Dataset of anaerobic acidogenic digestion for hydrogen production using xylose as substrate: Biogas production and metagenomic data.

This paper presents the raw data of biogas production and composition (relative pressures and concentrations of each of the biogas constituents) for batch experiments to evaluate the anaerobic digestion of xylose. Also, metagenomic sequencing data and analysis were reported. All data is available at Mendeley Data. 16S DNA sequencing data and metadata is available at MG-RAST (metagenomics.anl.gov/linkin.cgi?project = 9961). For further discussion, please refer to the scientific article entitled "Effect of acidic and thermal pretreatments on a microbial inoculum for hydrogen and volatile fatty acids production through xylose anaerobic acidogenic metabolism" (Mockaitis et al., 2020).

Characterization of the protein fraction of the extracellular polymeric substances of three anaerobic granular sludges.

Extracellular polymeric substances (EPS) play major roles in the efficacy of biofilms such as anaerobic granules, ranging from structural stability to more specific functions. The EPS of three granular anaerobic sludges of different origins were studied and compared. Particularly, the peptides from the protein fraction were identified by mass spectrometry. Desulfoglaeba and Treponema bacterial genera and Methanosaeta and Methanobacterium archaeal genera were prominent in all three sludges. Methanosaeta concilii proteins were the most represented in EPS of all three sludges studied. Principally, four proteins found in the three sludges, the S-layer protein, the CO-methylating acetyl-CoA synthase, an ABC transporter substrate-binding protein and the methyl-coenzyme M reductase, were expressed by Methanosaeta concilii. Mainly catabolic enzymes were found from the 45 proteins identified in the protein fraction of EPS. This suggests that EPS may have a role in allowing extracellular catabolic reactions.

Strategy to identify the causes and to solve a sludge granulation problem in methanogenic reactors: application to a full-scale plant treating cheese wastewater.

Granulation of biomass is at the basis of the operation of the most successful anaerobic systems (UASB, EGSB and IC reactors) applied worldwide for wastewater treatment. Despite of decades of studies of the biomass granulation process, it is still not fully understood and controlled. "Degranulation/lack of granulation" is a problem that occurs sometimes in anaerobic systems resulting often in heavy loss of biomass and poor treatment efficiencies or even complete reactor failure. Such a problem occurred in Mexico in two full-scale UASB reactors treating cheese wastewater. A close follow-up of the plant was performed to try to identify the factors responsible for the phenomenon. Basically, the list of possible causes to a granulation problem that were investigated can be classified amongst nutritional, i.e. related to wastewater composition (e.g. deficiency or excess of macronutrients or micronutrients, too high COD proportion due to proteins or volatile fatty acids, high ammonium, sulphate or fat concentrations), operational (excessive loading rate, sub- or over-optimal water upflow velocity) and structural (poor hydraulic design of the plant). Despite of an intensive search, the causes of the granulation problems could not be identified. The present case remains however an example of the strategy that must be followed to identify these causes and could be used as a guide for plant operators or consultants who are confronted with a similar situation independently of the type of wastewater. According to a large literature based on successful experiments at lab scale, an attempt to artificially granulate the industrial reactor biomass through the dosage of a cationic polymer was also tested but equally failed. Instead of promoting granulation, the dosage caused a heavy sludge flotation. This shows that the scaling of such a procedure from lab to real scale cannot be advised right away unless its operability at such a scale can be demonstrated.

Acidogenic digestion of food waste in a thermophilic leach bed reactor: Effect of pH and leachate recirculation rate on hydrolysis and volatile fatty acid production.

The effect of pH control (4, 5, 6, 7) on volatile fatty acids (VFA) production from food waste was investigated in a leach bed reactor (LBR) operated at 50°C. Stabilisation of pH at 7 resulted in hydrolysis yield of 530g soluble chemical oxygen demand (sCOD)/kg total volatile solids (TVS) added and VFA yield of 247gCOD/kg TVS added, which were highest among all pH tested. Butyric acid dominated the VFA mix (49-54%) at pH of 7 and 6, while acetate composed the primary VFA (41-56%) at pH of 4 and 5. A metabolic shift towards lactic acid production was observed at pH of 5. Improving leachate recirculation rate further improved the hydrolysis and degradation efficiency by 10-16% and the acidification yield to 340gCOD/kgTVS added. The butyric acid concentration of 16.8g/L obtained at neutral pH conditions is among the highest reported in literature.

