Inhibition Studies with 2-Bromoethanesulfonate Reveal a Novel Syntrophic Relationship in Anaerobic Oleate Degradation.

Degradation of long-chain fatty acids (LCFAs) in methanogenic environments is a syntrophic process involving the activity of LCFA-degrading bacteria and hydrogen-utilizing methanogens. If methanogens are inhibited, other hydrogen scavengers are needed to achieve complete LCFA degradation. In this work, we developed two different oleate (C18:1 LCFA)-degrading anaerobic enrichment cultures, one methanogenic (ME) and another in which methanogenesis was inhibited (IE). Inhibition of methanogens was attained by adding a solution of 2-bromoethanesulfonate (BrES), which turned out to consist of a mixture of BrES and isethionate. Approximately 5 times faster oleate degradation was accomplished by the IE culture compared with the ME culture. A bacterium closely related to Syntrophomonas zehnderi (99% 16S rRNA gene identity) was the main oleate degrader in both enrichments, in syntrophic relationship with hydrogenotrophic methanogens from the genera Methanobacterium and Methanoculleus (in ME culture) or with a bacterium closely related to Desulfovibrio aminophilus (in IE culture). A Desulfovibrio species was isolated, and its ability to utilize hydrogen was confirmed. This bacterium converted isethionate to acetate and sulfide, with or without hydrogen as electron donor. This bacterium also utilized BrES but only after 3 months of incubation. Our study shows that syntrophic oleate degradation can be coupled to desulfonation.IMPORTANCE In anaerobic treatment of complex wastewater containing fat, oils, and grease, high long-chain fatty acid (LCFA) concentrations may inhibit microbial communities, particularly those of methanogens. Here, we investigated if anaerobic degradation of LCFAs can proceed when methanogens are inhibited and in the absence of typical external electron acceptors, such as nitrate, iron, or sulfate. Inhibition studies were performed with the methanogenic inhibitor 2-bromoethanesulfonate (BrES). We noticed that, after autoclaving, BrES underwent partial hydrolysis and turned out to be a mixture of two sulfonates (BrES and isethionate). We found out that LCFA conversion proceeded faster in the assays where methanogenesis was inhibited, and that it was dependent on the utilization of isethionate. In this study, we report LCFA degradation coupled to desulfonation. Our results also showed that BrES can be utilized by anaerobic bacteria.

Moorella stamsii sp. nov., a new anaerobic thermophilic hydrogenogenic carboxydotroph isolated from digester sludge.

A novel anaerobic, thermophilic, carbon monoxide-utilizing bacterium, strain E3-O(T), was isolated from anaerobic sludge from a municipal solid waste digester. Cells were straight rods, 0.6-1 µm in diameter and 2-3 µm in length and grew as single cells or in pairs. Cells formed round terminal endospores. The temperature range for growth was 50-70 °C, with an optimum at 65 °C. The pH range for growth was 5.7-8.0, with an optimum at 7.5. Strain E3-O(T) had the ability to ferment various sugars, such as fructose, galactose, glucose, mannose, raffinose, ribose, sucrose and xylose, producing mainly H2 and acetate. In addition, the isolate was able to grow with CO as the sole carbon and energy source. CO oxidation was coupled to H2 and CO2 formation. The G+C content of the genomic DNA was 54.6 mol%. Based on 16S rRNA gene sequence analysis, this bacterium is most closely related to Moorella glycerini (97 % sequence identity). Based on the physiological features and phylogenetic analysis, it is proposed that strain E3-O(T) should be classified in the genus Moorella as a representative of a novel species, Moorella stamsii. The type strain of Moorella stamsii is E3-O(T) ( = DSM 26271(T) = CGMCC 1.5181(T)).

Sulfated glycosaminoglycan-like polymers are present in an acidophilic biofilm from a sulfidic cave.

