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Microalgae biomass can be a feedstock for anaerobic digestion, but the recalcitrance of the cell wall limits fully exploiting this source of organic matter. Alkaline pretreatments modify the structure of the cell wall facilitating bacterial attack and solubilizing the organic matter content. It is unknown whether alkaline hydrogen peroxide can help to improve biogas production from microalgae biomass compared to other alkaline pretreatments. This work focused on methane production at alkaline conditions (pH 9) with pretreated microalgal biomass (Scenedesmus obtusiusculus). Alkaline hydrogen peroxide (AHP) was used for the thermo-alkaline pretreatment and compared with pretreatments using NaOH or CaO. The pretreatment with 15% CaO solubilized the highest carbohydrates (22.1%) and proteins (18.3%), while the microalgal biomass pretreated with 4 M NaOH or 1.5% AHP attained the highest yields of methane (227.1 mL and 208.4 mL CH4/g VS, respectively), improving the methane potential between 34 and 46%, and producing biogas with methane content up to 91%. A preliminary techno-economic and energy analysis showed energy savings of 40% with 4 M NaOH and 31% with 1.5% AHP; therefore, AHP could be considered a suitable option for the pretreatment of microalgal biomass.

Sulfate-reducing microbial communities remain a suitable option for the remediation of acid mine drainage using several types of carrier materials and appropriate reactor configurations. However, acetate prevails as a product derived from the incomplete oxidation of most organic substrates by sulfate reducers, limiting the efficiency of the whole process. An established sulfate-reducing consortium, able to degrade acetate at initial acidic pH (3.0), was used to develop biofilms over granular activated carbon (GAC), glass beads, and zeolite as carrier materials. In batch assays using glycerol, biofilms successfully formed on zeolite, glass beads, and GAC with sulfide production rates of 0.32, 0.26, and 0.14 mmol H2S/L\textperiodcenteredd, respectively, but only with glass beads and zeolite, acetate was degraded completely. The planktonic and biofilm communities were determined by the 16S rRNA gene analysis to evaluate the microbial selectivity of the carrier materials. In total, 46 OTUs (family level) composed the microbial communities. Ruminococcaceae and Clostridiaceae families were present in zeolite and glass beads, whereas Peptococcaceae was mostly enriched on zeolite and Desulfovibrionaceae on glass beads. The most abundant sulfate reducer in the biofilm of zeolite was Desulfotomaculum sp., while Desulfatirhabdium sp. abounded in the planktonic community. With glass beads, Desulfovibrio sp. dominated the biofilm and the planktonic communities. Our results indicate that both materials (glass beads and zeolite) selected different key sulfate-reducing microorganisms able to oxidize glycerol completely at initial acidic pH, which is relevant for a future application of the consortium in continuous bioreactors to treat acidic streams., Netherlands Ministry of Education, Culture, and Science and the Netherlands Science Foundation through SIAM Gravitation grant 024.002.002, info:eu-repo/semantics/publishedVersion

Agave lechuguilla is a widely distributed plant in arid ecosystems. It has been suggested that its microbiome is partially responsible for its great adaptability to the oligotrophic environments of the Chihuahuan Desert. To lead the recruitment of beneficial rhizobacteria, the root exudates are essential; however, the amino acids contained within these compounds had been largely overlooked. Thus, we investigated how the variations of amino acids in the rhizosphere at different growth stages of A. lechuguilla affect the rhizobacterial community composition, its functions, and activity of the beneficial bacteria. In this regard, it was found that arginine and tyrosine were related to the composition of the rhizobacterial community associated to A. lechuguilla, where the most abundant genera were from the phylum Proteobacteria and Bacteroidetes. Moreover, Firmicutes was largely represented by Bacillus in the phosphorus-mineralizing bacteria community, which may indicate its great distribution and versatility in the harsh environments of the Chihuahuan Desert. In contrast, we found a high proportion of Unknown taxa of nitrogen-fixing bacteria, reflecting the enormous diversity in the rhizosphere of these types of plants that remains to be explored. This work also reports the influence of micronutrients and the amino acids methionine and arginine over the increased activity of the nitrogen-fixing and phosphorus-mineralizing bacteria in the rhizosphere of lechuguillas. In addition, the results highlight the multiple beneficial functions present in the microbiome that could help the host to tolerate arid conditions and improve nutrient availability.

Biogeochemical processes govern the transport and availability of arsenic in sediments. However, little is known about the transition from indigenous communities to cultivable consortia when exposed to high arsenic concentrations. Such cultivable communities could be exploited for arsenic bioremediation of waste streams and polluted sites. Thus, it is crucial to understand the dynamics and selective pressures that shape the communities during the development of customized bacterial consortia. First, from the arsenic partitioning of two sediments with high arsenic concentrations, we found that up to 55% of arsenic was bioavailable because it was associated with the soluble, carbonate, and ionically exchangeable fractions. Next, we prepared sediment enrichment cultures under arsenate- and sulfate-reducing conditions to precipitate arsenic sulfide biominerals and analyze the communities. The produced biominerals were used as the inoculum to develop bacterial consortia via successive transfers. Tracking of the 16S rRNA gene in the fresh sediments, sediment enrichments, biogenic minerals, and bacterial consortia revealed differences in the bacterial communities. Removing the sediment caused a substantial decrease in diversity and shifts toward the dominance of the Firmicutes phylum to the detriment of Proteobacteria. In agreement with the 16S rRNA gene results, the sequencing of the arrA gene confirmed the presence of phylotypes closely related to Desulfosporosinus sp. Y5 (100% similarity), highlighting the pivotal role of this genus in the removal of soluble arsenic. Here, we demonstrated for the first time that besides being important as arsenic sinks, the biogenic arsenic sulfide minerals are reservoirs of arsenic resistant/respiring bacteria and can be used to culture them.

A novel strategy to discontinuously increase the biomass concentration in a continuous stirred-tank reactor was evaluated to enhance the performance of dark fermentation. Different concentrations of biomass were evaluated at organic loading rates (OLR) ranging from 90 to 160 g lactose/L-d with a hydraulic retention time (HRT) of 6 h. The study revealed that the discontinuous increase of biomass enhanced the hydrogen (H2) production rates and carboxylic acids concentrations by 19–25% and 8–23%, respectively. In particular, a maximum H2 production rate of 30.8 L H2/L-d with carboxylic acids concentration of 20 g/L was reached at an OLR of 138 g lactose/L-d with a biomass concentration of 15 g volatile suspended solids/L. The analysis of microbial communities showed the co-dominance of Clostridium and lactic acid bacteria. Overall, the discontinuous increase of biomass was an effective strategy to improve the performance of suspended-biomass reactors operated at high OLR and low HRT.

