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Abstract This study investigated silage quality characteristics and ruminal fiber degradability of grass and straw ensiled with either anaerobic fungi (AF) supernatant with active fungal enzymes or mixed ruminal fluid as novel silage additives. Compared to control silages, AF supernatant improved the quality of grass and straw silages as evidenced by decreased pH, acetic acid concentration, and dry matter losses. Likewise, mixed ruminal fluid enhanced lactic acid fermentation, which further resulted in lower pH of the treated grass silage. The ruminal fiber degradability was determined using in situ incubations and, compared to controls, the cellulose degradability was higher for grass silage with AF supernatant, whereas ruminal degradability of straw silage was reduced by this treatment. In contrast, mixed ruminal fluid did not influence fiber degradability of silages in the rumen. Concluding, both novel additives improved silage quality, whereas only AF supernatant enhanced ruminal fiber degradability of grass silage and therefore may represent an approach for improving forage utilization by ruminants. Key points • Enzymes of anaerobic fungi supernatant improve quality of grass and straw silages. • Mixed ruminal fluid enhances lactic acid fermentation when ensiling grass and straw. • Enzymes of anaerobic fungi supernatant increase ruminal grass silage degradability.

Microbial community development within an anaerobic trickle bed reactor (TBR) during methanation of syngas (56% H2, 30% CO, 14% CO2) was investigated using three different nutrient media: defined nutrient medium (241 days), diluted digestate from a thermophilic co-digestion plant operating with food waste (200 days) and reject water from dewatered digested sewage sludge at a wastewater treatment plant (220 days). Different TBR operating periods showed slightly different performance that was not clearly linked to the nutrient medium, as all proved suitable for the methanation process. During operation, maximum syngas load was 5.33 L per L packed bed volume (pbv) & day and methane (CH4) production was 1.26 L CH4/Lpbv/d. Microbial community analysis with Illumina Miseq targeting 16S rDNA revealed high relative abundance (20–40%) of several potential syngas and acetate consumers within the generaSporomusa,Spirochaetaceae,RikenellaceaeandAcetobacteriumduring the process. These were the dominant taxa except in a period with high flow rate of digestate from the food waste plant. The dominant methanogen in all periods was a member of the genusMethanobacterium, whileMethanosarcinawas also observed in the carrier community. As in reactor effluent, the dominant bacterial genus in the carrier wasSporomusa. These results show that syngas methanation in TBR can proceed well with different nutrient sources, including undefined medium of different origins. Moreover, the dominant syngas community remained the same over time even when non-sterilised digestates were used as nutrient medium.Key points•Independent of nutrient source, syngas methanation above 1 L/Lpbv/D was achieved.•Methanobacterium and Sporomusa were dominant genera throughout the process.•Acetate conversion proceededviaboth methanogenesis and syntrophic acetate oxidation.Graphical abstract

Due to mounting impacts of climate change, particularly increased incidence of drought, hence water scarcity, it has become imperative to develop new technologies for recovering water from nutrient-rich, water-replete effluents other than sewage. Notably, anaerobic digestate could be harnessed for the purpose of water recovery by repurposing digestate-borne minerals as nutrients in fermentative processes. The high concentrations of ammonium, phosphate, sulfate, and metals in anaerobic digestate are veritable microbial nutrients that could be harnessed for bio-production of bulk and specialty chemicals. Tethering nutrient sequestration from anaerobic digestate to bio-product accumulation offers promise for concomitant water recovery, bio-chemical production, and possible phosphate recovery. In this review, we explore the potential of anaerobic digestate as a nutrient source and as a buffering agent in fermentative production of glutamine, glutamate, fumarate, lactate, and succinate. Additionally, we discuss the potential of synthetic biology as a tool for enhancing nutrient removal from anaerobic digestate and for expanding the range of products derivable from digestate-based fermentations. Strategies that harness the nutrients in anaerobic digestate with bio-product accumulation and water recovery could have far-reaching implications on sustainable management of nutrient-rich manure, tannery, and fish processing effluents that also contain high amounts of water. KEY POINTS: • Anaerobic digestate may serve as a source of nutrients in fermentation. • Use of digestate in fermentation would lead to the recovery of valuable water.

This work performed co-AD from the vinasse and filter cake (from 1G ethanol production) and deacetylation liquor (from the pre-treatment of sugarcane straw for 2G ethanol production) in a semi-Continuous Stirred Tank Reactor (s-CSTR) aiming to provide optimum operational parameters for continuous CH4 production. Using filter cake as co-substrate may allow the reactor to operate throughout the year, as it is available in the sugarcane off-season, unlike vinasse. A comparison was made from the microbial community of the seed sludge and the reactor sludge when CH4 production stabilized. Lactate, butyrate and propionate fermentation routes were denoted at the start-up of the s-CSTR, characterizing the acidogenic phase: the Oxidation-Reduction Potential (ORP) values ranged from -800 to -100 mV. Once the methanogenesis was initiated, alkalizing addition was no longer needed as its demand by the microrganisms was supplied by the alkali-characteriscs of the deacetylation liquor. The gradual increase of the applied Organic Load Rates (OLR) allowed stabilization of the methanogenesis from 3.20 gVS L-1 day-1: the highest CH4 yield (230 NmLCH4 gSV-1) and average organic matter removal efficiency (83% ± 13) was achieved at ORL of 4.16 gVS L-1day-1. The microbial community changed along the reactor operation, presenting different metabolic routes mainly due to the used lignocellulosic substrates. Bacteria from the syntrophic acetate oxidation (SAO) process coupled to hydrogenotrophic methanogenesis were predominant (∼ 2% Methanoculleus) during the CH4 production stability. The overall results are useful as preliminary drivers in terms of visualizing the co-AD process in a sugarcane biorefinery integrated to scale.KeypoitnsIntegration of 1G2G sugarcane ethanol biorefinery from co-digestion of its residues Biogas production from vinasse, filter cake and deacetylation liquor in a semi-CSTR Lignicellulosic substrates affected the biochemical routes and microbial community Biomol confirmed the stablismenht of thermophilic community from mesophilic sludge

