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Agricultural residues, such as lignocellulosic materials (LM), are the most attractive renewable bioenergy sources and are abundantly found in nature. Anaerobic digestion has been extensively studied for the effective utilization of LM for biogas production. Experimental investigation of physiochemical changes that occur during pretreatment is needed for developing mechanistic and effective models that can be employed for the rational design of pretreatment processes. Various-cutting edge pretreatment technologies (physical, chemical and biological) are being tested on the pilot scale. These different pretreatment methods are widely described in this paper, among them, microaerobic pretreatment (MP) has gained attention as a potential pretreatment method for the degradation of LM, which just requires a limited amount of oxygen (or air) supplied directly during the pretreatment step. MP involves microbial communities under mild conditions (temperature and pressure), uses fewer enzymes and less energy for methane production, and is probably the most promising and environmentally friendly technique in the long run. Moreover, it is technically and economically feasible to use microorganisms instead of expensive chemicals, biological enzymes or mechanical equipment. The information provided in this paper, will endow readers with the background knowledge necessary for finding a promising solution to methane production.

The biodegradation of organic aromatic compounds in subsurface environments is often hindered by limited dissolved oxygen. While oxygen supplementation can enhance in situ biodegradation, it poses financial and technical challenges. This study explores introducing low-oxygen concentrations in anaerobic environments for efficient contaminant removal, particularly in scenarios where coexisting pollutants are present. An innovative strategy of alternating nitrate-reducing and microaerobic conditions to stimulate biodegradation is proposed, utilizing nitrate initially to degrade easily-degradable compounds, and potentially reducing the need for additional oxygen. Batch experiments were conducted to assess the biodegradation of a BTEX, indene, indane, and naphthalene mixture using groundwater and sediments from an anaerobic contaminated aquifer. Two set-ups were incubated for 98 days to assess the redox transitions between microaerobic (oxygen concentrations < 0.5 mg O 2 L −1 ) and nitrate-reducing conditions, aiming to minimize external electron acceptor usage while maximizing degradation. Comparative experiments under fully aerobic and fully anaerobic (nitrate-reducing) conditions were conducted, revealing that under microaerobic conditions, all compounds were completely degraded, achieving removal efficiencies comparable to fully aerobic conditions. A pre-treatment phase involving nitrate-reducing conditions followed by microaerobic conditions showed more effective utilization of oxygen specifically for contaminant degradation compared to fully aerobic conditions. Contrarily, under fully anaerobic conditions, without oxygen addition, partial degradation of ethylbenzene was observed after 400 days, while other compounds remained. The outcomes of this study can provide valuable insights for refining strategies involving oxygen and nitrate dosages, thereby enhancing the efficacy of in situ bioremediation approaches targeting complex hydrocarbon mixtures within anaerobic subsurface environments. Key points • BTEX, indene, indane, and naphthalene mix biodegraded under microaerobic conditions • Subsurface microorganisms swiftly adapt from nitrate to microaerobic conditions • More oxygen directed to hydrocarbon biodegradation via a pre-anaerobic treatment

Microbial soil habitats are characterized by rapid shifts in substrate and nutrient availabilities, as well as chemical and physical parameters. One such parameter that can vary in soil is oxygen; thus, microbial survival is dependent on adaptation to this substrate. To better understand the metabolic abilities and adaptive strategies to oxygen-deprived environments, we combined genomics with transcriptomics of a model organism, Acidobacterium capsulatum, to explore the effect of decreasing, environmentally relevant oxygen concentrations. The decrease from 10 to 0.1 µM oxygen (3.6 to 0.036 pO2% present atmospheric level, respectively) caused the upregulation of the transcription of genes involved in signal transduction mechanisms, energy production and conversion and secondary metabolites biosynthesis, transport, and catabolism based on clusters of orthologous group categories. Contrary to established observations for aerobic metabolism, key genes in oxidative stress response were significantly upregulated at lower oxygen concentrations, presumably due to an NADH/NAD+ redox ratio imbalance as the cells transitioned into nanoxia. Furthermore, A. capsulatum adapted to nanoxia by inducing a respiro-fermentative metabolism and rerouting fluxes of its central carbon and energy pathways to adapt to high NADH/NAD+ redox ratios. Our results reveal physiological features and metabolic capabilities that allowed A. capsulatum to adapt to oxygen-limited conditions, which could expand into other environmentally relevant soil strains.

