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To achieve industrial-scale putrescine production, a high efficient bio-synthesis of putrescine involving arginase (ARG, EC 3.5.3.1) and L-ornithine decarboxylase was evaluated here. Among the four arginases tested, ARGBT from Bos Taurus showed the highest activity (1966 U/mg). Compared to the L-arginine decarboxylase (ADC) pathway, the strain expressing ARGBT and L-ornithine decarboxylase (SpeC) produced 28.7 g/L putrescine, a 38.6 % increase. Two pyridoxal phosphate (PLP) salvage pathways were evaluated, and the strain BL-PTac-PdxK co-expressed pyridoxal kinase (PdxK) performed better. D-Glucose was used as the co-substrate to improve the putrescine titer further. Under optimal conditions, 43.6 g/L putrescine was produced from 87.1 g/L L-arginine, and 76 g/L putrescine was synthesized on a 0.5 L scale. Using L-arginine fermentation broth (60 g/L) as the substrate, a titer of 30 g/L putrescine was achieved. This efficient biotransformation process presented here enables feasible industrial-scale putrescine production.

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BACKGROUND: 1,5-pentanediol (1,5-PDO) is a linear diol with an odd number of methylene groups, which is an important raw material for polyurethane production. In recent years, the chemical methods have been predominantly employed for synthesizing 1,5-PDO. However, with the increasing emphasis on environmentally friendly production, it has been a growing interest in the biosynthesis of 1,5-PDO. Due to the limited availability of only three reported feasible biosynthesis pathways, we developed a new biosynthetic pathway to form a cell factory in Escherichia coli to produce 1,5-PDO. RESULTS: In this study, we reported an artificial pathway for the synthesis of 1,5-PDO from lysine with an integrated cofactor and co-substrate recycling and also evaluated its feasibility in E.coli. To get through the pathway, we first screened aminotransferases originated from different organisms to identify the enzyme that could successfully transfer two amines from cadaverine, and thus GabT from E. coli was characterized. It was then cascaded with lysine decarboxylase and alcohol dehydrogenase from E. coli to achieve the whole-cell production of 1,5-PDO from lysine. To improve the whole-cell activity for 1,5-PDO production, we employed a protein scaffold of EutM for GabT assembly and glutamate dehydrogenase was also validated for the recycling of NADPH and α-ketoglutaric acid (α-KG). After optimizing the cultivation and bioconversion conditions, the titer of 1,5-PDO reached 4.03 mM. CONCLUSION: We established a novel pathway for 1,5-PDO production through two consecutive transamination reaction from cadaverine, and also integrated cofactor and co-substrate recycling system, which provided an alternative option for the biosynthesis of 1,5-PDO.

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The present study examines the impact of nitrogen sources (yeast extract, ammonium sulfate peptone, ammonium nitrate, urea, and sodium nitrate), salt solution (0.5 g/L MgSO4, 0.5 g/L KH2PO4, 0.3 g/L CaCl2), trace elements solution (0.1 g/L CuSO4, 0.1 g/L FeSO4, 0.02 g/L MnCl2, 0.02 g/L ZnSO4), operational parameters (temperature, aeration, agitation, initial pH and xylose concentration) and co- substrate supplementation (glucose, fructose, maltose, sucrose, and glycerol) on xylitol biosynthesis by Candida tropicalis ATCC 13803 using synthetic xylose. The significant medium components were identified using the Plackett Burman design followed by central composite designs to obtain the optimal concentration for the critical medium components in shaker flasks. Subsequently, the effect of operational parameters was examined using the One Factor At a Time method, followed by the impact of five co-substrates on xylitol biosynthesis in a 1 L bioreactor. The optimal media components and process parameters are as follows: peptone: 12.68 g/L, yeast extract: 6.62 g/L, salt solution (0.5 g/L MgSO4, 0.5 g/L KH2PO4, and 0.3 g/L CaCl2): 1.23 X (0.62 g/L, 0.62 g/L, and 0.37 g/L respectively), temperature: 30 °C, pH: 6, agitation: 400 rpm, aeration: 1 vvm, and xylose: 50 g/L. Optimization studies resulted in xylitol yield and productivity of 0.71 ± 0.004 g/g and 1.48 ± 0.018 g/L/h, respectively. Glycerol supplementation (2 g/L) further improved xylitol yield (0.83 ± 0.009 g/g) and productivity (1.87 ± 0.020 g/L/h) by 1.66 and 3.12 folds, respectively, higher than the unoptimized conditions thus exhibiting the potential of C. tropicalis ATCC 13803 being used for commercial xylitol production.