Anaerobic digestion of sewage sludge (SS) is one of the effective ways to reduce the waste generated from human life activities. To date, there are many reports to improve or repress methane production during the anaerobic digestion of SS. In the anaerobic digestion process, many microorganisms work positively or negatively, and as a result of their microbe-to-microbe interaction and regulation, methane production increases or decreases. In other words, understanding the complex control mechanism among the microorganisms and identifying the strains that are key to increase or decrease methane production are important for promoting the advanced production of bioenergy and beneficial compounds. In this mini-review, the literature on methane production in anaerobic digestion has been summarized based on the results of antibiotic addition, quorum sensing control, and inorganic substance addition. By optimizing the activity of microbial groups in SS, methane or acetate can be highly produced. KEY POINTS: • Bactericidal agents such as an antibiotic alter microbial community for enhanced CH4 production. • Bacterial interaction via quorum sensing is one of the key points for biofilm and methane production. • Anaerobic digestion can be altered in the presence of several inorganic materials.

Corynebacterium glutamicum, a gram-positive and facultative anaerobic bacterium, is widely used for the industrial production of amino acids, such as L-glutamate and L-lysine. C. glutamicum grows and produces amino acids under aerobic conditions. When restricted under anaerobic conditions, it produces organic acids, such as L-lactate and succinate, through metabolic shift. With the increasing threat of global warming, these organic acids have drawn considerable attention as bio-based plastic monomers. In addition to the organic acids, the anaerobic bioprocess is also used to produce other value-added compounds, including isobutanol, ethanol, 3-methyl-1-butanol, 2,3-butanediol, L-alanine, and L-valine. Therefore, C. glutamicum is now a versatile cell factory for producing a wide variety of useful chemicals under both aerobic and anaerobic conditions. The growth and metabolism of the bacterium depend on the oxygen levels, which modulate the rearrangement of the carbon flux by reprogramming gene expression patterns and intracellular redox states. Anaerobic cell growth and L-lysine production as well as aerobic succinate production have been demonstrated by engineering the metabolic pathways or supplying a terminal electron acceptor instead of oxygen. In this review, we discuss the physiological and metabolic changes in C. glutamicum associated with its application as a cell factory under different oxygen states. Physiological switching in bacteria is initiated with the sensing of oxygen availability. While such a sensor has not been identified in C. glutamicum yet, the molecular mechanism for oxygen sensing in related bacteria is also discussed. KEY POINTS: • C. glutamicum produces a wide variety of useful compounds under anaerobic conditions. • C. glutamicum is a versatile cell factory under both aerobic and anaerobic conditions. • Metabolic fate can be overcome by engineering metabolic pathways.

As a facultative anaerobe, Escherichia coli can activate various respiratory chains during anaerobic growth, among which the mode of anaerobic respiration with nitrate allows good energy conservation. NarL is one of the regulatory proteins in the Nar two-component system that regulates anaerobic respiration in E. coli. Previous studies have shown that NarL activates downstream gene regulation through phosphorylation. However, there are few studies on other protein translational modifications that influence the regulatory function of NarL. Herein, we demonstrate that acetylation modification exists on K188 and K192, the two lysine residues involved in contacting to DNA, and the degree of acetylation has significant effects on DNA-binding abilities, thus affecting the anaerobic growth of E. coli. In addition, NarL is mainly regulated by acetyl phosphate, but not by peptidyl-lysine N-acetyltransferase. These results indicate that non-enzymatic acetylation of NarL by AcP is one of the important mechanisms for the nitrate anaerobic respiratory pathway in response to environmental changes, which extends the idea of the mechanism underlying the response of intestinal flora to changes in the intestinal environment. KEY POINTS: • Acetylation was found in NarL, which was mainly mediated by AcP. • Non-enzymatic acetylation at K188 and K192 affects NarL binding ability. • Acetylation of NarL K188 and K192 regulates anaerobic nitrate growth of E. coli.

The ubiquitous signaling molecule autoinducer 2 (AI-2) is involved in intra- and interspecies communication, most notably between Gram-negative and Gram-positive bacteria. AI-2 accumulates during the exponential phase of the Escherichia coli (E. coli) monoculture and then rapidly decreases upon entry into the stationary phase. However, deleting both the genes encoding AI-2 synthase (LuxS) and the lsr operon regulator (LsrR) in the E. coli genome causes impaired AI-2 production and continuous AI-2 scavenging from the environment. This genetically-engineered E. coli mutant capable of quenching AI-2 quorum sensing (QS) system was utilized to evaluate the effect of AI-2 quenching on the anaerobic digestion of waste activated sludge (WAS) because the role of QS system via AI-2 in the process remains obscure. In this study, E. coli ∆luxS lsrR mutant cells were microencapsulated in sodium alginate beads and incubated with WAS anaerobically. After 15 days of anaerobic fermentation, the WAS containing double mutant cells produced significantly more methane than that of the parent E. coli cells. AI-2 quenching occurred concurrently with a shift of microbial communities that contribute to increasing acetate consumption by the Methanosarcina spp. resulting in an increase in methane production. KEY POINTS: • Impact of autoinducer 2 quenching in complex bacterial populations were determined. • Key microorganisms contributing to the increase of methane in WAS anaerobic digestion were found. • The AI-2 quenching is a potential regulatory in wastewater treatment and bioenergy research.

Recovery from stress is an important property for anaerobic digestion (AD). Although AD is quite adaptable with regard to waste composition, new substrates added to stable systems may cause process decline. We tested whether crude glycerol would cause stress to a thermophilic AD microbiome previously stabilized long-term on a low C/N ratio feedstock. Three-percent (v/v) crude glycerol was added to the basal substrate (poultry litter) for two hydraulic retention time (HRT) periods. This caused stress where biogas volume and methane percentage dramatically decreased and VFA levels increased. When the basal substrate was resumed, secondary inhibition occurred, resulting in even greater stress (biogas production ceased, methane 3.6%). Unassisted recovery of system processes required eight HRT periods. In contrast, crude glycerol applied at a lower organic loading rate did not cause inhibition. Crude glycerol caused changes in dominance in the microbial community (16S rRNA pyrotags). Although process resilience was slow, the recovery of digester functions occurred in conjunction with the recovery of community structure, particularly putative syntrophic acetate-oxidizing bacteria. KEY POINTS: • Crude glycerol caused stress in thermophilic co-digestion with poultry litter. • Unassisted resilience of digester functions (methane) required 8 HRT. • Syntrophic acetate-oxidizing bacteria implicated for keystone resilience functions. Graphical abstract.