Sulfated glycosaminoglycans (sGAG) are negatively charged extracellular polymeric substances that occur in biofilms from various environments. Yet, it remains unclear whether these polymers are acquired from the external environment or produced by microbes in the biofilm. To resolve this, we analyzed the presence of sGAGs in samples of an acidophilic biofilm collected from Sulfur Cave in Puturosu Mountain (Romania), an environment that is largely inaccessible to contamination. A maximum of 55.16 ± 2.06 µg sGAG-like polymers were recovered per mg of EPS. Enzymatic treatment with chondroitinase ABC resulted in a decrease of the mass of these polymers, suggesting the structure of the recovered sGAG is similar to chondroitin. Subsequent FT-IR analysis of these polymers revealed absorbance bands at 1230 cm-1, 1167 cm-1 and 900 cm-1, indicating a possible presence of polysaccharides and sulfate. Analysis of genomic sequences closely related to those predominant in the acidophilic biofilm, contained genes coding for sulfotransferase (an enzyme needed for the production of sGAG), which supports the hypothesis of microbial synthesis of sGAGs within the biofilm.

Strategies to suppress hydrogen-consuming microorganisms affect macro and micro scale structure and microbiology of granular sludge.

Treatment of anaerobic granules with heat and two chemical treatments, contacting with 2-bromoethanesulfonate (BES) and with BES + Chloroform, were applied to suppress hydrogen-consuming microorganisms. Three mesophilic expanded granular sludge bed (EGSB) reactors-R(Heat), R(BES), and R(BES + Chlo)--were inoculated with the treated sludges and fed with synthetic sugar-based wastewater (5 g(COD) L(-1), HRT 20-12 h). Morphological integrity of granules and bacterial communities were assessed by quantitative image analysis and 16S rRNA gene based techniques, respectively. Hydrogen production in R(Heat) was under 300 mL H(2) L(-1) day(-1), with a transient peak of 1,000 mL H(2) L(-1) day(-1) after decreasing HRT. In R(BES + Chlo) hydrogen production rate did not exceed 300 mL H(2) L(-1) day(-1) and there was granule fragmentation, release of free filaments from aggregates, and decrease of granule density. In R(BES), there was an initial period with unstable hydrogen production, but a pulse of BES triggered its production rate to 700 ± 200 mL H(2) L(-1) day(-1). This strategy did not affect granules structure significantly. Bacteria branching within Clostridiaceae and Ruminococcaceae were present in this sludge. This work demonstrates that, methods applied to suppress H(2)-consuming microorganisms can cause changes in the macro- and microstructure of granular sludge, which can be incompatible with the operation of high-rate reactors.

Short term changes in the abundance of nitrifying microorganisms in a soil-plant system simultaneously exposed to copper nanoparticles and atrazine.

Copper nanoparticles (NCu) may co-exist with other pollutants in agricultural soils, such as pesticides. However, this has been little evaluated yet. Thus, possible effects of the simultaneous applications of pesticides and NCu on biogeochemical cycles are expected, for example on the nitrogen cycle. Therefore, the aim of this work was to evaluate the effect of simultaneous application of the herbicide atrazine (ATZ) and NCu on the abundance of total bacteria and nitrifying communities: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Moreover, the ATZ dissipation was evaluated. A soil-plant system containing ATZ at field dose (3 mg a.i. kg-1) was mixed with two doses of NCu (0.05% or 0.15% w/w). Changes in the abundance of 16S rRNA and ammonia monooxygenase (amoA) genes of AOA and AOB were evaluated by real-time quantitative PCR (qPCR) at three sampling times (1, 15 and 30 days). The residual ATZ and nitrate production were also measured. The results showed significant differences in microbial composition and abundance over the 30 days of the experiment. Particularly, an initial decrease was observed in total bacterial abundance due to the presence of ATZ and NCu respect to ATZ alone (~60%). The abundance of AOA was also remarkably reduced (~85%), but these communities gradually recovered towards the end of the experiment. Conversely, AOB abundance initially increased (>100%) and remained mainly unaltered in soil exposed to ATZ and NCu 0.15% w/w, where nitrate formation was also constant. Moreover, NCu decreased the ATZ dissipation, which was translated in a 2-fold increase on the ATZ half-life values (T1/2). This study demonstrates that the simultaneous presence of NCu and ATZ may represent a risk for the total bacteria present in soil and sensitive microorganisms such as nitrifying communities, and changes in the dissipation of the pesticide could influence this process.