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The staggering increase in pollution associated with a sharp tightening in global energy demand is a major concern for organic substances. Renewable biofuel production through simultaneous waste reduction is a sustainable approach to meet this energy demand. This study co-fermentation of dairy whey and SCB was performed using mixed and pure bacterial cultures of Salmonella bongori, Escherichia coli, and Shewanella oneidensis by dark fermentation process for hydrogen production. The maximum H2 production was 202.7 ± 5.5 H2/mL/L, 237.3 ± 6.0 H2/mL/L, and 198 ± 9.9 H2/mL/L obtained in fermentation reactions containing dairy whey, solid and liquid hydrolysis of pretreated sugarcane bagasse as mono-substrates. The H2 production was greater in co-substrate by 347.3 ± 18.5 H2/mL/L under optimized conditions (pH 7.0, temperature 37 °C, substrate concentration 30:50 g/L) than expected in mono-substrate conditions, which confirms that co-fermentation of different substrates enhances the H2 potential. Fermentation medium during bio-H2 production under GC analysis has stated that using mixed cultures in dark fermentation favored acetic acid and butyric acid. Co-substrate degradation produces ethyl alcohol, benzoic acid, propionic acid, and butanol as metabolic by-products. The difference in the treated and untreated substrate and carbon enrichment in the substrates was evaluated by FT-IR analysis. The present study justifies that rather than the usage of mono-substrate for bio-H2 production, the co-substrate provided highly stable H2 production by mixed bacterial cultures. Fabricate the homemade single-chamber microbial fuel cell to generate electricity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03687-9.

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Ascorbic acid was introduced to enhance the performance of zero-valent iron (Fe(0)) in hydrogen production by photo fermentation of bean dregs and corn stover. The highest hydrogen production of 664.0 ± 5.3 mL and hydrogen production rate of 34.6 ± 0.1 mL/h was achieved at 150 mg/L ascorbic acid, which was 10.1% and 11.5% higher than that of 400 mg/L Fe(0) alone. The supplement of ascorbic acid to Fe(0) system accelerated the formation of Fe(Ⅱ) in solution due to its reducing and chelating ability. Hydrogen production of Fe(0) and ascorbic acid-Fe(0) (AA-Fe(0)) systems at different initial pH (5, 6, 7, 8 and 9) was studied. Result showed that hydrogen produced from AA-Fe(0) system was improved by 2.7-27.5% compared with Fe(0) system. The maximum hydrogen production of 767.5 ± 2.8 mL was achieved with initial pH 9 in the AA-Fe(0) system. This study provided a strategy for enhancing biohydrogen production.

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The potential of palm oil and derived wastewater pretreated by enzyme as co-substrates to accumulate polyhydroxyalkanoate (PHA) rich in short and medium-chain-length monomers under two feeding strategies was evaluated batchwise through mixed microbial cultures (MMCs) in activated sludge. A terpolymer with the maximum PHA content of 30.5 wt%, volumetric yield of 0.372 g COD/g COD and composition of ca. 84.7 âˆ¼ 97.4/0.5 âˆ¼ 1.6/2.1 âˆ¼ 13.7 (3-hydroxybutyrate/ 3-hydroxyvalerate/ 3-hydroxyoctanoate, %) was achieved as a result of co-substrate incorporation. From the perspective of economic benefits, PHA accumulated via adopting strategy of supplementing carbon source to the same initial concentration per cycle saved 42.7 % of carbon consumption, along with a reduction in culture time (72 h). The above discoveries signify that the combination of palm oil and derived wastewater plus MMCs provides an alternative to the plastics industries for a more sustainable and efficient utilization of biological resources and an economic PHA accumulation approach.