The recent periodic influx of massive quantities of pelagic Sargassum spp. (sargasso) into the Caribbean has posed ecological, social, and economic challenges to the region. Sustainable use of the biomass is crucial to mitigate negative impacts of beached algae. The current uses of sargasso in Mexico are reviewed, and a biorefinery approach is proposed to optimize its commercialization. The commercialization potential of sargasso in the Mexican Caribbean is analyzed using the strengths, weaknesses, opportunities, and threats (SWOT) analysis. The unpredictability of the influx to the Mexican shores is considerable, both in time and space, also, some areas receive consistently more sargasso than others. The lack of available technology, local infrastructure and regulations concerning sargasso are relevant, as is the urgent need to establish proper regulatory measures. In the context of the Mexican legal system, the category “special waste” can best be applied to sargasso that is collected from the beach or nearshore waters for the purpose of regulation. Lessons learnt from in Mexico may be applied elsewhere, with adaptations for each specific location. More importantly, mutual understanding of the constraints and possibilities of sargasso management in other countries, or territories, may facilitate the coordinated management of this transboundary macroalgae bloom.

Hydrolysis can be considered as the bottleneck for biofuel production from microalgae due to the recalcitrance of this type of biomass. Thermochemical pretreatment is an effective and widely used process to solubilize different types of biomass. Nevertheless, the variability of the cell wall composition, among different microalgae species, hinders the development of a suitable pretreatment. In this work, the most relevant factors for thermochemical pretreatment to solubilize organic matter from microalgal biomass (Scenedesmus obtusiusculus) were analyzed and identified. The aim was to obtain hydrolysates, rich in soluble organic matter, that allow attaining higher hydrogen and methane yields than with raw microalgae, using a factorial design to identify the significant factors of the thermochemical pretreatment. The results showed that temperature (100 °C) and HCl concentration (3%) were the most significant factors to achieve carbohydrates solubilization close to 100%. Further, reaction time (1.7 h) and biomass concentration (30 g total solids/L) were also important to achieve up to 60% chemical oxygen demand (COD) solubilization. Overall, the microalgal hydrolysates produced 48 NmL H2/g VS and 296 NmL CH4/g VS, which is 1.7- and 1.3-fold higher hydrogen and methane yields, respectively, than raw microalgae. These results are meaningful because high soluble carbohydrates and COD would allow the application of high organic loading rates in continuous reactors for biofuel production using renewable biomass such as microalgae.

Sediment historically impacted by acid mine drainage was exposed to different initial pH and electron donors to investigate the effect that both conditions had on the performance and fingerprint of...

BACKGROUND: Sulfatereducing microorganisms (SRM) can help to remediate acidic effluents containing metals. One drawback of sulfate reduction is that some SRM do not oxidize completely the substrate to CO2 and acetic acid may remain as a byproduct, affecting the process efficiency. Acidic environments are a potential source of sulfatereducers able to thrive acidic conditions. This work aimed to develop cultivable consortia of sulfatereducing microorganisms able to consume acetate at acidic pH and analyze their community composition. RESULTS Starting from sediment enrichments from a natural acidic source, by successive transfers and combinations of electron donors and pH we obtained seven sulfatereducing consortia. All the consortia consumed the acetate produced from the incomplete oxidation of the substrate (lactate or glycerol) and used 5375% of the reducing equivalents for sulfate reduction. The sulfide production rate of the consortia was between 0.220.26mmol/L·day in the range of pH 3 6, being slightly higher at acidic conditions (4 5). The microbial diversity of the consortia was dominated by 21 OTUs, including taxa of acetotrophic sulfate reducers (i.e., Desulfotomaculum and Desulfatirhabdium) and fermenting bacteria. CONCLUSION The consortia reported here have the potential to serve as inoculum for sulfatereducing bioreactors and could help to overcome acetate accumulation at low pH., This research was financially supported by Consejo Nacional de Ciencia y Tecnología, SEP-CONACYT Ciencia Básica grant 181809, and by the Netherlands Organization for Scientific Research (NWO) through SIAM Gravitation grant 024.002.002., info:eu-repo/semantics/publishedVersion

Sulfate-reducing microbial communities remain a suitable option for the remediation of acid mine drainage using several types of carrier materials and appropriate reactor configurations. However, acetate prevails as a product derived from the incomplete oxidation of most organic substrates by sulfate reducers, limiting the efficiency of the whole process. An established sulfate-reducing consortium, able to degrade acetate at initial acidic pH (3.0), was used to develop biofilms over granular activated carbon (GAC), glass beads, and zeolite as carrier materials. In batch assays using glycerol, biofilms successfully formed on zeolite, glass beads, and GAC with sulfide production rates of 0.32, 0.26, and 0.14 mmol H

Since late 2014, the Mexican Caribbean coast has periodically received massive, atypical influxes of pelagic Sargassum spp. (sargasso). Negative impacts associated with these influxes include mortality of nearshore benthic flora and fauna, beach erosion, pollution, decreasing tourism and high management costs. To understand the dynamics of the sargasso influx, we used Landsat 8 imagery (from 2016 to mid-2020) to record the coverage of sargasso in the sea off the Mexican Caribbean coastline, with a maximum reported in September 2018. Satellite image analysis also showed local differences in the quantity of beached sargasso along the coastline. Over the years, good practice for collection on the beach and for off-shore collection of sargasso have been established through trial and error, and the Mexican Government and hotel industry have spent millions of dollars on removal and off-shore detention of sargasso. Notwithstanding, sargasso also has various properties that could be harnessed in local industries. The stimulation of local industrial growth would offer alternatives to the dependence on tourism, as a circular economy, based on sargasso, is developed.

Agave tequilana bagasse is a suitable lignocellulosic residue for energy production. However, the presence of lignin and the heterogeneous structure of hemicellulose may hinder the availability of polysaccharides. In this work, the pretreatment of A. tequilana bagasse with alkaline hydrogen peroxide (AHP) followed by enzymatic saccharification with hemicellulases and cellulases was assessed for the removal of lignin and extraction of fermentable sugars, respectively. Results of the AHP pretreatment indicated that it is possible to attain up to 97% delignification and recover 88% of cellulose and hemicellulose after only 1.5 h of treatment. Regarding the saccharification process, the total sugar yield and productivity were both increased by 2-fold using an enzymatic mixture (cellulases + hemicellulases) compared to single enzyme hydrolysis (cellulases), evidencing synergism. Further evaluation of the hydrolyzates as substrate for hydrogen and methane production, resulted in yields 1.5 and 3.6-times (215.14 ± 13 L H2 and 393.4 ± 13 L CH4 per kg bagasse, respectively) superior to those obtained with hydrolyzates of non-pretreated bagasse processed with a single enzyme. Overall, using AHP pretreatment and subsequent hydrolysis with enzymatic mixtures improves the saccharification of A. tequilana bagasse enhancing the production of hydrogen and methane.