Anaerobic microbial LCFA degradation in bioreactors.

This paper reviews recent results obtained on long-chain fatty acids (LCFA) anaerobic degradation. Two LCFA were used as model substrates: oleate, a mono-unsaturated LCFA, and palmitate, a saturated LCFA, both abundant in LCFA-rich wastewaters. 16S rRNA gene analysis of sludge samples submitted to continuous oleate- and palmitate-feeding followed by batch degradation of the accumulated LCFA demonstrated that bacterial communities were dominated by members of the Clostridiaceae and Syntrophomonadaceae families. Archaeal populations were mainly comprised of hydrogen-consuming microorganisms belonging to the genus Methanobacterium, and acetate-utilizers from the genera Methanosaeta and Methanosarcina. Enrichment cultures growing on oleate and palmitate, in the absence or presence of sulfate, gave more insight into the major players involved in the degradation of unsaturated and saturated LCFA. Syntrophomonas-related species were identified as predominant microorganisms in all the enrichment cultures. Microorganisms clustering within the family Syntrophobacteraceae were identified in the methanogenic and sulfate-reducing enrichments growing on palmitate. Distinct bacterial consortia were developed in oleate and palmitate enrichments, and observed differences might be related to the different degrees of saturation of these two LCFA. A new obligately syntrophic bacterium, Syntrophomonas zehnderi, was isolated from an oleate-degrading culture and its presence in oleate-degrading sludges detected by 16S rRNA gene cloning and sequencing.

Carbendazim dissipation in the biomixture of on-farm biopurification systems and its effect on microbial communities.

The impact of repeated carbendazim (CARB) applications on the extent of CARB dissipation, the microbial diversity, the community level physiological profile (CLPP), and the enzymatic activity within the biomixture of an on-farm biopurification system was evaluated. After three successive CARB applications, the CARB dissipation efficiency was high; the efficiency of dissipation was 87%, 94% and 96% after each application, respectively. Although microbial enzymatic activity was affected significantly by CARB application, it could recover after each CARB pulse. Likewise, the numbers of cultivable bacteria, fungi and actinomycetes (as measured in CFUs) were slightly affected by the addition of CARB, but the inhibitory effect of the pesticide application was temporary. Denaturing gradient gel electrophoresis (DGGE) and Biolog Ecoplate assays demonstrated that the microbial populations remained relatively stable over time when compared to the control. The results obtained herein therefore demonstrate the high dissipation capacity of this biomixture and highlight the microbiological robustness of this biological system.

Atrazine dissipation and its impact on the microbial communities and community level physiological profiles in a microcosm simulating the biomixture of on-farm biopurification system.

The effects of repeated atrazine application (40 mg a.i.kg(-1)) on its degradation, microbial communities and enzyme activities were studied in a peat based biomixture composed by straw, soil and peat in the volumetric proportions of 2:1:1 that can be used in on-farm biopurification system. Atrazine removal efficiency was high (96%, 78% and 96%) after each atrazine application and did not show a lag phase. Microbial enzyme activities were reduced significantly with atrazine application but rapidly recovered. Microbial diversity obtained by BiologEcoplate was similar after the first and second atrazine application. However, an inhibitory effect was observed after the third application. After each atrazine application, culturable fungi were reduced, but rapidly recovered without significant changes in culturable bacteria and actinomycetes compared to the control. Denaturing gradient gel electrophoresis (DGGE) patterns demonstrated that microbial community structure remained relatively stable in time when compared to the controls. In conclusion, our results demonstrated that after successive ATZ applications, the peat based biomixture had a good degradation capacity. Moreover, microbiological assays demonstrated the robustness of the peat based biomixture from a microbiological point of view to support pesticide degradation.

Effects of pre-treatment and bioaugmentation strategies on the anaerobic digestion of chicken feathers.