Proteins, an important fraction of the organic matter in wastewater, typically enter a treatment facility as high molecular weight components. These components are degraded by extracellular protein hydrolytic enzymes, denoted as proteases. Adequate protein hydrolysis monitoring is crucial, since protein hydrolysis is often a rate-limiting step in wastewater treatment. However, current monitoring tools lack a high sample throughput and reliable quantification. Here, we present an improved assay for high-throughput protein hydrolysis rate measurements in wastewater treatment applications. A BODIPY FL casein model substrate was implemented in a microplate format for continuous fluorescent quantification. Case studies on a conventional and a high-rate aerobic municipal wastewater treatment plant and a lab-scale, two-stage, anaerobic reactor provided proof-of-concept. The assay presented in this study can help to obtain monitoring-based process insights, which will in turn allow improving biological performance of wastewater treatment installations in the future. KEY POINTS: • Protein hydrolysis is a crucial step in biological wastewater treatment. • Quantification of the protein hydrolysis rate enables in-depth process knowledge. • BODIPY FL casein is a suitable model substrate for a protein hydrolysis assay. • High sample throughput was obtained with fluorescent hydrolysis quantification. Graphical abstract. ispartof: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY vol:104 issue:18 pages:8037-8048 ispartof: location:Germany status: published

The salinity effect on anammox bacteria has been widely reported; however, rare studies describe the microbial dynamics of anammox-based process response to the introduction of real seawater at mainstream conditions. In this study, an anammox process at mainstream conditions without pre-enriching anammox bacteria was shifted to the feeds of a synthetic wastewater with a portion of seawater mixture. It achieved over 0.180 kg-N/(m3 day) of nitrogen removal rate with an additional seawater proportion of 20% in the influent. The bacterial biodiversity was significantly increased with the increase of seawater proportions. High relative abundance of anammox bacteria (34.24–39.92%) related to Ca. Brocadia was enriched and acclimated to the saline environment. However, the introduction of seawater caused the enrichment of nitrite-oxidizing Ca. Nitrospira, which was responsible for the deterioration of nitrogen removal efficiency. Possible adaptation metabolisms in anammox bacteria and other nitrogen transforming bacteria are discussed. These results highlight the importance of microbial diversity for anammox process under the saline environments of 20% and 40% seawater composition.

Protein turnover through de novo synthesis is critical for sustainable cellular functions. We previously found that glucose consumption rate in Corynebacterium glutamicum under anaerobic conditions increased at temperature higher than the upper limit of growth temperature. Here, we showed that production of lactic and succinic acids increased at higher temperature for long-term (48 h) anaerobic reaction in metabolically engineered strains. At 42 °C, beyond the upper limit of growth temperature range, biomass-specific lactic acid production rate was 8% higher than that at 30 °C, the optimal growth temperature. In contrast, biomass-specific succinic acid production rate was highest at 36 °C, 28% higher than that at 30 °C, although the production at 42 °C was still 23% higher than that at 30 °C. As enzymes are usually unstable at high temperatures, we investigated whether protein turnover of metabolic enzymes is required for the production of lactic and succinic acids under these conditions. Interestingly, when de novo protein synthesis was inhibited by addition of chloramphenicol, after 6 h, only succinic acid production was inhibited. Because glycolytic enzymes are involved in both lactic and succinic acids synthesis, enzymes in the anaplerotic pathway and the tricarboxylic acid (TCA) cycle leading to succinic acid synthesis were likely to be responsible for its decreased production. Among the five enzymes examined, the specific activity of only pyruvate carboxylase was drastically decreased after 48 h at 42 °C. Thus, the de novo synthesis of pyruvate carboxylase is required for long-term production of succinic acid. Graphical abstract KEY POINTS: • Long-term reaction for organic acids can be improved at temperature beyond ideal growth conditions. • De novo synthesis of pyruvate carboxylase is required for long-term succinic acid production.

Substrates with high sulfate levels pose problems for biogas production as they allow sulfate reducing bacteria to compete with syntrophic and methanogenic members of the community. In addition, the end product of sulfate reduction, hydrogen sulfide, is toxic and corrosive. Here we show how sulfate addition affects physiological processes in a thermophilic methanogenic system by analyzing the carbon flow and the microbial community with quantitative PCR and amplicon sequencing of the 16s rRNA gene. A sulfate addition of 0.5 to 3 g/L caused a decline in methane production by 73–92%, while higher sulfate concentrations had no additional inhibitory effect. Generally, sulfate addition induced a shift in the composition of the microbial community towards a higher dominance of Firmicutes and decreasing abundances of Bacteroidetes and Euryarchaeota. The abundance of methanogens (e.g., Methanoculleus and Methanosarcina) was reduced, while sulfate reducing bacteria (especially Candidatus Desulforudis and Desulfotomaculum) increased significantly in presence of sulfate. The sulfate addition had a significant impact on the carbon flow within the system, shifting the end product from methane and carbon dioxide to acetate and carbon dioxide. Interestingly, methane production quickly resumed, when sulfate was no longer present in the system. Despite the strong impact of sulfate addition on the carbon flow and the microbial community structure during thermophilic biogas production, short-term process disturbances caused by unexpected introduction of sulfate may be overcome due to the high resilience of the engaged microorganisms.

