Temperature Phased Anaerobic Digestion at the Intermediate Zone of 45 °C: Performances, Stability and Pathogen Deactivation

Temperature phased anaerobic digestion (TPAD) systems with conventional sequences (first stage of 55 ℃ and second stage of 35 ℃) have been widely studied. However, very limited studies were available on TPAD system with the first stage operated at the intermediate zone of 45 °C, mainly due to the notion that limited microbial activity occurs within this zone. The objective of this research was to evaluate the performance, stability and the capability of 45 °C TPAD in producing class A biosolids, in comparison to a conventional TPAD. Four combinations of TPAD systems were studied, 45 ℃ TPAD 2.5/10 (1st stage solids retention time (SRT) 2.5 days/2nd stage SRT 10 days), 45 ℃ TPAD 7.5/10, 55 ℃ TPAD 2.5/10 and 55 ℃ TPAD 7.5/10. Among all, 45 ℃ TPAD 7.5/10 was found to have the best performances, attributed to its high volatile solids (VS) destruction (58%), minimal acetate accumulation (127 mg/L), high methane yield (0.58 m3 CH4/kg VS removed), high COD destruction solid COD (sCOD; 74% and total COD (tCOD) 54%) and minimal free NH3 content (67.5 mg/L). As for stability, stable pH distribution, high alkalinity content and low VFA to alkalinity ratio, indicated a well-buffered system. Additionally, the system had also able to produce class A biosolids. Therefore, proved that TPAD system operated at the intermediate zone of 45 ℃ can perform better than the conventional TPAD, hence, highlighting its economic advantage.

2,3-Butanediol synthesis from glucose supplies NADH for elimination of toxic acetate produced during overflow metabolism

Overflow metabolism-caused acetate accumulation is a major problem that restricts industrial applications of various bacteria. 2,3-Butanediol (2,3-BD) synthesis in microorganisms is an ancient metabolic process with unidentified functions. We demonstrate here that acetate increases and then decreases during the growth of a bacterium Enterobacter cloacae subsp. dissolvens SDM. Both bifunctional acetaldehyde/ethanol dehydrogenase AdhE-catalyzed ethanol production and acetate-induced 2,3-BD biosynthesis are indispensable for the elimination of acetate generated during overflow metabolism. 2,3-BD biosynthesis from glucose supplies NADH required for acetate elimination via AdhE-catalyzed ethanol production. The coupling strategy involving 2,3-BD biosynthesis and ethanol production is widely distributed in bacteria and is important for toxic acetate elimination. Finally, we realized the co-production of ethanol and acetoin from chitin, the second most abundant natural biopolymer whose catabolism involves inevitable acetate production through the coupling acetate elimination strategy. The synthesis of a non-toxic chemical such as 2,3-BD may be viewed as a unique overflow metabolism with desirable metabolic functions.

Engineering Corynebacterium glutamicum for the Efficient Production of 3-Hydroxypropionic Acid from a Mixture of Glucose and Acetate via the Malonyl-CoA Pathway

3-Hydroxypropionic acid (3-HP) has been recognized as one of the top value-added building block chemicals, due to its numerous potential applications. Over the past decade, biosynthesis of 3-HP via the malonyl-CoA pathway has been increasingly favored because it is balanced in terms of ATP and reducing equivalents, does not require the addition of costly coenzymes, and can utilize renewable lignocellulosic biomass. In this study, gene mcr encoding malonyl-CoA reductase from Chloroflexus aurantiacus was introduced into Corynebacterium glutamicum ATCC13032 to construct the strain Cgz1, which accumulated 0.30 g/L 3-HP. Gene ldhA encoding lactate dehydrogenase was subsequently deleted to eliminate lactate accumulation, but this decreased 3-HP production and greatly increased acetate accumulation. Then, different acetate utilization genes were overexpressed to reuse the acetate, and the best candidate Cgz5 expressing endogenous gene pta could effectively reduce the acetate accumulation and produced 0.68 g/L 3-HP. To enhance the supply of the precursor acetyl-CoA, acetate was used as an ancillary carbon source to improve the 3-HP production, and 1.33 g/L 3-HP could be produced from a mixture of glucose and acetate, with a 2.06-fold higher yield than from glucose alone. Finally, to inhibit the major 3-HP competing pathway-fatty acid synthesis, 10 μM cerulenin was added and strain Cgz5 produced 3.77 g/L 3-HP from 15.47 g/L glucose and 4.68 g/L acetate with a yield of 187 mg/g substrate in 48 h, which was 12.57-fold higher than that of Cgz1. To our best knowledge, this is the first report on engineering C. glutamicum to produce 3-HP via the malonyl-CoA pathway. The results indicate that the innocuous biosafety level I microorganism C. glutamicum is a potential industrial 3-HP producer.