Bacterial degradation of crude oil is a promising strategy for reducing the concentration of hydrocarbons in contaminated environments. In the first part of this study, we report the enrichment of two bacterial consortia from deep sediments of the Gulf of Mexico with crude oil as the sole carbon and energy source. We conducted a comparative analysis of the bacterial community in the original sediment, assessing its diversity, and compared it to the enrichment observed after exposure to crude oil in defined cultures. The consortium exhibiting the highest hydrocarbon degradation was predominantly enriched with Rhodococcus (75%). Bacterial community analysis revealed the presence of other hydrocarbonoclastic members in both consortia. In the second part, we report the isolation of the strain Rhodococcus sp. GOMB7 with crude oil as a unique carbon source under microaerobic conditions and its characterization. This strain demonstrated the ability to degrade long-chain alkanes, including eicosane, tetracosane, and octacosane. We named this new strain Rhodococcus qingshengii GOMB7. Genome analysis revealed the presence of several genes related to aromatic compound degradation, such as benA, benB, benC, catA, catB, and catC; and five alkB genes related to alkane degradation. Although members of the genus Rhodococcus are well known for their great metabolic versatility, including the aerobic degradation of recalcitrant organic compounds such as petroleum hydrocarbons, this is the first report of a novel strain of Rhodococcus capable of degrading long-chain alkanes under microaerobic conditions. The potential of R. qingshengii GOMB7 for applications in bioreactors or controlled systems with low oxygen levels offers an energy-efficient approach for treating crude oil-contaminated water and sediments.

Lactate and isoprene are two common monomers for the industrial production of polyesters and synthetic rubbers. The present study tested the co-production of D-lactate and isoprene by engineered Escherichia coli in microaerobic conditions. The deletion of alcohol dehydrogenase (adhE) and acetate kinase (ackA) genes, along with the supplementation with betaine, improved the co-production of lactate and isoprene from the substrates of glucose and mevalonate. In fed-batch studies, microaerobic fermentation significantly improved the isoprene concentration in fermentation outlet gas (average 0.021 g/L), compared with fermentation under aerobic conditions (average 0.0009 g/L). The final production of D-lactate and isoprene can reach 44.0 g/L and 3.2 g/L, respectively, through fed-batch microaerobic fermentation. Our study demonstrated a dual-phase production strategy in the co-production of isoprene (gas phase) and lactate (liquid phase). The increased concentration of gas-phase isoprene could benefit the downstream process and decrease the production cost to collect and purify the bio-isoprene from the fermentation outlet gas. The proposed microaerobic process can potentially be applied in the production of other volatile bioproducts to benefit the downstream purification process.

Background Due to ecological concerns, alternative supply lines for fuel and bulk chemicals such as isopropanol are increasingly pursued. By implementing the formation pathways from natural producers like Clostridium beijerinckii and Clostridium aurantibutyricum , isopropanol can be produced in Escherichia coli . However, developing an industrially and economically feasible microbial production process requires a robust and efficient process strategy. Therefore, this study explores microaerobic conditions in combination with lactose and sour whey as sustainable carbon source as a basis for large-scale microbial isopropanol production. Results Different gas-liquid mass transfer regimes (affected by variations of the stirrer speed and ingas oxygen concentration) allowed the implementation of different microaerobic conditions characterized by their specific oxygen uptake rate (q O2 ) in cultivations with an isopropanol producing E. coli W strain on lactose. Under microaerobic conditions, the specific isopropanol production rate (q p, ipa ) exhibited a direct correlation with q O2 . Moreover, isopropanol production showed a pseudo growth-coupled behavior. Monitoring of the formation rates of various by-products such as acetone, lactate, acetate, pyruvate, formate and succinate allowed to identify a q O2 of 9.6 mmol g − 1 h − 1 in only slightly microaerobic cultivations as the best conditions for microbial isopropanol production. Additionally, the data suggests that a carbon bottleneck exists at the pyruvate node and the availability of the redox factor NADPH is crucial to shift the product balance from acetone to isopropanol. Finally, confirmation runs prove the effectiveness of the microaerobic production approach by yielding 8.2 g L − 1 (135.8 ± 13.3 mmol L − 1 ) and 20.6 g L − 1 (342.9 ± 0.4 mmol L − 1 ) isopropanol on lactose and whey, respectively, reaching a volumetric isopropanol formation rate of up to 2.44 g L − 1 h − 1 (40.6 mmol L − 1 h − 1 ). Conclusions This study identifies slightly microaerobic conditions (q O2 ~ 10 mmol g − 1 h − 1 ) as the currently best conditions for microbial isopropanol production on lactose and whey in E. coli W. In the future, optimizing the carbon flux around the pyruvate node, ensuring sufficient NADPH supply, and establishing a feedback control loop to control process variables affecting oxygen transfer to the culture, could make microbial isopropanol production feasible at an industrial scale.