Nitrite-dependent anaerobic methane oxidation (n-damo) acts as a crucial link between biogeochemical carbon and nitrogen cycles. Nevertheless, very few studies have characterized n-damo microorganisms in high-latitude permafrost regions. Therefore, this study investigated the vertical distribution and diversity of n-damo bacterial communities in soil from three forest types in the permafrost regions of the Daxing'an Mountains. A total of 11 and 8 operational taxonomic units (OTUs) of n-damo 16S rRNA and pmoA genes were observed, respectively. Remarkable spatial variations in n-damo bacteria community richness, diversity, and structure were observed at different soil depths. Moreover, the abundances of n-damo bacteria (16S rRNA and pmoA genes) varied between 1.55 × 10

The article aims to show the increased interest in the applications of vacuum in the area of environmental biotechnology and the lack of research related to the effects of vacuum on bacteria and microbial communities. Information on the impact of vacuum on bacteria is limited and often comes from unrelated research fields. In most cases (astrobiology research, food preservation technologies), the exposure of microorganisms in vacuum is permanent for the whole life of a cell. In environmental science applications, the exposure of microorganisms containing media such as sludge or soil in vacuum is rather persistent, and lower values of vacuum are used. Vacuum is used or proposed to be used in wastewater treatment, anaerobic digestion, sludge treatment, soil remediation and mining. Usually, vacuum is used to remove gases from the test medium, so a purely physical process is applied. However, most reports show the influence of vacuum on biological processes and its efficiency, as well as on the community structure.

Hydrogen-producing mixed cultures were subjected to a 48-h downward or upward temperature fluctuation from 55 to 35 or 75 °C. Hydrogen production was monitored during the fluctuations and for three consecutive batch cultivations at 55 °C to evaluate the impact of temperature fluctuations and bioaugmentation with synthetic mixed culture of known H2 producers either during or after the fluctuation. Without augmentation, H2 production was significantly reduced during the downward temperature fluctuation and no H2 was produced during the upward fluctuation. H2 production improved significantly during temperature fluctuation when bioaugmentation was applied to cultures exposed to downward or upward temperatures. However, when bioaugmentation was applied after the fluctuation, i.e., when the cultures were returned to 55 °C, the H2 yields obtained were between 1.6 and 5% higher than when bioaugmentation was applied during the fluctuation. Thus, the results indicate the usefulness of bioaugmentation in process recovery, especially if bioaugmentation time is optimised.

Kinetic parameters affecting effluent water quality including half saturation constant (Ks), maximum specific growth rate (μmax), and specific affinity ([Formula: see text], defined as μmax/Ks) were investigated using three types of anaerobic sludge (raw anaerobic digestion sludge referred to as unacclimated sludge, unacclimated sludge after endogenous decay, and sludge acclimated to low-strength wastewater in an anaerobic membrane bioreactor (AnMBR) for 360 days). Long-term acclimation to low-strength wastewater resulted in sludge with high specific affinity (1.6 × 10-3 L/mg COD/day for acclimated sludge compared to 4.1 × 10-4 L/mg COD/day for unacclimated sludge). The μmax values for unacclimated sludge and acclimated sludge were 0.08 and 0.07 day-1, respectively. The Ks values for unacclimated sludge and acclimated sludge were 194 ± 81 mg COD/L and 45 ± 13 mg COD/L, respectively. Although the Ks of unacclimated sludge after endogenous decay increased to 772 ± 74 mg COD/L, μmax increased to 0.35 day-1 as well, resulting in no statistically significant difference of [Formula: see text] between the two types of unacclimated sludge. Overall, [Formula: see text] is a better indicator than μmax or Ks alone for determining effluent water quality, as effluent substrate concentration is approximately inversely proportional to the specific affinity. 16S rRNA sequencing data analysis indicated a high abundance (85.8% of total archaea) of Methanosaeta in the microbial community after long-term acclimation. High [Formula: see text] associated with the enrichment of Methanosaeta appears to ensure successful anaerobic treatment of low-strength wastewater.

Although the niche differentiation of anammox bacteria has been extensively observed in a lab-scale reactor, the inherent mechanism for this ecological phenomenon is still elusive. Here, we combined the long-term reactor operation, genome-centered metagenome, and metatranscriptome analyses to gain insight into the substrate competition and niche differentiation of Candidatus Jettenia and Candidatus Brocadia. After 146 days of operation, we found the anammox bacterial population shifted from Ca. Jettenia to Ca. Brocadia in the immobilization-anaerobic baffled reactor (I-ABR) with the ammonium and nitrite concentrations of 30 mg/L. Importantly, the genome and transcript comparisons of Ca. Jettenia and Ca. Brocadia showed that Ca. Brocadia harbored more complete function in cell chemotaxis, flagellar assembly, and two-component system and more redundant function in nitrite reduction, in which the genes were also highly expressed. Ca. Brocadia out-competed Ca. Jettenia at the mainstream condition. Meanwhile, though the highest biomass concentration led to the highest nitrogen removal rate (NRR) in the first compartment (C1), the competition of Ca. Jettenia and Ca. Brocadia could also affect the NRR of different compartments through affecting the bacterial activity. Substrate competition of anammox bacteria led to higher transcript activity of Ca. Jettenia and Ca. Brocadia in the second (C2) and fourth (C4) compartments, respectively. Further, high transcript activity of Ca. Brocadia led to the higher NRR in C4. A comparison of metabolic potential based on the metagenome-assembled genome adds a different dimension for understanding the discrepantly physiological characteristics and competition of anammox bacteria for wastewater treatment.

Anaerobic ammonium oxidation (anammox) is an important scientific discovery in the field of wastewater treatment. This process is a sustainable option in nitrogen removal due to its energy-efficient and cost-effective advantage. Great effort has been made recently to remove ammonium from industrial and municipal wastewater via the anammox process with a preceding partial nitrification (PN) converting part of NH4+ to NO2-. Anammox process is seldom involved in the nitrate removal. Nitrate (NO3-), one of the main nitrogen compounds produced from various industries, is typically converted to nitrogen gas via denitrification process where a large amount of carbon source is consumed. Within this context, we reviewed the current technologies for high-strength nitrate wastewater treatment. It is found that nitrite accumulation often occurs during nitrate reduction, and its accumulating level would be increased at certain conditions (i.e., low C/N ratio and high pH). Hence, this provides a great opportunity to employ the anammox process to further convert nitrite in a more sustainable way. In this review, we highlight a new approach for industrial nitrate wastewater treatment via partial denitrification coupled with anammox process (PD-A). We also discuss the conditions to achieve successful PD-A process, economic and environmental benefits, and potential challenges as well as the future perspectives in practical application.