Biomethanation of Syngas Using Anaerobic Sludge: Shift in the Catabolic Routes with the CO Partial Pressure Increase.

Syngas generated by thermal gasification of biomass or coal can be steam reformed and purified into methane, which could be used locally for energy needs, or re-injected in the natural gas grid. As an alternative to chemical catalysis, the main components of the syngas (CO, CO2, and H2) can be used as substrates by a wide range of microorganisms, to be converted into gas biofuels, including methane. This study evaluates the carboxydotrophic (CO-consuming) methanogenic potential present in an anaerobic sludge from an upflow anaerobic sludge bed (UASB) reactor treating waste water, and elucidates the CO conversion routes to methane at 35 ± 3°C. Kinetic activity tests under CO at partial pressures (pCO) varying from 0.1 to 1.5 atm (0.09-1.31 mmol/L in the liquid phase) showed a significant carboxydotrophic activity potential for growing conditions on CO alone. A maximum methanogenic activity of 1 mmol CH4 per g of volatile suspended solid and per day was achieved at 0.2 atm of CO (0.17 mmol/L), and then the rate decreased with the amount of CO supplied. The intermediary metabolites such as acetate, H2, and propionate started to accumulate at higher CO concentrations. Inhibition experiments with 2-bromoethanesulfonic acid (BES), fluoroacetate, and vancomycin showed that in a mixed culture CO was converted mainly to acetate by acetogenic bacteria, which was further transformed to methane by acetoclastic methanogens, while direct methanogenic CO conversion was negligible. Methanogenesis was totally blocked at high pCO in the bottles (≥1 atm). However it was possible to achieve higher methanogenic potential under a 100% CO atmosphere after acclimation of the sludge to CO. This adaptation to high CO concentrations led to a shift in the archaeal population, then dominated by hydrogen-utilizing methanogens, which were able to take over acetoclastic methanogens, while syntrophic acetate oxidizing (SAO) bacteria oxidized acetate into CO2 and H2. The disaggregation of the granular sludge showed a negative impact on their methanogenic activity, confirming that the acetoclastic methanogens were the most sensitive to CO, and a contrario, the advantage of using granular sludge for further development toward large-scale methane production from CO-rich syngas.

Acidogenic fermentation of Scenedesmus sp.-AMDD: Comparison of volatile fatty acids yields between mesophilic and thermophilic conditions.

This study compared the acidogenic fermentation of Scenedesmus sp.-AMDD at laboratory-scale, under mesophilic (35°C) and thermophilic conditions (55°C). Preliminary batch tests were performed to evaluate best conditions for volatile fatty acid (VFA) production from microalgal biomass, with respect to the inoculum, pH and nutrients. The use of bovine manure as inoculum, the operating pH of 4.5 and the addition of a nutrient mix, resulted in a high VFA production of up to 222mgg(-1) total volatile solid (TVS), with a butyrate share of 27%. Both digesters displayed similar hydrolytic activity with 0.38±0.02 and 0.42±0.03 g soluble chemical oxygen demand (COD)g(-1) TVS for the digesters operated at 35 and 55°C, respectively. Mesophilic conditions were more favorable for VFA production, which reached 171±5, compared to 88±12 mg soluble CODg(-1) TVS added under thermophilic conditions (94% more). It was shown that in both digesters, butyrate was the predominant VFA.

Direct Interspecies Electron Transfer in Anaerobic Digestion: A Review.

Direct interspecies electrons transfer (DIET) is a syntrophic metabolism in which free electrons flow from one cell to another without being shuttled by reduced molecules such as molecular hydrogen or formate. As more and more microorganisms show a capacity for electron exchange, either to export or import them, it becomes obvious that DIET is a syntrophic metabolism that is much more present in nature than previously thought. This article reviews literature related to DIET, specifically in reference to anaerobic digestion. Anaerobic granular sludge, a biofilm, is a specialized microenvironment where syntrophic bacterial and archaeal organisms grow together in close proximity. Exoelectrogenic bacteria degrading organic substrates or intermediates need an electron sink and electrotrophic methanogens represent perfect partners to assimilate those electrons and produce methane. The granule extracellular polymeric substances by making the biofilm matrix more conductive, play a role as electrons carrier in DIET.