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The study aimed to comprehensively determine P extraction efficiency and co-digestion of food waste (FW) and primary settled-nightsoil sludge (PSNS) process performance influenced by different hydraulic retention times (4, 7, 10, and 15 days) and mixture ratios of FW:PSNS in substrates (100:0, 75:25, 50:50, 25:75, and 0:100). P-transformation was evaluated to identify P fractionation in both supernatant and sludge accumulated in reactors. The results showed that anaerobic co-digestion was inhibited by the accumulation of undigested feedstock due to higher %PSNS found in AD4 (25FW:75PSNS) and AD5 (100PSNS). A more stable process was found in AD2 (75FW:25PSNS) under hydraulic retention time (HRT) 15 days in which COD removal efficiency and P release were 97.2 and 80.2%, respectively. This recommended condition allowed a high organic loading rate (OLR) at 12 gVS/L/day resulting in the highest biogas yield of 0.93 L/L/day. Distribution of P data demonstrated that most of P in feedstock was deposited and accumulated in sediment up to 97.8%. Poor biodegradability resulting from using shortened HRT led to high increased P-solid content in effluent. In addition, available P in effluents and accumulated P-solids in sediment obtained from the AcoD process has the potential to serve as sources for P recovery.

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The melibiose permease MelB is a well-studied Na+-coupled transporter of the major facilitator superfamily. However, the symport mechanism of galactosides and cations is still not fully understood, especially at structural levels. Here, we use single-molecule force spectroscopy to investigate substrate-induced structural changes of MelB from Salmonella typhimurium. In the absence of substrate, MelB equally populates two different states, from which one shows higher mechanical structural stability with additional stabilization of the cytoplasmic middle-loop C3. In the presence of either melibiose or a coupling Na+-cation, however, MelB increasingly populates the mechanically less stable state, which shows a destabilized middle-loop C3. In the presence of both substrate and co-substrate, this mechanically less stable state of MelB is predominant. Our findings describe how both substrates guide MelB transporters to populate two different mechanically stabilized states, and contribute mechanistic insights to the alternating-access action for the galactoside/cation symport catalyzed by MelB.

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Methane production characteristics of anaerobic co-digestion of pig manure (PM) and fermented liquid feed (FLF) were investigated in a continuous digester under mesophilic conditions. The experiment followed three phases. PM alone was digested in phase I. In phases II and III, PM and FLF were mixed in a ratio of 95:5 and 90:10 (% v/v), respectively. The specific methane yields (SMYs) during phases I, II, and III were 238, 278, and 326.8 mLCH4·gVS-1-added, respectively. It was due to the effect of balancing the feedstock carbon-to-nitrogen ratio by adding FLF. This improvement can also be attributed to the readily biodegradable compounds in the FLF. The higher SMY obtained in this study showed a positive synergistic effect in the anaerobic co-digestion of PM and FLF. The results also indicate that adding the FLF positively affected and maintained a constant pH level, avoiding volatile fatty acid accumulation and ammonia inhibition in the anaerobic digestion (AD). Thus, this study provides valuable information regarding the usage of unused or wasted FLF as a co-substrate for the practical AD of PM. The production of fermented liquid additives such as FLF to improve the methane production from the AD of PM is a potential novel alternative to food waste recycling in Japan, besides compost and animal feeding.