Strain development is critical for microbial production of bio-based chemicals. The stereo-complex form of polylactic acid, a complex of poly-L- and poly-D-lactic acid, is a promising polymer candidate due to its high thermotolerance. Here, we developed Corynebacterium glutamicum strains producing high amounts of L- and D-lactic acid through intensive metabolic engineering. Chromosomal overexpression of genes encoding the glycolytic enzymes, glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, triosephosphate isomerase, and enolase, increased L- and D-lactic acid concentration by 146% and 56%, respectively. Chromosomal integration of two genes involved in the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-dehydro-3-deoxyphosphogluconate aldolase), together with a gene encoding glucose-6-phosphate dehydrogenase from Zymomonas mobilis, to bypass the carbon flow from glucose, further increased L- and D-lactic acid concentration by 11% and 44%, respectively. Finally, additional chromosomal overexpression of a gene encoding NADH dehydrogenase to modulate the redox balance resulted in the production of 212 g/L L-lactic acid with a 97.9% yield and 264 g/L D-lactic acid with a 95.0% yield. The optical purity of both L- and D-lactic acid was 99.9%. Because the constructed metabolically engineered strains were devoid of plasmids and antibiotic resistance genes and were cultivated in mineral salts medium, these strains could contribute to the cost-effective production of the stereo-complex form of polylactic acid in practical scale.

Anaerobic fermentation is a favorable process for microbial production of bulk chemicals like ethanol and organic acids. Low productivity is the bottleneck of several anaerobic processes which has significant impact on the technique competitiveness of production strain. Improving growth rate of production strain can speed up the total production cycle and may finally increase productivity of anaerobic processes. In this work, evolutionary engineering of wild-type strain Escherichia coli W3110 was adopted to improve anaerobic growth in mineral medium. Significant increases in exponential growth rate and stationary cell density were achieved in evolved strain WE269, and a 96.5% increase in lactate productivity has also been observed in batch fermentation of this strain with M9 minimal medium. Then, an engineered strain for lactate production (BW100) was constructed by using WE269 as a platform and 98.3 g/L lactate (with an optical purity of D-lactate above 95%) was produced in a 5-L bioreactor after 48 h with a productivity of 2.05 g/(L·h). Finally, preliminary investigation demonstrated that mutation in sucD (sucD M245I) (encoding succinyl-CoA synthetase); ilvG (ilvG Δ1bp) (encoding acetolactate synthase 2 catalytic subunit), and rpoB (rpoB T1037P) (encoding RNA polymerase β subunit) significantly improved anaerobic growth of E. coli. Double-gene mutation in ilvG and sucD resumed most of the growth potential of evolved strain WE269. This work suggested that improving anaerobic growth of production host can increase productivity of organic acids like lactate, and specific mutation-enabled improved growth may also be applied to metabolic engineering for production of other bulk chemicals.

Anaerobic ammonium oxidation (anammox) has been widely applied for biological nitrogen removal in freshwater systems, and there is a potential for its extension in saline water systems. In this study, the abundance and biodiversity of anammox bacteria were investigated in both saline and freshwater full-scale sewage treatment plants (STPs). The anammox bacteria were widely found in four tested STPs with abundance of 105–107 copies per mL of 16S rRNA gene. Phylogenetic results showed that Ca. Scalindua and Ca. Brocadia dominated in saline and freshwater STPs, respectively. Ca. Kuenenia dominated in one of freshwater STPs. However, redundancy discriminate analysis (RDA) indicates the distribution of Ca. Kuenenia in both saline and freshwater conditions. To further elucidate these observations, the Monod model was integrated with Gauss equation for the evaluation of salinity-induced kinetics. Model results reveal that when nitrite concentration (SNO2−) is higher than nitrite affinity constant (KNO2−), salinity (over ~ 3.0%) is responsible for Candidatus Scalindua dominance over Candidatus Kuenenia. Conversely, in nitrite-depleted conditions (KNO2− ≥ SNO2−), high nitrite affinity leads to the predominance of Ca. Scalindua in all salinities. This study provides fundamental insights into saline anammox applications.

Conductive iron oxides (CIO) have been proved recently to facilitate the anaerobic microbial syntrophy based on the direct interspecies electron transfer (DIET) in batch experiments. However, the effect of CIO was always insignificant in anaerobic digestion (AD) reactor especially when the DIET-based syntrophic partners were absent. In this study, the effect of magnetite on performance of AD system with sucrose as a sole carbon source was investigated, but limited enhancement was achieved during the first 36-day operation. The short-term effect of ethanol addition was further studied in the magnetite-amended AD reactor, and results showed that the AD reactor with 10gFe/L micro-sized magnetite (R3) achieved higher performance of COD removal and methane proportion compared with the other reactors (R1 without magnetite; R2 with 2gFe/L micro-sized magnetite; R4 with 2gFe/L nano-sized magnetite). Meanwhile, the pyridoxine in extracellular polymeric substances (EPS) and conductivity of anaerobic sludge from R3 increased more significantly than those of the others. Analysis of high-throughput sequencing indicated that the abundance of archaea increased in sludge from R3 and Methanosarcina responsible for DIET was dominant (63.64%). Additionally, the abundance of potential electroactive bacteria Chloroflexi in R3 was 7.57-fold, 3.61-fold and 7.37-fold as that of R1, R2 and R4, respectively. These results demonstrated that the electroactive microbes and methanogens could be enriched efficiently in anaerobic sludge via synergetic effect of magnetite addition and ethanol short-term stimulation.