Specific inhibitors for identifying pathways for methane production from carbon monoxide by a nonadapted anaerobic mixed culture.

Specific inhibitors such as 2-bromoethanesulfonate (BES) and vancomycin were employed in activity batch tests to decipher metabolic pathways that are preferentially used by a mixed anaerobic consortium (sludge from an anaerobic digester) to transform carbon monoxide (CO) into methane (CH4). We first evaluated the inhibitory effect of both BES and vancomycin on the microbial community, as well as the efficiency and stability of vancomycin at 35 °C, over time. The activity tests with CO2-H2, CO, glucose, acetate, formate, propionate, butyrate, methanol, and ethanol showed that vancomycin does not inhibit some Gram-negative bacteria, and 50 mmol/L BES effectively blocks CH4 production in the sludge. However, when sludge was incubated with propionate, butyrate, methanol, or ethanol as the sole energy and carbon source, methanogenesis was only partially inhibited by BES. Separate tests showed that 0.07 mmol/L vancomycin is enough to maintain its inhibitory efficiency and stability in the population for at least 32 days at 35 °C. Using the inhibitors above, it was demonstrated that CO conversion to CH4 is an indirect, 2-step process, in which the CO is converted first to acetate and subsequently to CH4.

The role of Carboxydothermus hydrogenoformans in the conversion of calcium phosphate from amorphous to crystalline state.

Two previously unknown modes of biomineralization observed in the presence of Carboxydothermus hydrogenoformans are presented. Following the addition of NaHCO3 and the formation of an amorphous calcium phosphate precipitate in a DSMZ medium inoculated with C. hydrogenoformans, two distinct crystalline solids were recovered after 15 and 30 days of incubation. The first of these solids occurred as micrometric clusters of blocky, angular crystals, which were associated with bacterial biofilm. The second solid occurred as 30-50 nm nanorods that were found scattered among the organic products of bacterial lysis. The biphasic mixture of solids was clearly dominated by the first phase. The X-ray diffractometry (XRD) peaks and Fourier transform infrared spectroscopy (FTIR) spectrum of this biphasic material consistently showed features characteristic of Mg-whitlockite. No organic content or protein could be identified by dissolving the solids. In both cases, the mode of biomineralization appears to be biologically induced rather than biologically controlled. Since Mg is known to be a strong inhibitor of the nucleation and growth of CaP, C. hydrogenoformans may act by providing sites that chelate Mg or form complexes with it, thus decreasing its activity as nucleation and crystal growth inhibitor. The synthesis of whitlockite and nano-HAP-like material by C. hydrogenoformans demonstrates the versatility of this organism also known for its ability to perform the water-gas shift reaction, and may have applications in bacterially mediated synthesis of CaP materials, as an environmentally friendly alternative process.

Growth profile of Carboxydothermus hydrogenoformans on pyruvate.

Carboxydothermus hydrogenoformans is a thermophilic anaerobic strain most widely known for its ability to produce hydrogen (H2) when grown on carbon monoxide (CO). Although relatively well studied, growth characterization on pyruvate has never been assessed. The present work fully characterizes growth of the bacterium on pyruvate as a sole carbon source. C. hydrogenoformans demonstrated a growth rate of 0.03 h-1, with pyruvate consumption ranging between 0.21 and 0.48 mol · g-1 volatile suspended solid · d-1. A lag phase was also observed when switching from pyruvate to CO. When grown simultaneously on pyruvate and CO, pyruvate consumption was initiated upon CO depletion. This was attributed to pyruvate oxidation inhibition by CO, and not to a diauxic phenomenom. The strain also showed homoacetogenic activity.

Anaerobic co-digestion of dairy manure with mulched switchgrass for improvement of the methane yield.

