluene sulfonate stimulated the growth of Ms. mazei MM on acetate. The sulfate-reducing strain Desulfovibrio sp. SR1 utilized p-toluene sulfonate as an electron acceptor. The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.     

Evidence for syntrophic butyrate metabolism under sulfate-reducing conditions in a hydrocarbon-contaminated aquifer

The importance of syntrophy in the degradation of butyrate in an aquifer where sulfate reduction was shown to be an important terminal electron-accepting process was assessed. Hydrocarbon-contaminated aquifer sediments coupled butyrate degradation to sulfate reduction and methane production. Butyrate degradation in methanogenic microcosms was inhibited by the addition of 2-bromoethanesulfonic acid, and was restored by the addition of 10 mM sulfate and a hydrogen- and formate-using sulfate reducer, but not by the addition of 10 mM sulfate alone. Molybdate addition inhibited butyrate degradation in sulfate-reducing microcosms. The addition of CO, which inhibits hydrogenases, to sulfate-reducing microcosms inhibited butyrate metabolism and caused the hydrogen partial pressure to increase to levels that would make syntrophic butyrate degradation via sulfate reduction energetically unfavorable (-5 to +3 kJ mol(-1) ). DNA extracted from the most probable number cultures and contaminated sediments contained sequences related to members of the families Syntrophomonadaceae and Syntrophaceae, whose members are known to syntrophically degrade fatty acids, as well as sequences related to uncultured Firmicutes, Desulfobulbaceae, Desulfobacteriaceae, and Desulfovibrionaceae. These data show that contaminated sediments degraded butyrate syntrophically coupled to methane production and sulfate reduction.