In the pursuit of renewable sources for biogas generation, microalgae biomass constitutes a promising alternative. In this work, thermally pretreated Scenedesmus obtusiusculus was evaluated as source of biomass for methane production, in systems operated under batch and semi-batch regime. In batch experiments, methane production yields ranging from 109.3 to 192.7 mL CH4/g VSadded were observed with thermally-pretreated (98 °C, 6 h) microalgae biomass at concentrations between 1 and 10 g tCOD/L. On the other hand, the system operated under semi-batch mode for 50 days attained a volumetric methane production rate of 1.15 L CH4/L-d with a methane yield of 177.9 mL CH4/g VSadded at an organic loading rate of 10 g tCOD/L-d and a reaction time of 12 h. These results demonstrate that methane production from microalgae biomass in a semi-batch reactor is possible at low reaction time (12 h) and high organic loading rate using a moderate-temperature pretreatment for the microalgae biomass.

Anthraquinone-2,6-disulfonate (AQDS) was used as an electron acceptor to promote the establishment of a community of quinone-reducing microorganisms from an anaerobic sludge using acetate and glucose as energy sources. The enriched and control (not enriched with AQDS) sludge were used for the direct and mediated biotransformation of congo red. For the mediated reduction, AQDS was used soluble and immobilized on granular activated carbon (GAC). The adsorption capacity of AQDS on GAC was 87.6 mg/g, which improved the electron-accepting capacity of the material 2.26-times compared to the unmodified GAC. The rate of decolorization of congo red with the enriched sludge was 2.94-times higher than the value achieved with the control sludge for the direct biotransformation. For the mediated biotransformation using AQDS immobilized, the rates with the enriched sludge were improved 3.79- and 1.13-times respect to the incubations with the control sludge lacking AQDS and with AQDS immobilized, respectively. The microbial analysis revealed a change in the bacterial composition of the enriched sludge compared to the control sludge. In contrast, the profiles of the archaeal composition remained the same in the two types of sludge. The results presented in this study confirm the potential use of quinone-reducing microorganisms enriched to improve the redox biotransformation of electron-accepting pollutants (e.g. azo dyes) by direct reduction and mediated with soluble and immobilized redox mediators.

High arsenic concentrations have been detected in alluvial aquifers of arid and semi-arid zones in Mexico. This work describes the potential of microbial arsenate reduction of the indigenous community present in sediments from an arsenic contaminated aquifer. Microcosms assays were conducted to evaluate arsenate and sulfate-reducing activities of the native microbiota. Two different sediments were used as inoculum in the assays amended with lactate (10mM) as electron donor and with sulfate and arsenate (10mM each) as electron acceptors. Sediments were distinguished by their concentration of total arsenic 238.3±4.1mg/kg or 2263.1±167.7mg/kg, which may be considered as highly contaminated sediments with arsenic. Microbial communities present in both sediments were able to carry out arsenate reduction, accomplished within 4days, with the corresponding formation of arsenite; sulfate reduction took place as well. Both reducing activities occurred without previous acclimation period or enrichment, even at potential inhibitory concentrations of arsenate as high as 750mg/L (10mM). The formation of a yellowish colloidal precipitate was evident when both reducing processes occurred in the microcosm, which contributed to remove between 52 and 90.9% of As(III) from the liquid phase by bioprecipitation of arsenic as arsenic sulfide.

The start-up of microbial fuel cells (MFCs) requires biofilm development onto electrodes and acclimation of the microbial community to specific substrates. This process is time-consuming and shows low effectivity when complex substrates are utilized. The varying proportions and complex mixtures of volatile fatty acids (VFAs) in bioprocess effluents makes it difficult to predict the power output of VFA-based MFCs when this technology is coupled to a previous bioprocess. In this work, the effect of the degree of reduction of single VFAs on MFC potential was investigated, and a novel acclimation procedure involving the feeding of gradually more complex VFA mixtures is proposed. The power output obtained with butyric acid (6.1 ± 2 mW m−2) was higher than that obtained with propionic acid (5.0 ± 1 mW m−2), as expected based on their degree of reduction. The MFC potential generated by an acetic acid-fed MFC reached 60 mV, but an acetic-butyric acid mixture reduced the cell potential by one order of magnitude. The subsequent addition of propionic acid to the acetic-butyric mixture increased the MFC potential from 1.9 ± 0.1 mV to 35 ± 10 mV in sequential feeding cycles. Consequently, the feeding sequence of acetic, acetic-butyric, and acetic-butyric-propionic acids is proposed as an efficient strategy for the acclimation of MFCs to complex substrates.

The effect of two different inoculum pretreatments, thermal and cell wash-out (A1 and A2, respectively) on the performance of anaerobic fluidized bed reactors for hydrogen production was determined. The reactors were operated for 112 days under the same operational conditions using glucose as substrate at increasing organic loading rates and decreasing hydraulic retention times. Both treatments were effective avoiding methanogenesis. Reactor A2 showed better performance and stability than reactor A1 in each one of the different operational conditions. Cell wash-out treatment produced higher hydrogen volumetric production rates and yields than thermal treatment (7 L H2/L-d, 3.5 mol H2/mol hexose, respectively). DGGE analysis revealed that the microbial communities developed were affected by the inoculum treatment. Organisms from the genera Clostridium and Lactobacillus predominated in both reactors, with their relative abundances linked to hydrogen production. Resilience was observed in both reactors after a period of starvation.

It is well known that activated sludge treatment systems generate a lot of surplus sludge having environmental and economic impacts. Although several approaches have been proposed for the treatment/reuse of the excess of sludge, there are few studies focused on decreasing the biomass yield without affecting the metabolic activity. This work reports the effect of low magnitude electrical fields (0.07 to 0.2 V/cm) on the growth yield of a pure strain of Pseudomonas denitrificans (used as model microorganism). Cell potentials between 0.2 and 0.57 V were measured during 24 h to the aerobic culture; biomass production and substrate consumption were evaluated at regular intervals. Results indicated that the substrate (lactate) consumption efficiency increased with the applied potential, up to 100%, while the yield diminished 31% (0.34 g biomass/g lactate consumed) at 0.7 V vs. NHE. Bioenergetics showed that the fraction of electron equivalents toward biomass synthesis decreased from 0.68 (when no potential was applied) to 0.47 at 0.57 V, pointing out the redirection of the energy flow toward maintenance to cope with the stress caused by the imposed voltage. Therefore, the electrical stimulus could be used as control of biomass growth in aerobic wastewater treatment lines.

