Magnetite particles accelerate methanogenic degradation of highly concentrated acetic acid in anaerobic digestion process.

The anaerobic digestion (AD) process has become significant for its capability to convert organic wastewater into biogas, a valuable energy source. Excessive acetic acid accumulation in the anaerobic digester can inhibit methanogens, ultimately leading to the deterioration of process performance. Herein, the effect of magnetite particles (MP) as an enhancer on the methanogenic degradation of highly-concentrated acetate (6 g COD/L) was examined through long-term sequential AD batch tests. Bioreactors with (AM) and without (AO) MP were compared. AO experienced inhibition and its methane production rate (qm) converged to 0.45 L CH4/g VSS/d after 10 sequential batches (AO10, the 10th batch in a series of the sequential batch tests conducted using bioreactors without MP addition). In contrast, AM achieved 3-425% higher qm through the sequential batches, indicating that MP could counteract the inhibition caused by the highly-concentrated acetate. MP addition to inhibited bioreactors (AO10) successfully restored them, achieving qm of 1.53 L CH4/g VSS/d, 3.4 times increase from AO10 after 8 days lag time, validating its potential as a recovery strategy for inhibited digesters with acetate accumulation. AM exhibited higher microbial populations (1.8-3.8 times) and intracellular activity (9.3 times) compared to AO. MP enriched Methanosaeta, Peptoclostridium, Paraclostridium, OPB41, and genes related to direct interspecies electron transfer and acetate oxidation, potentially driving the improvement of qm through MP-mediated methanogenesis. These findings demonstrated the potential of MP supplementation as an effective strategy to accelerate acetate-utilizing methanogenesis and restore an inhibited anaerobic digester with high acetate accumulation.

Metabolic engineering for the synthesis of polyesters: A 100-year journey from polyhydroxyalkanoates to non-natural microbial polyesters.

As concerns increase regarding sustainable industries and environmental pollutions caused by the accumulation of non-degradable plastic wastes, bio-based polymers, particularly biodegradable plastics, have attracted considerable attention as potential candidates for solving these problems by substituting petroleum-based plastics. Among these candidates, polyhydroxyalkanoates (PHAs), natural polyesters that are synthesized and accumulated in a range of microorganisms, are considered as promising biopolymers since they have biocompatibility, biodegradability, and material properties similar to those of commodity plastics. Accordingly, substantial efforts have been made to gain a better understanding of mechanisms related to the biosynthesis and properties of PHAs and to develop natural and recombinant microorganisms that can efficiently produce PHAs comprising desired monomers with high titer and productivity for industrial applications. Recent advances in biotechnology, including those related to evolutionary engineering, synthetic biology, and systems biology, can provide efficient and effective tools and strategies that reduce time, labor, and costs to develop microbial platform strains that produce desired chemicals and materials. Adopting these technologies in a systematic manner has enabled microbial fermentative production of non-natural polyesters such as poly(lactate) [PLA], poly(lactate-co-glycolate) [PLGA], and even polyesters consisting of aromatic monomers from renewable biomass-derived carbohydrates, which can be widely used in current chemical industries. In this review, we present an overview of strain development for the production of various important natural PHAs, which will give the reader an insight into the recent advances and provide indicators for the future direction of engineering microorganisms as plastic cell factories. On the basis of our current understanding of PHA biosynthesis systems, we discuss recent advances in the approaches adopted for strain development in the production of non-natural polyesters, notably 2-hydroxycarboxylic acid-containing polymers, with particular reference to systems metabolic engineering strategies.

A Na+-coupled C4-dicarboxylate transporter (Asuc_0304) and aerobic growth of Actinobacillus succinogenes on C4-dicarboxylates.

Actinobacillus succinogenes, which is known to produce large amounts of succinate during fermentation of hexoses, was able to grow on C4-dicarboxylates such as fumarate under aerobic and anaerobic conditions. Anaerobic growth on fumarate was stimulated by glycerol and the major product was succinate, indicating the involvement of fumarate respiration similar to succinate production from glucose. The aerobic growth on C4-dicarboxylates and the transport proteins involved were studied. Fumarate was oxidized to acetate. The genome of A. succinogenes encodes six proteins with similarity to secondary C4-dicarboxylate transporters, including transporters of the Dcu (C4-dicarboxylate uptake), DcuC (C4-dicarboxylate uptake C), DASS (divalent anion : sodium symporter) and TDT (tellurite resistance dicarboxylate transporter) family. From the cloned genes, Asuc_0304 of the DASS family protein was able to restore aerobic growth on C4-dicarboxylates in a C4-dicarboxylate-transport-negative Escherichia coli strain. The strain regained succinate or fumarate uptake, which was dependent on the electrochemical proton potential and the presence of Na(+). The transport had an optimum pH ~7, indicating transport of the dianionic C4-dicarboxylates. Transport competition experiments suggested substrate specificity for fumarate and succinate. The transport characteristics for C4-dicarboxylate uptake by cells of aerobically grown A. succinogenes were similar to those of Asuc_0304 expressed in E. coli, suggesting that Asuc_0304 has an important role in aerobic fumarate uptake in A. succinogenes. Asuc_0304 has sequence similarity to bacterial Na(+)-dicarboxylate cotransporters and contains the carboxylate-binding signature. Asuc_0304 was named SdcA (sodium-coupled C4-dicarboxylate transporter from A. succinogenes).