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Melatonin exerts its effects through a series of target proteins/receptors and enzymes. Its antioxidant capacity might be due to its capacity to inhibit a quinone reductase (NQO2) at high concentration (50 µM). Demonstrating the existence of a complex between a compound and a protein is often not easy. It requires either that the compound is an inhibitor-and the complex translates by an inhibition of the catalytic activity-or the compound is radiolabeled-and the complex translates in standard binding approaches, such as in receptology. Outside these two cases, the detection of the protein:small molecule complexes by mass spectrometry has recently been made possible, thanks to the development of so-called native mass spectrometry. Using this approach, one can measure masses corresponding to an intact noncovalent complex between a compound and its target, usually after titration or competition experiments. In the present chapter, we detail the characterization of NQO2:melatonin interaction using native mass spectrometry.

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The presence of organic co-substrate in groundwater and soils is inevitable, and much remains to be learned about the roles of organic co-substrates during pyrite-based denitrification. Herein, an organic co-substrate (acetate) was added to a pyrite-based denitrification system, and the impact of the organic co-substrate on the performance and bacterial community of pyrite-based denitrification processes was evaluated. The addition of organic co-substrate at concentrations higher than 48 mg L-1 inhibited pyrite-based autotrophic denitrification, as no sulfate was produced in treatments with high organic co-substrate addition. In contrast, both competition and promotion effects on pyrite-based autotrophic denitrification occurred with organic co-substrate addition at concentrations of 24 and 48 mg L-1. The subsequent validation experiments suggested that competition had a greater influence than promotion when organic co-substrate was added, even at a low concentration. Thiobacillus, a common chemolithoautotrophic sulfur-oxidizing denitrifier, dominated the system with a relative abundance of 13.04% when pyrite served as the sole electron donor. With the addition of organic co-substrate, Pseudomonas became the dominant genus, with 60.82%, 61.34%, 70.37%, 73.44%, and 35.46% abundance at organic matter concentrations of 24, 48, 120, 240, and 480 mg L-1, respectively. These findings provide an important theoretical basis for the cultivation of pyrite-based autotrophic denitrifying microorganisms for nitrate removal in soils and groundwater.

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In this study, the performance and mechanism of nitrogen removal were investigated in a zero-valent iron-mediated nitrogen removal system operated in co-substrate mode with sodium acetate as the organic carbon source. The results showed that the additional organic matter had the capacity to promote NH4+-N and total inorganic nitrogen (TIN) removal with efficiencies of 91.09% and 84.10%, and increases of 60.06% and 75.32% compared with the control group, respectively. The organic matter also stimulated the production of extracellular polymer substances that reduced the passivation and toxicity of iron to microorganisms. The ammonia oxidation activity was 2.5 times higher than that in the control group, and the anammonia oxidation activity and denitrification activity were substantially higher than in the control group with TIN removal efficiencies of 1.02 and 1.19 mgN/(gVSS·d), respectively. In addition, the organic matter increased the enrichment of the heterotrophic denitrification bacterium Diaphorobacter and facultative iron salt-based bacterium Dechloromonas.

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Using co-substrates to enhance the metabolic activity of microbes is an effective way for high-molecular-weight polycyclic aromatic hydrocarbons removal in petroleum-contaminated environments. However, the long degradation period and exhausting substrates limit the enhancement of metabolic activity. In this study, Altererythrobacter sp. N1 was screened from petroleum-contaminated soil in Shengli Oilfield, China, which could utilize pyrene as the sole carbon source and energy source. Saturated aromatic fractions and crude oils were used as in-situ co-substrates to enhance pyrene degradation. Enzyme activity was influenced by the different co-substrates. The highest degradation rate (75.98%) was achieved when crude oil was used as the substrate because strain N1 could utilize saturated and aromatic hydrocarbons from crude oil simultaneously to enhance the degrading enzyme activity. Moreover, the phthalate pathway was dominant, while the salicylate pathway was secondary. Furthermore, the Rieske-type aromatic cyclo-dioxygenase gene was annotated in the Altererythrobacter sp. N1 genome for the first time. Therefore, the co-metabolism of pyrene was sustained to achieve a long degradation period without the addition of exogenous substrates. This study is valuable as a potential method for the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons.