Agave bagasse is the lignocellulosic residue of the tequila industry and a potential feedstock for hydrogen production (H2) via dark fermentation after proper saccharification. Highly specialized enzymes for hydrolysis of lignocellulosic biomass are costly, hindering their application in continuous processes. This work investigated the use of a generic cellulase (Cellulase 50 XL), to prepare the hydrolysates from agave bagasse, either alone or combined with a highly specialized hemicellulase (Viscozyme L). The combination of Cellulase 50 XL and Viscozyme L increased the release of both total carbohydrates and chemical oxygen demand (COD) in 28.5% and 22.2%, respectively compared with Cellulase 50 XL. Conversely, the maximum volumetric H2 production rate (VHPR, 9.9 L H2/L-d) reached with the Cellulase 50 XL hydrolysates in continuous stirred tank reactors at an organic loading rate of 100 g COD/L-d, is the highest VHPR reported so far with enzymatic agave bagasse hydrolysates obtained with non-specialized enzyme. The estimated cost of H2 production using Cellulase 50 XL at lab-scale was 0.7 USD/L H2. From a systematic comparison of H2 productivities and lab-scale costs with similar studies it was concluded that: i) the composition, and not only the concentration of total carbohydrates plays a critical role in the performance of H2 production, ii) Cellulase 50 XL was one of the best cost-effective alternatives despite being designed for food sectors and not for the hydrolysis of lignocellulosic biomass. Overall, considering H2 productivities and cost of H2 production, Cellulase 50 XL hydrolysates resulted an appropriate option for cost-effective continuous H2 production.

Over the past decades, biofuels from microalgae have gained increasing attention. In the present work, methane production from mono-digestion of thermochemical microalgal hydrolysate and co-digestion with cheese whey was evaluated in an anaerobic sequencing batch reactor. The reactor was fed with 5 g total chemical oxygen demand (TCOD)/L and operated either at alkaline or neutral pH. The average methane content in the biogas was higher at alkaline pH (83 %) than at neutral pH (61 %), whereas the methane yield from co-digestion was similar at alkaline (233 N mL/g TCOD) and neutral pH (226 N mL/g TCOD), being 1.8-fold higher than the mono-digestion of the microalgal hydrolysate. After increasing stepwise the organic load up to 12 g TCOD/L, the methane production rate reached peaks around 1000 N mL/L-d, being between 200−800 mL/L-d in those stages with lower organic loads. The methane production from the particulate fraction of the microalgal hydrolysate and its co-digestion with cheese whey demonstrated that the particulate fraction was indeed used to produce methane, but co-digestion did not improve methane yield. The results showed that microalgal hydrolysate could be efficiently co-digested with a carbon-rich substrate enhancing methane production.

The intensive use of groundwater by the gold mining industry is an important sustainability concern, especially in arid and semiarid regions where groundwater is a scarce resource. Alternatives, such as water reuse, treatment and recycling, have been implemented to overcome this issue. The potential use of reclaimed wastewater in gold heap leaching, without decreasing the process efficiency, has not yet been considered. Hence, this review focuses on the physicochemical, biological, and chemical features (organic matter, microbial loads, metal ions, and anions) of reclaimed wastewater that may limit its use in gold heap leaching.

Hydrogen production by fermentation with granular anaerobic biomass has been regarded as one alternative to obtain clean energy. However critical aspects such as process conditions to obtain hydrogenogenic granules have been scarcely investigated. In this work two inoculum pretreatments and different upward liquid velocities (ULV) in expanded granular sludge bed (EGSB) reactors were applied to determine the influence of these parameters on the formation, structure and specific hydrogenogenic activity (SHA) of the granules. Heat pretreated inoculum produced granules with greater manipulation resistance compared to those produced with wash-out pretreatment. Furthermore, the increase of ULV (2.5–4.5 m/h) generated bigger granules with a granule size distribution with a trend to a normal distribution and higher protein to carbohydrate ratios. Mass balances showed that propionate was the main metabolite, nonetheless, its production decreased substantially as the ULV applied increased, indicating a selective wash-out of propionate-producing bacteria. In the opposite way, SHA values were higher as the ULV applied increased as a result of selective enrichment and better mass transfer conditions. The whole results indicated that the inoculum pretreatment and hydrodynamic conditions play a key role in the formation, structure and in the biological properties of hydrogen-producing granules in EGSB reactors.

International audience; Hydrogen (H-2) produced by dark fermentation is an alternative to fulfill the requirements of the transportation sector and to be a complementary source in the forthcoming electricity grid. However, the dark fermentative H-2 production is limited by the accumulation of H-2 in the fermentation broth. In continuous stirred-tank reactors (CSTR), such phenomenon is associated with poor mass transfer conditions. Nevertheless, this parameter has been scarcely considered to enhance H-2 production. In this research, the effect of the H-2 mass transfer conditions on the productivity and efficiency of continuous H-2 production was evaluated using a series of CSTR operated at H-2 mass transfer coefficients (kLa) ranging from 1.04 to 4.23 1/h. The results showed that volumetric H-2 production rate (VHPR) and H-2 yield increased 74 and 78%, respectively, due to enhanced mass transfer conditions. This behavior was driven by 53% decrease of the dissolved H-2 concentration. More specifically, the maximum VHPR of 7.66 L/L-d with a H-2 yield of 1.1 mol H-2/mol hexose was obtained at a k(L)a= 4.23 1/h. Furthermore, 16S-DGGE analysis and sequencing revealed that Clostridium and Lactobacillus were the dominant bacterial genera in continuous operation. In particular, Clostridium increased its occurrence at kLa of 2.72-4.23 1/h as a response to lower dissolved H-2 concentrations. The novelty of this work relies on the demonstration that mass transfer conditions control not only the H-2 accumulation and reactor performance (VHPR and H-2 yield), but they also influence the metabolic pathways and the composition of the microbial community.