Anammox bacteria have attracted attention due to their apparent importance in saving energy and reducing organic chemical demands. Here, we report the detection and quantification of anammox bacteria with an improved primer set in a validation wastewater treatment plant. The improved primer set was shown to detect a broad range of anammox bacteria (47.3%) facilitating more accurate analyses of nitrogen removal mechanisms. Nitrogen removal efficiency and dominant nitrogen removal mechanisms were compared in the modification-Johannesburg (Mod-JHB), modified Ludzack-Ettinger (MLE) single-feed, and anoxic-oxic-anoxic-oxic (AOAO) step-feed modes. In the Mod-JHB configuration, simultaneous nitrification and denitrification (SND) and anammox were found to be responsible for more than 80% of total inorganic nitrogen (TIN) removal (98.5 ± 0.8% of TIN removal). Decrease of anoxic SRT from 5 to 2.5 days did not have any obvious effect on nitrogen removal or the abundance of functional microorganisms. Microbial batch tests demonstrated that both partial nitrification and dissimilatory nitrate reduction to ammonium (DNRA) were responsible for maintaining the anammox process. Short SRT (2 days) in the aerobic zone may explain the presence of partial nitrification. This study provides insights to the analysis of nitrogen removal mechanisms in validation wastewater treatment plants (WWTPs) aiming for high nitrogen removal efficiency.

Abstract Complex substrates, like proteins, carbohydrates, and lipids, are major components of domestic wastewater, and yet their degradation in biofilm-based wastewater treatment technologies, such as aerobic granular sludge (AGS), is not well understood. Hydrolysis is considered the rate-limiting step in the bioconversion of complex substrates, and as such, it will impact the utilization of a large wastewater COD (chemical oxygen demand) fraction by the biofilms or granules. To study the hydrolysis of complex substrates within these types of biomass, this paper investigates the anaerobic activity of major hydrolytic enzymes in the different sludge fractions of a full-scale AGS reactor. Chromogenic substrates were used under fully mixed anaerobic conditions to determine lipase, protease, α-glucosidase, and β-glucosidase activities in large granules (>1 mm in diameter), small granules (0.2–1 mm), flocculent sludge (0.045–0.2 mm), and bulk liquid. Furthermore, composition and hydrolytic activity of influent wastewater samples were determined. Our results showed an overcapacity of the sludge to hydrolyze wastewater soluble and colloidal polymeric substrates. The highest specific hydrolytic activity was associated with the flocculent sludge fraction (1.5–7.5 times that of large and smaller granules), in agreement with its large available surface area. However, the biomass in the full-scale reactor consisted of 84% large granules, making the large granules account for 55–68% of the total hydrolytic activity potential in the reactor. These observations shine a new light on the contribution of large granules to the conversion of polymeric COD and suggest that large granules can hydrolyze a significant amount of this influent fraction. The anaerobic removal of polymeric soluble and colloidal substrates could clarify the stable granule formation that is observed in full-scale installations, even when those are fed with complex wastewaters. Key points • Large and small granules contain >70% of the hydrolysis potential in an AGS reactor. • Flocculent sludge has high hydrolytic activity but constitutes • AGS has an overcapacity to hydrolyze complex substrates in domestic wastewater. Graphical abstract

Renewable energy can boost the growing population's need and rapid budgetary development. To reduce fossil fuel consumption is the initial purpose of renewable and sustainable energy, producing valuable bio-based products. The fermenters, using for pretreatment of swine manure, and involvement of swine carcasses are reported to enhance organic loading rate followed by improved biogas yield on household digesters. The compositions such as animal residues, pathogenic microbes, pharmaceutical residues and nutrient compositions including undigested feed are still confused. Therefore, it is mandatory to optimize and stabilize anaerobic practice and digestate filtration purification for consequential fertilizer consumption. The effective bio-methane recovery from energy-rich compounds is challenging due to slow degradation procedures. The pretreatment procedure could enhance lipid depolymerization and improve anaerobic fermentation. This article deeply focuses on biodegradation of swine manure. The components of this manure were evaluated and established several approaches to improve biogas production. Furthermore, recycling of co-digestates was discussed in detail as fertilizer consumption including hygienic aspects of manure and pretreatment strategies of biomass residues. KEY POINTS: • Co-digestion of manure and carcasses enhance bio-methane production. • Removel of ammonia from biogas digester may improve bio-methane gas. • A strong antimicrobial influence has been reported on biogas production.

Among processes that control microbial community assembly, microbial invasion has received little attention until recently, especially in the field of anaerobic digestion. However, knowledge of the principles regulating the taxonomic and functional stability of microbial communities is key to truly develop better predictive models and effective management strategies for the anaerobic digestion process. To date, available studies focus on microbial invasions in digesters feed with activated sludge from municipal wastewater treatment plants. Herein, this review summarizes the importance of invasions for anaerobic digestion management, the ecological theories about microbial invasions, the traits of activated sludge microorganisms entering the digesters, and the resident communities of anaerobic reactors that are relevant for invasions and the current knowledge about the success and impacts of invasions, and discusses the research needs on this topic. The initial data indicate that the impact of invasions is low and only a small percentage of the mostly aerobic microorganisms present in the activated sludge feed are able to become stablished in the anaerobic digesters. However, there are still numerous unknowns about microbial invasions in anaerobic digestion including the influence of anaerobic feedstocks or process perturbances that new approaches on microbial ecology could unveil. KEY POINTS: • Microbial invasions are key processes to develop better strategies for digesters management. • Knowledge on pathogen invasions can improve anaerobic digestion microbial safety. • To date, the number of successful invasions on anaerobic digesters from activated sludge organisms is low. • Feed organisms detected in digesters are mostly inactive residual populations. • Need to expand the range of invaders and operational scenarios studied.