Anaerobic digestion of raw chicken feather waste and its co-digestion with poultry litter were assessed in batch assays. Following, two strategies were evaluated to improve methane production from chicken feathers: (i) waste pre-hydrolysis through thermochemical treatment using lime and sodium hydroxide, and (ii) amendment of digestion broth with the proteolytic bacterium Fervidobacterium pennivorans. Anaerobic digestion of the raw waste (2.5% total solids) allowed a specific methane production of 123 ± 3 L CH(4) kg(-1) VS. Pre-treatment and bioaugmentation strategies did not improve methane production from feather waste, despite the significant increase in waste solubilisation, from 45 ± 5% up to 64 ± 1% using F. pennivorans and up to 96% after pre-treatment with 2g NaOH g(-1) waste. These results indicate that conversion of soluble organic matter to methane, and not the hydrolysis rate, was the limiting step for the anaerobic digestion of chicken feather waste.

Thermochemical pre- and biological co-treatments to improve hydrolysis and methane production from poultry litter.

The biochemical methane potential (BMP) of raw poultry litter waste was assessed in batch assays. Biological co-treatment with Clostridium cellulolyticum, Caldicellulosiruptor saccharolyticum and Clostridium thermocellum as bioaugmentation strains, and thermochemical pre-treatments with lime and sodium hydroxide performed at different temperatures and pressures were applied as strategies to improve the BMP by favouring the hydrolysis of the cellulolytic material in the waste. Anaerobic digestion of the raw waste allowed a specific methane production of 145 ± 14 LCH(4)kg(-1)VS, with 1% total solids and 0.72 g VS(inoculum)g(-1)VS(waste). The pre- and co-treatments contributed to a significant increase (up to 74%) in the waste solubilisation when using C. saccharolyticum, but methane production did not improve considerably. Therefore, the conversion of soluble organic matter to methane was the limiting step of the anaerobic digestion process of poultry litter waste.

Methane production from oleate: assessing the bioaugmentation potential of Syntrophomonas zehnderi.

The potential for improving long-chain fatty acids (LCFA) conversion to methane was evaluated by bioaugmenting a non-acclimated anaerobic granular sludge with Syntrophomonas zehnderi. Batch bioaugmentation assays were performed with and without the solid microcarrier sepiolite, using 1 mM oleate as sole carbon and energy source. When S. zehnderi was added to the anaerobic sludge methane production from oleate was faster. High methane yields, i.e. 89 ± 5% and 72 ± 1%, were observed in bioaugmented assays in the absence and presence of sepiolite, respectively. Sepiolite stimulated a faster methane production from oleate and prevented the accumulation of acetate. Acetoclastic activity was affected by oleate in the absence of sepiolite, where methane production rate was 26% lower than in assays with microcarrier.

Mineralization of LCFA associated with anaerobic sludge: Kinetics, enhancement of methanogenic activity, and effect of VFA.

Long-chain fatty acids (LCFA) associated with anaerobic sludge by mechanisms of precipitation, adsorption, or entrapment can be biodegraded to methane. The mineralization kinetics of biomass-associated LCFA were established according to an inhibition model based on Haldane's enzymatic inhibition kinetics. A value around 1,000 mg COD-LCFA..g VSS(-1) was obtained for the optimal specific LCFA content that allowed the maximal mineralization rate. For sludge with specific LCFA contents of 2,838 +/- 63 and 4,571 +/- 257 mg COD-LCFA..g VSS(-1), the specific methanogenic activities in the presence of acetate, butyrate, and H(2)/CO(2) were significantly enhanced after the mineralization of the biomass-associated LCFA. For sludge with a specific LCFA content near the optimal value defined by the kinetic model, the effect of adding VFA to the medium was studied during the mineralization of the biomass-associated LCFA. Different patterns were obtained for each individual substrate. Acetate and butyrate were preferentially consumed by the consortium, but in the case of propionate no evidence of a sequential consumption pattern could be withdrawn. It was concluded that LCFA do not exert a bactericidal neither a permanent toxic effect toward the anaerobic consortia. A discussion is addressed to the relative roles of a reversible inhibitory effect and a transport limitation effect imposed by the LCFA surrounding the cells.