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Chiral and enantiopure amines can be produced by enantioselective transaminases via kinetic resolution of amine racemates. This transamination reaction requires stoichiometric amounts of co-substrate. A dual-enzyme recycling system overcomes this limitation: l-amino acid oxidases (LAAO) recycle the accumulating co-product of (S)-selective transaminases in the kinetic resolution of racemic amines to produce pure (R)-amines. However, availability of suitable LAAOs is limited. Here we use the heterologously produced, highly active fungal hcLAAO4 with broad substrate spectrum. H2 O2 as byproduct of hcLAAO4 is detoxified by a catalase. The final system allows using sub-stoichiometric amounts of 1 mol% of the transaminase co-substrate as well as the initial application of l-amino acids instead of α-keto acids. With an optimized protocol, the synthetic potential of this kinetic resolution cascade was proven at the preparative scale (>90 mg) by the synthesis of highly enantiomerically pure (R)-methylbenzylamine (>99 %ee) at complete conversion (50 %).

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Both co-cultivation and co-substrate addition strategies have exhibited massive potential in microalgae-based antibiotic bioremediation. In this study, glucose and sodium acetate were employed as co-substrate in the cultivation of microalgae-bacteria consortium for enhanced sulfadiazine (SDZ) and sulfamethoxazole (SMX) removal. Glucose demonstrated a two-fold increase in biomass production with a maximum specific growth rate of 0.63 ± 0.01 d-1 compared with sodium acetate. The supplementation of co-substrate enhanced the degradation of SDZ significantly up to 703 ± 18% for sodium acetate and 290 ± 22% for glucose, but had almost no effect on SMX. The activities of antioxidant enzymes, including peroxidase, superoxide dismutase and catalase decreased with co-substrate supplementation. Chlorophyll a was associated with protection against sulfonamides and chlorophyll b might contribute to SDZ degradation. The addition of co-substrates influenced bacterial community structure greatly. Glucose enhanced the relative abundance of Proteobacteria, while sodium acetate improved the relative abundance of Bacteroidetes significantly.

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In this study, four groups of laboratory scale experiments were performed by adding sodium acetate (SA), phthalic acid (PA), and SA-PA to river sediment to observe the microbial response and biodegradation efficiency of polycyclic aromatic hydrocarbons (PAHs). The results showed that the amount of total organic carbon consumed and the amount of sulfate reduction were both positively correlated (p < 0.01) with the biodegradation efficiency of the sum (∑) PAHs (∼40.5%). The lower the number of rings, the more PAHs were biodegraded, with an efficiency of 63.0% for ∑ (2 + 3) ring PAHs. Based on high-throughput sequencing and molecular ecological network analysis, it was found that the combined stimulation of SA and PA not only increased the relative abundance of PAHs-degrading bacterial (eg., Proteobacteria, Desulfobacterota, Campilobacterota and Firmicutes), but also had a strengthening effect on microbes in sediments. The altered microbial structure caused a variation in metabolic functions, which increased the amino acid metabolism to 12.2%, thus increasing the positive correlations among genera and improving the connectivity of the microbial network (p < 0.01). These changes may be responsible for the enhanced biodegradation of PAHs under SA-PA dosing in comparison to SA or PA dosing alone. This study revealed that the microbial community was stimulated by the combined addition of SA and PA, and indicated its role in enhancing biodegradation of PAHs in contaminated river sediments.