The owners of farm-scale anaerobic digesters are relying on off-farm wastes or energy crops as a co-digestion feedstock with animal manure in order to increase their production of methane and thus revenues. Switchgrass represents an interesting feedstock for Canadian digesters owners as it is a high-yielding low-maintenance perennial crop, well adapted to northern climate. Methane potential assays in batch tests showed methane production of 19.4 ± 3.6, 28.3 ± 1.7, 37.3 ± 7.1 and 45.7 ± 0.8 L kg(-1), for raw manure, blended manure, manure and mulched switchgrass, manure and pretreated switchgrass, respectively. Two 6-L lab-scale anaerobic digesters were operated for 130 days in order to assess the benefit of co-digesting switchgrass with bovine manure (digester #2), at a 20% wet mass fraction, compared with a manure-only operation (digester #1) The digesters were operated at an hydraulic retention time of 37 ± 6 days and at loads of 2.4 ± 0.6 and 2.6 ± 0.6 kg total volatile solids (TVS) L(-1) day(-1) for digesters #1 (D1) and #2 (D2), respectively. The TVS degradation reached 25 and 39%, which resulted in a methane production of 1.18 ± 0.18 and 2.19 ± 0.31 L day(-1) for D1 and D2, respectively. The addition of 20% on a wet mass ratio of switchgrass to a manure digester increased its methane production by 86%. The co-digestion of switchgrass in a 500 m(3) manure digester could yield up to 10.2 GJ day(-1) of purified methane or 1.1 MWh day(-1) of electricity.

Kinetics of CO conversion into H2 by Carboxydothermus hydrogenoformans.

The objective of this study was to improve the biological water-gas shift reaction for producing hydrogen (H(2)) by conversion of carbon monoxide (CO) using an anaerobic thermophilic pure strain, Carboxydothermus hydrogenoformans. Specific hydrogen production rates and yields were investigated at initial biomass densities varying from 5 to 20 mg volatile suspended solid (VSS) L(-1). Results showed that the gas-liquid mass transfer limits the CO conversion rate at high biomass concentrations. At 100-rpm agitation and at CO partial pressure of 1 atm, the optimal substrate/biomass ratio must exceed 5 mol CO g(-1) biomass VSS in order to avoid gas-liquid substrate transfer limitation. An average H(2) yield of 94 ± 3% and a specific hydrogen production rate of ca. 3 mol g(-1) VSS day(-1) were obtained at initial biomass densities between 5 and 8 mg VSS(-1). In addition, CO bioconversion kinetics was assessed at CO partial pressure from 0.16 to 2 atm, corresponding to a dissolved CO concentration at 70°C from 0.09 to 1.1 mM. Specific bioactivity was maximal at 3.5 mol CO g(-1) VSS day(-1) for a dissolved CO concentration of 0.55 mM in the culture. This optimal concentration is higher than with most other hydrogenogenic carboxydotrophic species.

Integration of microalgae cultivation with industrial waste remediation for biofuel and bioenergy production: opportunities and limitations.

There is currently a renewed interest in developing microalgae as a source of renewable energy and fuel. Microalgae hold great potential as a source of biomass for the production of energy and fungible liquid transportation fuels. However, the technologies required for large-scale cultivation, processing, and conversion of microalgal biomass to energy products are underdeveloped. Microalgae offer several advantages over traditional 'first-generation' biofuels crops like corn: these include superior biomass productivity, the ability to grow on poor-quality land unsuitable for agriculture, and the potential for sustainable growth by extracting macro- and micronutrients from wastewater and industrial flue-stack emissions. Integrating microalgal cultivation with municipal wastewater treatment and industrial CO(2) emissions from coal-fired power plants is a potential strategy to produce large quantities of biomass, and represents an opportunity to develop, test, and optimize the necessary technologies to make microalgal biofuels more cost-effective and efficient. However, many constraints on the eventual deployment of this technology must be taken into consideration and mitigating strategies developed before large scale microalgal cultivation can become a reality. As a strategy for CO(2) biomitigation from industrial point source emitters, microalgal cultivation can be limited by the availability of land, light, and other nutrients like N and P. Effective removal of N and P from municipal wastewater is limited by the processing capacity of available microalgal cultivation systems. Strategies to mitigate against the constraints are discussed.

Electricity generation from carbon monoxide and syngas in a microbial fuel cell.

Electricity generation in microbial fuel cells (MFCs) has been a subject of significant research efforts. MFCs employ the ability of electricigenic bacteria to oxidize organic substrates using an electrode as an electron acceptor. While MFC application for electricity production from a variety of organic sources has been demonstrated, very little research on electricity production from carbon monoxide and synthesis gas (syngas) in an MFC has been reported. Although most of the syngas today is produced from non-renewable sources, syngas production from renewable biomass or poorly degradable organic matter makes energy generation from syngas a sustainable process, which combines energy production with the reprocessing of solid wastes. An MFC-based process of syngas conversion to electricity might offer a number of advantages such as high Coulombic efficiency and biocatalytic activity in the presence of carbon monoxide and sulfur components. This paper presents a discussion on microorganisms and reactor designs that can be used for operating an MFC on syngas.