Anaerobic wastewater treatment offers several advantages; however, the effluent of anaerobic digesters still contains high levels of ammonium and dissolved methane that need to be removed before these effluents can be discharged to surface waters. The simultaneous anaerobic removal of methane and ammonium by denitrifying (N-damo) methanotrophs in combination with anaerobic ammonium-oxidizing (anammox) bacteria could be a potential solution to this challenge. After a molecular survey of a wastewater plant treating brewery effluent, indicating the presence of both N-damo and anammox bacteria, we started an anaerobic bioreactor with a continuous supply of methane, ammonium, and nitrite to enrich these anaerobic microorganisms. After 14 months of operation, a stable enrichment culture containing two types of ‘CandidatusMethylomirabilis oxyfera’ bacteria and two strains of ‘Ca. Brocadia’-like anammox bacteria was achieved. In this community, anammox bacteria converted 80% of the nitrite with ammonium, while ‘Ca. Methylomirabilis’ contributed to 20% of the nitrite consumption. The analysis of metagenomic 16S rRNA reads and fluorescence in situ hybridization (FISH) correlated well and showed that, after 14 months, ‘Ca. Methylomirabilis’ and anammox bacteria constituted approximately 30 and 20% of the total microbial community. In addition, a substantial part (10%) of the community consisted ofPhycisphaera-related planctomycetes. Assembly and binning of the metagenomic sequences resulted in high-quality draft genome of two ‘Ca. Methylomirabilis’ species containing the marker genespmoCAB,xoxF, andnirSand putative NO dismutase genes. The anammox draft genomes most closely related to ‘Ca.Brocadia fulgida’ included the marker geneshzsABC,hao, andhdh. Whole-reactor and batch anaerobic activity measurements with methane, ammonium, nitrite, and nitrate revealed an average anaerobic methane oxidation rate of 0.12 mmol h−1 L−1and ammonium oxidation rate of 0.5 mmol h−1 L−1. Together, this study describes the enrichment and draft genomes of anaerobic methanotrophs from a brewery wastewater treatment plant, where these organisms together with anammox bacteria can contribute significantly to the removal of methane and ammonium in a more sustainable way.Key points•An enrichment culture containing both N-damo and anammox bacteria was obtained.• Simultaneous consumption of ammonia, nitrite, and methane under anoxic conditions.•In-depth metagenomic biodiversity analysis of inoculum and enrichment culture.

The anaerobic biodegradation of phenol has been realised in a sequencing batch reactor (SBR) under anaerobic conditions with phenol as sole carbon and energy source and with glucose as co-substrate. A step-change increase of phenol loading (from 100 up to 2000 mg/L of phenol concentration in the feed solution) has been applied during the acclimation phase in order to progressively induce the development of a specialised microbial consortium. This approach, combined with the dynamic sequence of operations characterising SBRs and with the high biomass retention time, led to satisfactory phenol and COD removal efficiencies with values 70% for the highest phenol input (2000 mg/L) fed as the single carbon and energy source. Analysis of removal efficiencies and biodegradation rates suggested that the use of glucose as co-substrate did not induce a significant improvement in process performance. Kinetic tests have been performed at different initial phenol (400-1000 mg/L) and glucose (1880-0 mg/L) concentrations to kinetically characterise the developed biomass: estimated kinetic constants are suitable for application and no inhibitory effect due to high concentrations of phenol has been observed in all investigated conditions. The microbial community has been characterised at different operating conditions through molecular tools: results confirm the successful adaptation-operation approach of the microbial consortium showing a gradual increase in richness and diversity and the occurrence and selection of a high proportion of phenol-degrading genera at the end of the experimentation. Key Points • Anaerobic phenol removal in the range of 70-99% in a sequencing batch reactor. • Negligible effect of co-substrate on removal efficiencies and biodegradation rates. • No biomass inhibition due to phenol concentration in the range of 400-1000 mg/L. • Increasing phenol loads promoted the culture enrichment of phenol-degrading genera.

Cell proliferation is achieved through numerous enzyme reactions. Temperature governs the activity of each enzyme, ultimately determining the optimal growth temperature. The synthesis of useful chemicals and fuels utilizes a fraction of available metabolic pathways, primarily central metabolic pathways including glycolysis and the tricarboxylic acid cycle. However, it remains unclear whether the optimal temperature for these pathways is correlated with that for cell proliferation. Here, we found that wild-type Corynebacterium glutamicum displayed increased glycolytic activity under non-growing anaerobic conditions at 42.5 °C, at which cells do not proliferate under aerobic conditions. At this temperature, glucose consumption was not inhibited and increased by 28% compared with that at the optimal growth temperature of 30 °C. Transcriptional analysis revealed that a gene encoding glucose transporter (iolT2) was upregulated by 12.3-fold compared with that at 30 °C, with concomitant upregulation of NCgl2954 encoding the iolT2-regulating transcription factor. Deletion of iolT2 decreased glucose consumption rate at 42.5 °C by 28%. Complementation of iolT2 restored glucose consumption rate, highlighting the involvement of iolT2 in the accelerating glucose consumption at an elevated temperature. This study shows that the optimal temperature for glucose metabolism in C. glutamicum under anaerobic conditions differs greatly from that for cell growth under aerobic conditions, being beyond the upper limit of the growth temperature. This is beneficial for fuel and chemical production not only in terms of increasing productivity but also for saving cooling costs. KEY POINTS: • C. glutamicum accelerated anaerobic glucose consumption at elevated temperature. • The optimal temperature for glucose consumption was above the upper limit for growth. • Gene expression involved in glucose transport was upregulated at elevated temperature. Graphical abstract.

Simultaneous denitrification and anammox process are newly developed wastewater treatment processes with high-nitrogen removal efficiency, but little information is available regarding its optimal operational conditions and molecular mechanisms. In this study, a lab-scale up-flow anaerobic sludge blanket bioreactor fed with nitrate and ammonia was continuously operated for 180 days to establish the simultaneous denitrification and anammox system, so as to investigate the changing patterns of nitrogen removal efficiency and functional bacteria and genes at varying substrate loadings. Results showed that the optimal removal efficiency of total nitrogen (TN) reached 92.47%, corresponding to TN loading removal rate of 0.9 kg-N m−3 day−1 that was achieved at low influent substrate concentrations and short hydraulic retention time (HRT) (4 h). High-throughput sequencing of 16S rRNA gene amplicons revealed that the predominant denitrifying and anammox bacteria in the coupling system were Thauera (relative abundance of 25.30%) and Candidatus Brocadia (relative abundance of 6.85%), respectively. Quantitative real-time PCR showed that the anammox genes (hzsA, hzsB, and hzo) and denitrifying genes (narG, napA, and nirS) demonstrated high abundance in the optimized bioreactor. Batch experiments were also conducted to determine the temporal difference in the expression of the anammox and denitrifying genes. NarG was found to play a crucial role in nitrate reduction to produce nitrite for anammox bacteria, while nirS was identified as the key genes for nitrite reduction to produce nitric oxide, which acted as an important role in both denitrification and anammox reaction.