Sequential hydrolysis of oat straw and hydrogen (H2) production from hydrolysates and hydrolysates constituents were investigated. One acid-enzymatic and two acid-alkaline-enzymatic procedures were assessed to measure their capability to convert hemicellulose and cellulose from oat straw to sugars. Acid hydrolysis, using HCl, resulted effective to depolymerize hemicellulose (81%) and also facilitated the activity of cellulases over remaining fiber. When alkaline hydrolysis, using KOH/NaClO2/KOH or NaOH/H2O2, were applied, the depolymerization of cellulose was only slightly increased. Total sugar recoveries ranged from 69 to 79% in the different sequential hydrolysis tested. Hence, hydrolysates from sequential acid-enzymatic hydrolysis were used as substrates for H2 production in batch assays. The enzymatic hydrolysate produced a higher H2 molar yield (2.39 mol H2/mol reducing sugars) than the acid hydrolysate (1.1 mol H2/mol reducing sugars). It was found that lower H2 production from the acid hydrolysate was partially due to a lower H2 yield from arabinose and not to measured microbial inhibitors (furfural, hydroxymethylfurfural, vanillin and syringaldehyde). Also, it was found that the commercial enzyme (Celluclast 1.5 L) was easily fermented, and greatly contributed to the H2 production. This is the first study that provides experimental evidence of H2 production from fermentation of a commercial enzyme.

Hydrogen production by dark fermentation is one of the most promising methods for obtaining clean energy. Inoculum pretreatments allow the selection of bacteria that have better performance in hydrogen production, because the selection of pretreatment limits the presence of some species while favoring others. In order to elucidate the inoculum pretreatment influence during the operation of two EGSB reactors, two pretreatments were assayed: heat shock and cell wash-out. Different organic loading rates (24–60 g glucose/L d) and hydraulic retention times (10–4 h) were applied to both reactors to determine population dynamics along 100 days of operation. Reactors exhibited differences in both volumetric hydrogen production rate and molar yield but with cell wash-out pretreatment showing better performance than heat shock pretreatment. Maximum molar yield (0.92 mol H 2 /mol hexose) and volumetric hydrogen production rate (4.23 L H 2 /L d) were obtained with organic loading rates of 36 g glucose/L d at HRT of 10 h in EGSB reactor inoculated with cell wash-out pretreated sludge. The microbial community of the reactors samples was analyzed by 16S rRNA genes profiles and the predominant bands were excised and their DNA sequence determined. Clostridium and representatives of Enterobacteriaceae were dominant, with a strong presence of Lactobacillus genus. The whole result indicates that the inoculum pretreatment has a strong initial effect during early stages of fermentation, after which the operating conditions have a greater impact on reactor performance.

The hydrogen (H-2) production efficiency in dark fermentation systems is strongly dependent on the occurrence of metabolic pathways derived from the selection of microbial species that either consume molecular H-2 or outcompete hydrogenogenic bacteria for the organic substrate. In this study, the effect of organic loading rate (OLR) on the H-2 production performance, the metabolic pathways, and the microbial community composition in a continuous system was evaluated. Two bacterial genera, Clostridium and Streptococcus, were dominant in the microbial community depending on the OLR applied. At low OLR (14.7-44.1 g(Lactose)/L-d), Clostridium sp. was dominant and directed the system towards the acetate-butyrate fermentation pathway, with a maximum H-2 yield of 2.14 mol(H2)/mol(Hexose) obtained at 29.4 g(Lactose)/L-d. Under such conditions, the volumetric hydrogen production rate (VHPR) was between 3.2 and 11.6 L-H2/L-d. In contrast, relatively high OLR (58.8 and 88.2 g(Lactose)/L-d) favored the dominance of Streptococcus sp. as co-dominant microorganism leading to lactate production. Under these conditions, the formate production was also stimulated serving as a strategy to dispose the surplus of reduced molecules (e.g., NADH(2) (+)), which theoretically consumed up to 5.72 L-H2/L-d. In such scenario, the VHPR was enhanced (13.7-14.5 L-H2/L-d) but the H-2 yield dropped to a minimum of 0.74 mol(H2)/mol(Hexose) at OLR = 58.8 g(Lactose)/L-d. Overall, this research brings clear evidence of the intrinsic occurrence of metabolic pathways detrimental for biohydrogen production, i.e., lactic acid fermentation and formate production, suggesting the use of low OLR as a strategy to control them.

Several studies have demonstrated that gypsum (CaSO4·2H2O) and calcite (CaCO3) can be important hosts of arsenic in contaminated hydrogeological systems. However, the extent to which microbial reducing processes contribute to the dissolution and transformation of carbonate and sulfate minerals and, thereby, to arsenic mobilization is poorly understood. These processes are likely to have a strong impact on arsenic mobility in iron-poor environments and in reducing aquifers where iron oxyhydroxides become unstable. Anoxic batch bioassays with arsenate (As(V)) coprecipitated with calcite, gypsum, or ferrihydrite (Fe(OH)3) were conducted in the presence of sulfate or molybdate to examine the impact of bioprocesses (i.e. As(V), sulfate, and Fe(III)-reduction) on arsenic dissolution, speciation, and eventual remineralization. Microbial reduction of As(V)-bearing calcite caused an important dissolution of arsenite, As(III), which remained in solution up to the end of the experiment (30 days). The reduction of As(V) from gypsum-As(V) also led to the release of As(III), which was subsequently remineralized, possibly as arsenic sulfides. The presence of sulfate triggered arsenic dissolution in the bioassays with ferrihydrite-As(V). This study showed that although gypsum and calcite have a lower capacity to bind arsenic, compared to iron oxides, they can play a critical role in the biogeochemical cycle of arsenic in natural calcareous and gypsiferous systems depleted of iron since they can be a source of electron acceptors for reducing bioprocesses.

Methane occurrence is a common concern in hydrogen producing reactors. This study presents the analysis of the microbial community structure during the application of operational strategies to decrease methane production, in three different up-flow anaerobic sludge blanket hydrogen-producing reactors. Cloning and denaturing gradient gel electrophoresis approach were used to establish the presence of homoacetogens, methanogens and hydrogen producers. The results showed that homoacetogenic organisms related to Blautia hydrogenotrophica and Oscillibacter valericigenes, and the hydrogen producer Enterobacter aerogenes where favored during pH decreasing strategies (5.6 to 4.5). The increment of the organic loading rate from 20 to 30 g chemical oxygen demand/L-d, selected hydrogen producers similar to Clostridium tyrobutyricum, Citrobacter freundii and E. aerogenes ; further increments caused inhibition of hydrogen production due to the high undissociated acids concentration. Methane production was inhibited completely only when the biomass of the reactor was heat treated for a second time, this strategy selected hydrogen producers capable to sporulate, but homoacetogens were also favored. In all reactors the methanogenic activity was attributed to hydrogenotrophs related to the genera Methanobrevibacter and Methanobacterium .