Domestication of the novel alcohologenic acetogen Clostridium sp. AWRP: from isolation to characterization for syngas fermentation

Background Gas-fermenting acetogens have received a great deal of attention for their ability to grow on various syngas and waste gas containing carbon monoxide (CO), producing acetate as the primary metabolite. Among them, some Clostridium species, such as C. ljungdahlii and C. autoethanogenum, are of particular interest as they produce fuel alcohols as well. Despite recent efforts, alcohol production by these species is still unsatisfactory due to their low productivity and acetate accumulation, necessitating the isolation of strains with better phenotypes. Results In this study, a novel alcohol-producing acetogen (Clostridium sp. AWRP) was isolated, and its complete genome was sequenced. This bacterium belongs the same phylogenetic group as C. ljungdahlii, C. autoethanogenum, C. ragsdalei, and C. coskatii based on 16S rRNA homology; however, the levels of genome-wide average nucleotide identity (gANI) for strain AWRP compared with these strains range between 95 and 96%, suggesting that this strain can be classified as a novel species. In addition, strain AWRP produced a substantial amount of ethanol (70–90 mM) from syngas in batch serum bottle cultures, which was comparable to or even exceeded the typical values obtained using its close relatives cultivated under similar conditions. In a batch bioreactor, strain AWRP produced 119 and 12 mM of ethanol and 2,3-butanediol, respectively, while yielding only 1.4 mM of residual acetate. Interestingly, the alcohologenesis of this strain was strongly affected by oxidoreduction potential (ORP), which has not been reported with other gas-fermenting clostridia. Conclusion Considering its ethanol production under low oxidoreduction potential (ORP) conditions, Clostridium sp. AWRP will be an interesting host for biochemical studies to understand the physiology of alcohol-producing acetogens, which will contribute to metabolic engineering of those strains for the production of alcohols and other value-added compounds from syngas.

Pseudomonas aeruginosa Ethanol Oxidation by AdhA in Low-Oxygen Environments

ABSTRACT Pseudomonas aeruginosa has a broad metabolic repertoire that facilitates its coexistence with different microbes. Many microbes secrete products that P. aeruginosa can then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen-limiting conditions P. aeruginosa utilizes AdhA, an NAD-linked alcohol dehydrogenase, as a previously undescribed means for ethanol catabolism. In a rich medium containing ethanol, AdhA, but not the previously described PQQ-linked alcohol dehydrogenase, ExaA, oxidizes ethanol and leads to the accumulation of acetate in culture supernatants. AdhA-dependent acetate accumulation and the accompanying decrease in pH promote P. aeruginosa survival in LB-grown stationary-phase cultures. The transcription of adhA is elevated by hypoxia and under anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown that lasR mutants, which lack an important quorum sensing regulator, have higher levels of Anr-regulated transcripts under low-oxygen conditions than their wild-type counterparts. Here, we show that a lasR mutant, when grown with ethanol, has an even larger decrease in pH than the wild type (WT) that is dependent on both anr and adhA. The large increase in AdhA activity is similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism in P. aeruginosa by AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification under anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation in P. aeruginosa. IMPORTANCE Ethanol is a common product of microbial fermentation, and the Pseudomonas aeruginosa response to and utilization of ethanol are relevant to our understanding of its role in microbial communities. Here, we report that the putative alcohol dehydrogenase AdhA is responsible for ethanol catabolism and acetate accumulation under low-oxygen conditions and that it is regulated by Anr.

Pseudomonas aeruginosaethanol oxidation by AdhA in low oxygen environments

Pseudomonas aeruginosahas a broad metabolic repertoire that facilitates its co-existence with different microbes. Many microbes secrete products thatP. aeruginosacan then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen limiting conditionsP. aeruginosautilizes AdhA, an NAD-linked alcohol dehydrogenase, as a previously undescribed means for ethanol catabolism. In a rich medium containing ethanol, AdhA, but not the previously described PQQ-linked alcohol dehydrogenase, ExaA, oxidizes ethanol and leads to the accumulation of acetate in culture supernatants. AdhA-dependent acetate accumulation, and the accompanying decrease in pH, promotesP. aeruginosasurvival in LB-grown stationary phase cultures. The transcription ofadhAis elevated by hypoxia and in anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown thatlasRmutants have higher levels of Anr-regulated transcripts in low oxygen conditions compared to their wild type counterparts. Here, we show that alasRmutant, when grown with ethanol, has an even larger decrease in pH than WT that is dependent on bothanrandadhA. The large increase in AdhA activity similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism inP. aeruginosaby AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification in anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation inP. aeruginosa.ImportanceEthanol is a common product of microbial fermentation, and thePseudomonas aeruginosaresponse to and utilization of ethanol is relevant to our understanding of its role in microbial communities. Here, we report that the putative alcohol dehydrogenase, AdhA, is responsible for ethanol catabolism and acetate accumulation in low oxygen conditions and that it is regulated by Anr.