Effects of the induction of anoxia in photobioreactor on effective cultivation of Scenedesmus acuminatus under mixotrophic cultivation mode.

The purpose of this study was to investigate the optimum conditions of several factors (i.e. types and concentration of acetate, aeration rate, pH control) for maximizing the mixotrophic cultivation of Scenedesmus acuminatus using acetate as an organic carbon source. When acetate was used, dissolved oxygen (DO) was quickly consumed and resulted in an anoxic condition for 52 h. Then, DO increased quickly by photosynthetic reaction. Whenever we put acetate in a reactor after DO was recovered to higher than 7 mg/L, cells were quickly grown via cell respiration, which subsequently resulted in an anoxic condition. Compared to aeration, ammonium acetate, ammonium acetate with aeration tests, the highest maximum biomass productivity of 0.73 g/L/d was obtained for pH control test with ammonium acetate dosage. From this study, we found that DO was essential for the fast assimilation of acetate and depleted DO was quickly regenerated for pH control test. From this fact, we found that pH control test with ammonium acetate dosage was the best cultivation method for Scenedesmus acuminatus under mixotrophic condition. These findings could be a useful reference for maximizing the cultivation of S. acuminatus in industrial-scale applications.

High-affinity l-malate transporter DcuE of Actinobacillus succinogenes catalyses reversible exchange of C4-dicarboxylates.

Actinobacillus succinogenes is a natural succinate producer, which is the result of fumarate respiration. Succinate production from anaerobic growth with C4 -dicarboxylates requires transporters catalysing uptake and efflux of C4 -dicarboxylates. Transporter Asuc_1999 (DcuE) found in A. succinogenes belongs to the Dcu family and was considered the main transporter for fumarate respiration. However, deletion of dcuE affected l-malate uptake of A. succinogenes rather than fumarate uptake. DcuE complemented anaerobic growth of Escherichia coli on l-malate or fumarate; thus, the transporter was characterized in E. coli heterologously. Time-dependent uptake and competitive inhibition assays demonstrated that l-malate is the most preferred substrate for uptake by DcuE. The Vmax of DcuE for l-malate was 20.04 µmol/gDW·min with Km of 57 µM. The Vmax for l-malate was comparable to that for fumarate, whereas the Km for l-malate was 8 times lower than that for fumarate. The catalytic efficiency of DcuE for l-malate was 7.3-fold higher than that for fumarate, showing high efficiency and high affinity for l-malate. Furthermore, DcuE catalysed the reversible exchange of three C4 -dicarboxylates - l-malate, fumarate and succinate - but the preferred substrate for uptake was l-malate. Under physiological conditions, the C4 -dicarboxylates were reduced to succinate. Therefore, DcuE is proposed as the l-malate/succinate antiporter in A. succinogenes.

Transcriptome analysis and anaerobic C4 -dicarboxylate transport in Actinobacillus succinogenes.

A global transcriptome analysis of the natural succinate producer Actinobacillus succinogenes revealed that 353 genes were differentially expressed when grown on various carbon and energy sources, which were categorized into six functional groups. We then analyzed the expression pattern of 37 potential C4 -dicarboxylate transporters in detail. A total of six transporters were considered potential fumarate transporters: three transporters, Asuc_1999 (Dcu), Asuc_0304 (DASS), and Asuc_0270-0273 (TRAP), were constitutively expressed, whereas three others, Asuc_1568 (DASS), Asuc_1482 (DASS), and Asuc_0142 (Dcu), were differentially expressed during growth on fumarate. Transport assays under anaerobic conditions with [14 C]fumarate and [14 C]succinate were performed to experimentally verify that A. succinogenes possesses multiple C4 -dicarboxlayte transport systems with different substrate affinities. Upon uptake of 5 mmol/L fumarate, the systems had substrate specificity for fumarate, oxaloacetate, and malate, but not for succinate. Uptake was optimal at pH 7, and was dependent on both proton and sodium gradients. Asuc_1999 was suspected to be a major C4 -dicarboxylate transporter because of its noticeably high and constitutive expression. An Asuc_1999 deletion (∆1999) decreased fumarate uptake significantly at approximately 5 mmol/L fumarate, which was complemented by the introduction of Asuc_1999. Asuc_1999 expressed in Escherichia coli catalyzed fumarate uptake at a level of 21.6 µmol·gDW-1 ·min-1 . These results suggest that C4 -dicarboxylate transport in A. succinogenes is mediated by multiple transporters, which transport various types and concentrations of C4 -dicarboxylates.