BACKGROUND Previous studies have reported that complexity of the substrate, type of inoculum and reactor configuration strongly affect fermentative hydrogen production. Therefore, the aim of this study was to evaluate the effect of simple (glucose, xylose and sucrose) and complex (acid and enzymatic oat straw hydrolysates) substrates on the continuous hydrogen production in a trickling bed reactor. Novel inoculum (triticale silage) and biofilm support configuration (vertically organized PET tubing) were tested. RESULTS The enzymatic hydrolysate was a suitable substrate for hydrogen production, since its hydrogen molar yield was similar to that obtained with glucose, mean values of 1.6 and 1.7 mol H2 mol−1 sugar consumed were obtained, respectively. In contrast, hydrogen was not produced from the acid hydrolysate. The highest hydrogen production rate (840 mL H2 L−1 h−1) was obtained with glucose at an organic loading rate of 160 g COD L−1 d−1. In spite of the high organic loading rate applied, clogging due to excessive biomass growth was not observed. Finally, PCR-DGGE analysis revealed that bacteria from Clostridium genus were the putative responsible for hydrogen production. CONCLUSION This work demonstrates that continuous hydrogen production is affected by complexity of the substrate. Furthermore, it also shows the feasibility of using triticale silage as inoculum in hydrogen production systems. © 2014 Society of Chemical Industry

One of the problems in fermentative hydrogen producing reactors, inoculated with pre-treated anaerobic granular sludge, is the eventual methane production by hydrogen-consuming methanogens. In this study, strategies such as reduction of pH and HRT, organic shock loads and repeated biomass heat treatment were applied to hydrogenogenic UASB reactors fed with cheese whey, that showed methane production after certain time of continuous operation (between 10 and 60 days). The reduction of pH to 4.5 not only decreased methane production but also hydrogen production. Organic shock load (from 20 to 30 g COD/L-d) was the more effective strategy to decrease the methane production rate (75%) and to increase the hydrogen production rate (172%), without stopping reactor operation. Repeated heat treatment of the granular sludge was the only strategy that inhibited completely methane production, leading to high volumetric hydrogen production rates (1.67 L H2/L-d), however this strategy required stopping reactor operation; in addition homoacetogenesis, another hydrogen-consuming pathway, was not completely inhibited. This work demonstrated that it was possible to control the methane activity in hydrogen producing reactors using operational strategies.

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Feasibility of hydrogen production from acid and enzymatic oat straw hydrolysates was evaluated in an anaerobic sequencing batch reactor at 35 °C and constant substrate concentration (5 g chemical oxygen demand/L). In a first experiment, hydrogen production was replaced by methane production. Selective pressures applied in a second experiment successfully prevented methane production. During this experiment, initial feeding with glucose/xylose, as model substrates, promoted biomass granulation. Also, the highest hydrogen molar yield (HMY, 2 mol H 2 /mol sugar consumed) and hydrogen production rate (HPR, 278 mL H 2 /L-h) were obtained with these model substrates. Gradual substitution of glucose/xylose by acid hydrolysate led to disaggregation of granules and lower HPR and HMY. When the model substrates were completely substituted by enzymatic hydrolysate, the HMY and HPR were 0.81 mol H 2 /mol sugar consumed and 29.6 mL H 2 /L-h, respectively. Molecular analysis revealed a low bacterial diversity in the stages with high hydrogen production and vice versa. Furthermore, Clostridium pasteurianum was identified as the most abundant species in stages with a high hydrogen production. Despite that feasibility of hydrogen production from hydrolysates was demonstrated, lower performance from hydrolysates than from model substrates was obtained.

The anaerobic degradation of azo dyes under anaerobic conditions is possible but at a slow rate. Redox mediators (quinones, activated carbon) are used to improve the reduction rate. The aim of this work was to use activated carbon fiber (ACF) as a redox mediator for the anaerobic reduction of the azo dye methyl red. ACF was chemically modified with 8 M HNO3 to increase its redox-mediating capacity and used in chemical and anaerobic biological batch assays for the reduction of methyl red. ACF increased its redox-mediating capacity up to 3-fold in chemical assays; in biological assays ACF increased the reduction rate up to 8-fold compared to controls without ACF. However, since the ACF served as support for biomass, a biofilm formed on the fiber significantly reduced its redox-mediating capacity; substrate consumption suggested that the electron transport from ACF to methyl red was the rate-limiting step in the process. These results are the first evidence of the role of ACF as a redox mediator in the reductive decolorization of methyl red, in addition to the effect of biofilm attached to ACF on methyl red reduction. Due to the versatile characteristics of ACF and its redox-mediating capacity, carbon fibers could be used in biological wastewater treatment systems to accelerate the reductive transformation of pollutants commonly found in industrial effluents.

Sulfate reduction is an appropriate approach for the treatment of effluents with sulfate and dissolved metals. In sulfate-reducing reactors, acetate may largely contribute to the residual organic matter, because not all sulfate reducers are able to couple the oxidation of acetate to the reduction of sulfate, limiting the treatment efficiency. In this study, we investigated the diversity of a bacterial community in the biofilm of a laboratory scale down-flow fluidized bed reactor, which was developed under sulfidogenic conditions at an influent pH between 4 and 6. The sequence analysis of the microbial community showed that the 16S rRNA gene sequence of almost 50% of the clones had a high similarity with Anaerolineaceae. At second place, 33% of the 16S rRNA phylotypes were affiliated with the sulfate-reducing bacteria Desulfobacca acetoxidans and Desulfatirhabdium butyrativorans, suggesting that acetotrophic sulfate reduction was occurring in the system. The remaining bacterial phylotypes were related to fermenting bacteria found at the advanced stage of reactor operation. The results indicate that the acetotrophic sulfate-reducing bacteria were able to remain within the biofilm, which is a significant result because few natural consortia harbor complete oxidizing sulfate-reducers, improving the acetate removal via sulfate reduction in the reactor.