Potential of wastewater-treating anaerobic granules for biomethanation of synthesis gas.

Gasification of biomass produces a mixture of gas (mainly carbon monoxide (CO), carbon dioxide (CO(2)), and hydrogen (H(2))) called synthesis gas, or syngas, by thermal degradation without combustion. Syngas can be used for heat or electricity production by thermochemical processes. This project aims at developing an alternative way to bioupgrade syngas into biogas (mainly methane), via anaerobic fermentation. Nonacclimated industrial granular sludge to be used as reactor inoculum was initially evaluated for mesophilic carboxydotrophic methanogenesis potential in batch tests at 4 and 8 mmol CO/g VSS.d, in the absence and presence of H(2) and CO(2), respectively. Granular sludge was then introduced into a 30 L gas-lift reactor and supplied with CO, to study the production of methane and other metabolites, at different gas dilutions as well as feeding and recirculation rates. A maximal CO conversion efficiency of 75%, which was gas-liquid mass transfer limited, occurred at a CO partial pressure of 0.6 atm combined with a gas recirculation ratio of 20:1. The anaerobic granule potential for methanogenesis from CO was likely hydrogenotrophic, combined with CO-dependent H(2) formation, either under mesophilic or thermophilic conditions. Thermophilic conditions provide the anaerobic granules with a CO-bioconversion potential significantly larger (5-fold) than under mesophilic conditions, so long as the gas-liquid transfer is alleviated.

Genomic analysis of carbon monoxide utilization and butanol production by Clostridium carboxidivorans strain P7.

Increasing demand for the production of renewable fuels has recently generated a particular interest in microbial production of butanol. Anaerobic bacteria, such as Clostridium spp., can naturally convert carbohydrates into a variety of primary products, including alcohols like butanol. The genetics of microorganisms like Clostridium acetobutylicum have been well studied and their solvent-producing metabolic pathways characterized. In contrast, less is known about the genetics of Clostridium spp. capable of converting syngas or its individual components into solvents. In this study, the type of strain of a new solventogenic Clostridium species, C. carboxidivorans, was genetically characterized by genome sequencing. C. carboxidivorans strain P7(T) possessed a complete Wood-Ljungdahl pathway gene cluster, involving CO and CO(2) fixation and conversion to acetyl-CoA. Moreover, with the exception of an acetone production pathway, all the genetic determinants of canonical ABE metabolic pathways for acetate, butyrate, ethanol and butanol production were present in the P7(T) chromosome. The functionality of these pathways was also confirmed by growth of P7(T) on CO and production of CO(2) as well as volatile fatty acids (acetate and butyrate) and solvents (ethanol and butanol). P7(T) was also found to harbour a 19 Kbp plasmid, which did not include essential or butanol production related genes. This study has generated in depth knowledge of the P7(T) genome, which will be helpful in developing metabolic engineering strategies to improve C. carboxidivorans's natural capacity to produce potential biofuels from syngas.

Electrolytic methanogenic-methanotrophic coupling for tetrachloroethylene bioremediation: proof of concept.

Coupling of methanogenic and methanotrophic catabolisms was performed in a single-stage technology equipped with a water electrolysis cell placed in the effluent recirculation loop. The electrolysis-generated hydrogen served as an electron donor for both bicarbonate reduction into CH4 and reductive dechlorination, while the O2 and CH4, supported the cometabolic oxidation of chlorinated intermediates left over by the tetrachloroethylene (PCE) transformation. The electrolytical methanogenic/methanotrophic coupled (eMaMoC) process was tested in a laboratory-scale setup at PCE loads ranging from 5 to 50 micromol/L(rx) x d (inlet concentrations from 4 to 11 mg/L), and at various hydraulic residence times (HRT). Degradation followed essentially a reductive dechlorination pathway from PCE to cis-1,2-dichloroethene (DCE), and an oxidative pathway from DCE to CO2. PCE reductive dechlorination to DCE was consistently over 98% while a maximum oxidative DCE mineralization of 89% was obtained at a load of 4.3 micromol PCE/ L(rx) x d and an HRT of 6 days. Controlling dissolved oxygen concentrations within a relatively low range (2-3 mg/L) seemed instrumental to sustain the overall degradation capacity. Degradation kinetics were further evaluated: the apparent half-saturation constant (K(s)) had to be set relatively high (29 microM) for the simulated data to best fit the experimental ones. In spite of such kinetic limitations, the eMaMoC system, while fueled by water electrolysis, was effective in building and sustaining a functional methanogenic/methanotrophic consortium capable of significant PCE mineralization in a single-stage process. Hence, degradation standards are within reach so long as the methanotrophic DCE-oxidizing potential, including substrate affinity, are optimized and HRT accordingly adjusted.