Abstract The aim of this study was to investigate the relationship between system removal performance and microbial community structure in a novel full-scale microaerobic-anoxic-oxic (M/A/O) system for coking wastewater (CWW) treatment. The results showed that 93% of chemical oxygen demand (COD) and 99% of NH4+-N removal efficiency were achieved via the M/A/O process, meanwhile, main organic pollutants in CWW, including phenolic compounds, heterocyclic compounds and polycyclic aromatic hydrocarbons were basically removed. Four dominant phyla of Proteobacteria, Firmicutes, Bacteroidetes and Nitrospirae were demonstrated to be distributed in the system and played significant roles in the M/A/O biological treatment process. The major function of the M process was to partly remove the biodegradable substances such as phenols and hydrolyze the refractory contaminants such as N-heterocyclic compounds to improve the biological oxygen demand/COD (BOD5) ratio and release ammonia. This work illustrated the structure and function of the microbial community in the M/A/O system and provided a new choice for high-strength CWW treatment. This work illustrated the structure and function of a microbial community in a novel full-scale M/A/O system and provided a new choice for high-strength coking wastewater treatment.

Background Ralstonia eutropha H16, a facultative chemolitoautotroph, is an important workhorse for bioindustrial production of useful compounds such as polyhydroxyalkanoates (PHAs). Despite the extensive studies to date, some of its physiological properties remain not fully understood. Results This study demonstrated that the knallgas bacterium exhibited altered PHA production behaviors under slow-shaking condition, as compared to its usual aerobic condition. One of them was a notable increase in PHA accumulation, ranging from 3.0 to 4.5-fold in the mutants lacking of at least two NADPH-acetoacetyl-CoA reductases (PhaB1, PhaB3 and/or phaB2) when compared to their respective aerobic counterpart, suggesting the probable existence of ( R )-3HB-CoA-providing route(s) independent on PhaBs. Interestingly, PHA production was still considerably high even with an excess nitrogen source under this regime. The present study further uncovered the conditional activation of native reverse β -oxidation (rBOX) allowing formation of ( R )-3HHx-CoA, a crucial precursor for poly(3-hydroxybutyrate- co -3-hydroxyhexanoate) [P(3HB- co -3HHx)], solely from glucose. This native rBOX led to the natural incorporation of 3.9 mol% 3HHx in a triple phaB -deleted mutant (∆ phaB1 ∆ phaB1 ∆ phaB2-C2 ) . Gene deletion experiments elucidated that the native rBOX was mediated by previously characterized ( S )-3HB-CoA dehydrogenases (PaaH1/Had), β-ketothiolase (BktB), ( R )-2-enoyl-CoA hydratase (PhaJ4a), and unknown crotonase(s) and reductase(s) for crotonyl-CoA to butyryl-CoA conversion prior to elongation. The introduction of heterologous enzymes, crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) along with ( R )-2-enoyl-CoA hydratase (PhaJ) aided the native rBOX, resulting in remarkably high 3HHx composition (up to 37.9 mol%) in the polyester chains under the low-aerated condition. Conclusion These findings shed new light on the robust characteristics of Ralstonia eutropha H16 and have the potential for the development of new strategies for practical P(3HB- co -3HHx) copolyesters production from sugars under low-aerated conditions. Graphical Abstract

Aerobic granular sludge is an interesting alternative to the conventional activated sludge (CAS) system and modified-Ludzack–Ettinger (MLE) process for biological wastewater treatment, as it allows a more cost-effective and simultaneous removal of carbon (C) and nitrogen (N) compounds in a single stage. In this study, (micro)aerobic C and N removal from synthetic urban wastewater was investigated in a continuous-double-column-upflow aerobic granular sludge blanket (UAGSB) system. The UAGSB reactor was operated under different dissolved oxygen (DO) ranges (0.01–6.00 mg∙L−1), feed C/N ratios (4.7–13.6), and hydraulic retention times (HRTs) (6–24 h). At a DO range of 0.01–0.30 mg∙L−1, feed C/N ratio of 13.6, and HRT of 24 h, the UAGSB achieved the highest chemical oxygen demand (COD), N-NH4+, and total inorganic nitrogen (TIN) removal efficiencies of 86, 99, and 84%, respectively. A preliminary assessment of the energy and economic savings associated with the process investigated was also carried out. The impact of capital and operating costs mainly related to the energy consumption of the aeration was taken into account. The assessment reveals that the capital and energy expenses of the UAGSB reactor would result in cost savings of around 14 and 7%, respectively, compared with a MLE system.

Aerobic protozoa can maintain fully aerobic metabolic rates even at very low O^sub 2^-tensions; this is related to their small sizes. Many -- or perhaps all -- protozoa show particular preferences for a given range of O^sub 2^-tensions. The reasons for this and the role for their distribution in nature are discussed and examples of protozoan biota in O^sub 2^-gradients in aquatic systems are presented. Facultative anaerobes capable of both aerobic and anaerobic growth are probably common within several protozoan taxa. It is concluded that, further progress in this area is contingent on physiological studies of phenotypes.