BACKGROUND Sulfate-reducing fluidized bed reactors represent an alternative for the treatment of wastewaters that contain dissolved metals. However, the low acetate consumption achieved through sulfate reduction affects the organic matter removal efficiency. The aim of this present work was to develop a sulfidogenic biofilm able to consume acetate via sulfate reduction within a short start-up period (21 days). Three experiments were conducted in a down-flow fluidized bed reactor with different acetate/lactate proportions in the feed (50/50, 80/20 and 90/10). Reduction of the influent pH from 6.0 to 4.0 was also studied at the higher acetate content. RESULTS Organic matter oxidation efficiency was similar in the three experiments (∼67%), nonetheless the sulfate reduction rate was higher in the experiments with 80 and 90% acetate in the feed (744 and 730 mg L−1 d−1). Acetate oxidation via sulfate reduction was highest (39% of inlet) at the ratio 90/10, at which the biofilm specific sulfate reducing activity with acetate was 4.3 times higher than that developed at 50/50. Influent pH reduction to 4.0 was not detrimental to acetate consumption via sulfate reduction, which was 56%. Analysis of the biofilm through DGGE found a similar community in the three experiments and the presence of acetotrophic microorganisms affiliated to Desulfobacca acetoxidans. CONCLUSIONS Limiting the substrate (lactate) was an appropriate strategy to enrich acetate-consuming sulfate reducers and improve the low acetate removal efficiency that sulfate-reducing reactors face. © 2012 Society of Chemical Industry

Different technologies have been implemented for the treatment of acid mine drainage. Among these are technologies such as geochemical barriers and sulfidogenic reactors, which use biogenic sulfide (produced by sulfate-reducing bacteria) for metallic stabilization. Because both processes involve microorganisms, it is important to have a clear understanding of the factors that influence their activity in different toxic environments. Given that microbial communities isolated from polluted sites could have a higher tolerance to toxic ions, two consortia with sulfate-reducing activity were isolated from different soils impacted by mining activities. These soils had different total (401 and 19,300 mg Pb kg−1), mobile (54 and 1,415 mg Pb kg−1) and bioaccessible (316 and 3,175 mg Pb kg−1) concentrations of lead. The kinetics of biogenic sulfide production (BSP) for both consortia were monitored in a batch reactor after they were exposed to different initial lead concentrations. These lead concentrations were established based on the results of lead mobility tests. The estimated BSP rates and biomass concentrations of both consortia showed different responses to the presence of lead. Results highlighted that lead sulfide precipitation on microbial cell is a tolerance mechanism identified and this one is triggered for the lead bioaccessible concentrations threshold in soil. These results could be useful for the designing of processes based on sulfate reducing activity for the removal or stabilization of metal present in water or soil, respectively.

The effect of different operational strategies and inoculum structure (granules and disaggregated granules) during the start-up of four up-flow anaerobic sludge blanket hydrogenogenic reactors was investigated. The more stable volumetric hydrogen production rates obtained were 0.38 and 0.36 L H2/L-d, in reactors operated with a constant organic loading rate (OLR) with both inoculum structures, whereas in reactors operated with an increasing OLR methane started to be produced earliest in time. Specific hydrogenogenic activity results proved that the disaggregated inoculum produced a more active biomass than the granular one, but not granule formation was evident. The methane hydrogenotrophic activity was the main limitation of the systems evaluated. In the reactors inoculated with disaggregated sludge the start-up strategy did not influence the bacterial DGGE fingerprint, in contrast to the reactors started-up with granular sludge; members of the Clostridium genus were always present. The results demonstrated that operational conditions during the start-up period are crucial for the production of hydrogenogenic biomass.

This study investigated the inhibition effect of iron, cadmium and sulfide on the substrate utilization rate of sulfate reducing granular sludge. A series of batch experiments in a UASB reactor were conducted with different concentrations of iron (Fe2+, 4.0–8.5 mM), cadmium (Cd2+, 0.53–3.0 mM) and sulfide (4.2–10.6 mM), the reactor was fed with ethanol at 1 g chemical oxygen demand (COD)/L and sulfate to yield a COD/SO42− (g/g) ratio of 0.5. The addition of iron, up to a concentration of 8.1 mM, had a positive effect on the substrate utilization rate which increased 40% compared to the rate obtained without metal addition (0.25 g COD/g VSS-d). Nonetheless, iron concentration of 8.5 mM inhibited the specific substrate utilization rate by 57% compared to the substrate utilization rate obtained in the batch amended with 4.0 mM Fe2+ (0.44 g COD/g VSS-d). Cadmium had a negative effect on the specific substrate utilization rate at the concentrations tested; at 3.0 mM Cd2+ the substrate utilization rate was inhibited by 44% compared with the substrate utilization rate without metal addition. Cadmium precipitation with sulfide did not decrease the inhibition of cadmium on sulfate reduction. These results could have important practical implications mainly when considering the application of the sulfate reducing process to treat effluents with high concentrations of sulfate and dissolved metals such as iron and cadmium.

An anaerobic down-flow fluidized bed reactor was inoculated with granular sludge and started-up with sulfate containing synthetic wastewater to promote the formation of a biofilm enriched in sulfate-reducing bacteria (SRB), to produce biogenic sulfide. The start-up was done in two stages operating the reactor in batch for 45 days followed by 85 days of continuous operation. Low-density polyethylene was used as support. The biofilm formation was followed up by biochemical and electron microscopy analyses and the composition of the community was examined by 16S rDNA sequence analysis. Maximum immobilized volatile solids (1.2 g IVS/L(support)) were obtained after 14 days in batch regime. During the 85 days of continuous operation, the reactor removed up to 80% of chemical oxygen demand (COD), up to 28% of the supplied sulfate and acetate was present in the effluent. Sulfate-reducing activity determined in the biofilm with ethanol or lactate as substrate was 11.7 and 15.3 g COD/g IVS per day, respectively. These results suggested the immobilization of sulfate reducers that incompletely oxidize the substrate to acetate; the phylogenetic analysis of the cloned 16S rDNA gene sequences showed high identity to the genus Desulfovibrio that oxidizes the substrates incompletely. In contrast, in the granular sludge used as inoculum a considerable number of clones showed homology to Methanobacterium and just few clones were close to SRB. The starting-up approach allowed the enrichment of SRB within the diverse community developed over the polyethylene support.