International audience; Strictly anaerobic bacteria of the Clostridium genus have attracted great interest as potential cell factories for molecular hydrogen production purposes. In addition to being a useful approach to this process, dark fermentation has the advantage of using the degradation of cheap agricultural residues and industrial wastes for molecular hydrogen production. However, many improvements are still required before large-scale hydrogen production from clostridial metabolism is possible. Here we review the literature on the basic biological processes involved in clostridial hydrogen production, and present the main advances obtained so far in order to enhance the hydrogen productivity, as well as suggesting some possible future prospects.

Anaerobic ammonium oxidation (anammox)-based nitrogen removal saves aeration energy and organic carbon costs, attributed to its anaerobic and autotrophic nature. However, due to the slow growth of anaerobic ammonium oxidation bacteria (AnAOB), drawbacks including long startup time and sensitivity to toxins still hamper the application of anammox-based processes. To cope with the slow growth of AnAOB, various bioreactor configurations have been investigated for the capability of retaining anammox biomass, among which, the expanded granular sludge bed (EGSB) reactor is a promising option. In this study, two laboratory-scale EGSB reactors were used to gain insights of microbial population and their response to amending biofilm-carriers, aiming to enhance the biomass retention of AnAOB. The respective ammonium and nitrite removal efficiencies were up to over 90%, and the overall nitrogen removal efficiency (NRE) was stable at over 70%, in the EGSB reactor amended with carriers (CEGSB). Compared to the control EGSB, CEGSB's observed performance was more stable during the 236-day operational period. The abundance of AnAOB reached 22% in the EGSB and 49% in the CEGSB. It was also observed that Ca. Brocadia (14.25%) and Asahi BRW2 (33.19%) coexisted in the CEGSB. The dynamics of major metabolisms and functional genes involved in nitrogen conversion were further observed by FAPROTAX based on the taxonomic data, providing more insights into the functions of the microbial communities.

Lignocellulose is a widely available renewable carbon source and a promising feedstock for the production of various chemicals in biorefineries. However, its recalcitrant nature is a major hurdle that must be overcome to enable economic conversion processes. Deconstruction of lignocellulose is part of the global carbon cycle, and efficient microbial degradation systems have evolved that might serve as models to improve commercial conversion processes. Biofilms—matrix encased, spatially organized clusters of microbial cells and the predominating lifestyle in nature—have been recognized for their essential role in the degradation of cellulose in nature, e.g., in soils or in the digestive tracts of ruminant animals. Cellulolytic biofilms allow for a high concentration of enzymes at the boundary layer between the solid substrate and the liquid phase and the more complete capture of hydrolysis products directly at the hydrolysis site, which is energetically favorable. Furthermore, enhanced expression of genes for carbohydrate active enzymes as a response to the attachment on solid substrate has been demonstrated for cellulolytic aerobic fungi and anerobic bacteria. In natural multispecies biofilms, the vicinity of different microbial species allows the creation of efficient food webs and synergistic interactions thereby, e.g., avoiding the accumulation of inhibiting metabolites. In this review, these topics are discussed and attempts to realize the benefits of biofilms in targeted applications such as the consolidated bioprocessing of lignocellulose are highlighted. Key Points Multispecies biofilms enable efficient lignocellulose destruction in the biosphere. Cellulose degradation by anaerobic bacteria often occurs by monolayered biofilms. Fungal biofilms immobilize enzymes and substrates in an external digestion system. Surface attached cultures typically show higher expression of cellulolytic enzymes.

Each year, large quantities of waste cooking oils are produced worldwide which are currently reused mainly for biodiesel production. Since lipids have a very high potential for biomethanation, the production of biogas is a possible alternative for the recycling of edible used oils. The digestion of fats is hindered mainly by their hydrophobicity, which implies a biphasic system with problems of floating and foaming of the oily materials, and by the accumulation of long-chain fatty acids, which are toxic to microbial consortia. The objectives of this review were to highlight the recycling potential of waste cooking oil to biogas production and to facilitate the application of the technology by identifying solutions to overcome biological and engineering limits to its diffusion. Particular attention was paid to the microbial populations involved, to the process factors whose control is important to improve the digestion of fats such as lipid concentration, pH, temperature, and agitation, and to technological solutions whose application also aims to improve digestion, such as pretreatment of raw materials and co-digestion of fats with other feedstocks. The state of the art in reactor designs suitable for lipid digestion was also examined.

Multi-walled carbon nanotubes (MWCNTs) released into the sewage may cause negative and/or positive effects on the treatment system. The objective of this study was to explore over 110 days' effect of MWCNTs on the performance of anaerobic granular sludge and microbial community structures in an upflow anaerobic sludge blanket (UASB) reactor. The results showed that MWCNTs had no significant effect on the removal of chemical oxidation demand (COD) and ammonia in UASB reactor, but the total phosphorus (TP) removal efficiency increased by 29.34%. The biogas production of the reactor did not change. The anaerobic granular sludge tended to excrete more EPS to resist the effects of MWCNTs during the long-term impact. Illumina MiSeq sequencing of 16S rRNA gene revealed that MWCNTs did not affect the microbial diversity, but altered the composition and structure of microbial community in the reactor. In this process, Saccharibacteria replaced Proteobacteria as the highest abundant bacterial phylum. MWCNTs promoted the differentiation of methanogen structure, resulting in increase of Methanomassiliicoccus, Methanoculleus, and the uncultured WCHA1-57. These results indicated that MWCNTs impacted the performance of UASB reactor and the structures of the microbial community in anaerobic granular sludge.