Evaluating limiting steps of anaerobic degradation of food waste based on methane production tests.

This research adapted a batch test for biochemical methane production (BMP) to follow the degradation of complex compounds such as proteins and vegetable oils. The test measured the transformation of albumin and olive oil into methane under mesophilic and thermophilic conditions and assess limiting step in the overall degradation process. The thermophilic sludge used for the BMP tests was adapted during ten month from mesophilic sludge while being fed food waste. As compared to acetic acid, the specific rate of transformation of albumin and olive oil into methane reached 22 and 51%, respectively, under mesophilic conditions. Acetoclastic methanogenesis was not the limiting step in the presence of albumin or olive oil (and its monomer-like molecules such as amino acids, glycerol and oleic acid). Rather, the degradation of albumin was restricted by the presence of proteins. The thermophilically adapted sludge showed good proteolytic activity, but its acetoclastic methanogens were unable to degrade olive oil, because of the inhibitory effect of oleic acid.

Thermophilic adaptation of a mesophilic anaerobic sludge for food waste treatment.

As opposed to mesophilic, thermophilic anaerobic digestion of food waste can increase the biogas output of reactors. To facilitate the transition of anaerobic digesters, this paper investigated the impact of adapting mesophilic sludge to thermophilic conditions. A 5L bench scale reactor was seeded with mesophilic granular sludge obtained from an up-flow anaerobic sludge blanket digester. After 13 days of operation at 35 degrees C, the reactor temperature was instantaneously increased to 55 degrees C and operated at this temperature until day 21. The biomass was then fed food waste on days 21, 42 and 63, each time with an F/M (Food/Microorganism) ratio increasing from 0.12 to 4.43 gVS/gVSS. Sludge samples were collected on days 0, 21, 42 and 63 to conduct substrate activity tests, and reactor biogas production was monitored during the full experimental period. The sludge collected on day 21 demonstrated that the abrupt temperature change had no pasteurization effect, but rather lead to a biomass with a fermentative activity of 3.58 g Glucose/gVSS/d and a methanogenic activity of 0.47 and 0.26 g Substrate/gVSS/d, related respectively, to acetoclastic and hydrogenophilic microorganisms. At 55 degrees C, an ultimate gas production (Go) and a biodegradation potential (Bo) of 0.2-1.4 L(STP)/gVS(fed) and of 0.1-0.84 L(STP) CH(4)/gVS(fed) were obtained, respectively. For the treatment of food waste, a fully adapted inoculum was developed by eliminating the initial time-consuming acclimatization stage from mesophilic to thermophilic conditions. The feeding stage was initiated within 20 days, but to increase the population of thermophilic methanogenic microorganisms, a substrate supply program must be carefully observed.

Temperature-based control of an anaerobic reactor using a multi-model observer-based estimator.

This study presents a temperature-based control strategy for the stabilization of an anaerobic reactor during organic overloads. To prove feasibility of the proposed approach the rate of methane production was followed in batch activity tests and reactor runs during mesophilic-thermophilic transitions. Within the first 0.25-6 h of temperature augmentation, an increase in the rate of methane production was observed with higher rates measured under thermophilic (above 40 degrees C) conditions. However, 24 h after startup both in batch tests and reactor runs, the rate of methane production under thermophilic conditions was inferior to that under optimal mesophilic conditions (35 degrees C). Following these results, a control strategy based on short-term augmentation of the reactor temperature was proposed and tested in a 10 L UASB reactor. The control strategy employed a multi-model observer-based estimator to stabilize the effluent COD concentration during organic overloads. The temperature-based control resulted in an increased methanization rate and improved reactor stability overall.