BACKGROUND: Biological sulfate removal is a process based on the biological sulfur cycle that consists of two steps: (1) production of sulfide by sulfate reduction; and (2) biological or physico-chemical sulfide oxidation to elemental sulfur (S0). The objective of this work was to transform soluble sulfur (sulfate) into insoluble sulfur (elemental sulfur) coupling sulfate reduction and sulfide oxidation in one reactor. To accomplish this, a 2.3 L down-flow fluidized bed reactor was used. Lactate was supplied as electron donor, sulfate and oxygen (air) were the electron acceptors. RESULTS: After 55 days of batch operation a biofilm with sulfate reducing and sulfide oxidizing activities was developed over a plastic support. Continuous operation for 90 days at a down-flow superficial velocity of 7.7 m h−1 and 30 °C, showed that sulfate reduction amounted to 72–77% and carbon removal to 20–31%. Under low aeration rates (2.3 L d−1) 50% of the sulfate was transformed to elemental sulfur, when aeration increased to 5.4 L d−1 elemental sulfur recovery was only 30% and sulfide in the effluent amounted to 27% of the sulfur fed. CONCLUSION: It was possible to obtain elemental sulfur through a coupled anaerobic/aerobic process in one reactor using lactate, sulfate and oxygen (air) as substrates. The development of a biofilm with sulfate reducing and sulfide oxidizing activities was the key of the process. Copyright © 2007 Society of Chemical Industry

In recent years, anaerobic digestion has been recognized as a suitable alternative for tequila vinasses treatment due to its high energy recovery and chemical oxygen demand (COD) removal efficiency. However, key factors such as the lack of suitable monitoring schemes and the presence of load disturbances, which may induce unstable operating conditions in continuous systems, have limited its application at full scale. Therefore, the aim of this work was to evaluate the anaerobic sequencing batch reactor (AnSBR) configuration in order to provide a low cost and easy operation alternative for the treatment of these complex effluents. In particular, the AnSBR was evaluated under different pH–temperature combinations: 7 and 32 °C; 7 and 38 °C; 8 and 32 °C and 8 and 38 °C. Results showed that the AnSBR configuration was able to achieve high COD removal efficiencies (around 85%) for all the tested conditions, while the highest methane yield was obtained at pH 7 and 38 °C (0.29 L/g COD added). Furthermore, high robustness was found in all the AnSBR experiments. Therefore, the full-scale application of the AnSBR technology for the treatment of tequila vinasses is quite encouraging, in particular for small and medium size tequila industries that operate under seasonal conditions.

Graphene oxide (GO) is reported for the first time as electron shuttle to increase the redox conversion of the azo compound, reactive red 2 (RR2, 0.5mM), and the nitroaromatic, 3-chloronitrobenzene (3CNB, 0.5mM). GO (5mgL(-1)) increased 10-fold and 7.6-fold the reduction rate of RR2 and 3CNB, respectively, in abiotic incubations with sulfide (2.6mM) as electron donor. GO also increased by 2-fold and 3.6-fold, the microbial reduction rate of RR2 by anaerobic sludge under methanogenic and sulfate-reducing conditions, respectively. Deep characterization of GO showed that it has a proper size distribution (predominantly between 450 and 700nm) and redox potential (+50.8mV) to promote the reduction of RR2 and 3CNB. Further analysis revealed that biogenic sulfide plays a major role on the GO-mediated reduction of RR2. GO is proposed as an electron shuttle to accelerate the redox conversion of recalcitrant pollutants, such as nitro-benzenes and azo dyes.

Humus constitutes a very abundant class of organic compounds that are chemically heterogeneous and widely distributed in terrestrial and aquatic environments. Evidence accumulated during the last decades indicating that humic substances play relevant roles on the transport, fate, and redox conversion of organic and inorganic compounds both in chemically and microbially driven reactions. The present review underlines the contribution of humus-reducing microorganisms in relevant environmental processes such as biodegradation of recalcitrant pollutants and mitigation of greenhouse gases emission in anoxic ecosystems, redox conversion of industrial contaminants in anaerobic wastewater treatment systems, and on the microbial production of nanocatalysts and alternative energy sources.

The present study reports a novel treatment concept combining the redox-mediating capacity of immobilized humic substances with the biodegrading activity of anaerobic sludge for the simultaneous removal of two representative pollutants of textile wastewaters (e.g., phenol and Reactive Red 2 (RR2)) in a high-rate anaerobic reactor. The use of immobilized humic substances (1 g total organic carbon (TOC) L(-1), supported on an anion exchange resin) in an upflow anaerobic sludge blanket (UASB) reactor increased the decolorization efficiency of RR2 (~90 %), extent of phenol oxidation (~75 %), and stability as compared to a control UASB reactor operated without immobilized humic substances, which collapsed after 120 days of dye introduction (50-100 mg L(-1)). Increase in the concentration of immobilized humic substances (2 g TOC L(-1)) further enhanced the stability and efficiency of the UASB reactor. Detection of aniline in the effluent as RR2 reduction product confirmed that reduction of RR2 was the major mechanism of dye removal. This is the first demonstration of immobilized humic substances serving as effective redox mediators for the removal of recalcitrant pollutants from wastewater in a high-rate anaerobic bioreactor. The novel treatment concept could also be applicable to remove a wide variety of contaminants susceptible to redox conversion, which are commonly found in different industrial sectors.

Methanogens have been reported in complex microbial communities from hypersaline environments, but little is known about their phylogenetic diversity. In this work, methane concentrations in environmental gas samples were determined while methane production rates were measured in microcosm experiments with competitive and non-competitive substrates. In addition, the phylogenetic diversity of methanogens in microbial mats from two geographical locations was analyzed: the well studied Guerrero Negro hypersaline ecosystem, and a site not previously investigated, namely Laguna San Ignacio, Baja California Sur, Mexico. Methanogenesis in these microbial mats was suspected based on the detection of methane (in the range of 0.00086 to 3.204 %) in environmental gas samples. Microcosm experiments confirmed methane production by the mats and demonstrated that it was promoted only by non-competitive substrates (trimethylamine and methanol), suggesting that methylotrophy is the main characteristic process by which these hypersaline microbial mats produce methane. Phylogenetic analysis of amino acid sequences of the methyl coenzyme-M reductase (mcrA) gene from natural and manipulated samples revealed various methylotrophic methanogens belonging exclusively to the family Methanosarcinaceae. Moderately halophilic microorganisms of the genus Methanohalophilus were predominant (60 % of mcrA sequences retrieved). Slightly halophilic and marine microorganisms of the genera Methanococcoides and Methanolobus, respectively, were also identified, but in lower abundances.

Extremophilic anaerobes are widespread in saline, acid, alkaline, and high or low temperature environments. Carbon is essential to living organisms and its fixation, degradation, or mineralization is driven by, up to now, six metabolic pathways. Organisms using these metabolisms are known as autotrophs, acetotrophs or carbon mineralizers, respectively. In anoxic and extreme environments, besides the well-studied Calvin–Benson–Bassham cycle, there are other five carbon fixation pathways responsible of autotrophy. Moreover, regarding carbon mineralization, two pathways perform this key process for carbon cycling. We might imagine that all the pathways can be found evenly distributed in microbial biotopes; however, in extreme environments, this does not occur. This manuscript reviews the most commonly reported anaerobic organisms that fix carbon and mineralize acetate in extreme anoxic habitats. Additionally, an inventory of autotrophic extremophiles by biotope is presented.