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The objective of this research was to evaluate anaerobic co-digestion of guinea pig manure (GP) with Andean agricultural residues such as amaranth (AM), quinoa (QU) and wheat (TR) in batch biodigesters under mesophilic conditions (37 0C) for 40 days. As microbial inoculum, sewage treatment sludge was used in two inoculum/substrate ratios (ISR of 1 and 2). In terms of methane production, the best results occurred in treatments containing AM and QU as co-substrate and an ISR of 2. Thus, the highest methane production yield in the GP:AM biodigesters (25:75) and GP:QU (25:75) with 341.86 mlCH4/g VS added and 341.05 mlCH4/g VS added, respectively. On the other hand, the results showed that methane production with an ISR of 2 generated higher yields for guinea pig waste and the methane fraction of the biogas generated was in a range from 57 to 69%. Methane production kinetics from these raw materials was studied using five kinetic models: modified Gompertz, logistic equation, transfer, cone and Richards. The cone model adjusted best to the experimental values ​​with those observed with r2 of 0.999 and RMSE of 1.16 mlCH4/g VS added. Finally, the highest biodegradability (experimental yield/theoretical yield) was obtained in the GP-AM biodigesters (25:75) with 67.92%.

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This study investigated the effects of representative co-substrate (corncob particles) and additive (brick granules) alone on antibiotic resistome of swine manure during composting and subsequent compost application. For relative abundances, four antibiotic resistance gene (ARG) types encoding resistance to aminoglycoside, multidrug, florfenicol-chloramphenicol-amphenicol-fluoroquinolone-quinolone, and sulfonamide increased remarkably during composting, whereas all the ARG types decreased after compost application. Interestingly, much more ARG subtypes (50.1% in total) were reduced in corncob addition treatment. Meanwhile, the addition of corncob particles lowered the relative abundance and diversity of ARGs more significantly. Microbial community exhibited conspicuous changes across the manure, compost, and soil samples where the dominant genera were completely different. Procrustes test proved the co-occurrence and driving effect of microbial community on resistome variation, especially in corncob addition treatment during composting. Network analysis demonstrated that the dissipation of the dominant genera such as Ruminofilibacter, Luteimonas, and Pseudidiomarina in the composts after application contributed greatly to the reduction in ARG relative abundance. Besides, the low abundance of mobile genetic elements (MGEs) in soil also accounted for the attenuation of ARGs to some extent. Our findings clearly proved that co-composting materials can further affect the attenuation of antibiotic resistome in soil application, which can help in understanding the spread and control of ARGs during agricultural process.

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The feasibility of producing polyhydroxyalkanoate (PHA) from pretreated waste wood hydrolysate and volatile fatty acids (VFAs) from sewage fermented products as co-substrate feedstock through mixed microbial cultures (MMCs) process was explored. The results showed that the addition of co-substrate shortened the cycle of PHA reaching the maximum and increased the proportion of 3-hydroxyvalerate (3HV) monomer. Compared with N-excess supply, almost 1.6 times increased PHA accumulation was realized under N-limitation, and simultaneously the highest proportion of 3HV monomer with 51% was also obtained. Additionally, PHA production in S1400 reactor reached a maximum value of about 3088 mg COD/L with culture time to 36 h. The microbial community also displayed a high diversity, which was composed of 65 bacterial genera. It is a novel attempt to accumulate PHA from pretreated waste wood hydrolysate and VFAs co-substrate through MMCs, providing an effective green approach to reduce its expensive cost and achieve mass production.

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Integrating microalgae culture and wastewater purification is a promising technology for sustainable bioresource production. However, the challenge is that toxins in wastewater usually limit risk elimination and cause poor bioresource production. Easy-to-biodegrade substrates could alleviate the resistant stress on a bacterial community but we know little about how they function with microalgae. In this study, we tested if Easy-to-biodegrade substrates could simultaneously promote Chlorella to degrade antibiotic amoxicillin (AMO) and produce bioresources. Sodium acetate (NaAC) was used as the representative co-substrate. The results showed NaAC could enhance AMO removal by 76%. The ß-lactam structure was destroyed and detoxified to small molecules, due to the up-regulation of hydrolase, oxidoreductase, reductase, and transferase. Chlorella biomass production increased by 36%. The genes encoding the glutathione metabolism and peroxisome pathways were significantly up-regulated to alleviate the antibiotic stress, and the DNA replication pathway was activated. As a result, the production of lipid, carbohydrate, and protein was enhanced by 61%, 122%, and 34%, respectively. This study provides new insights for using microalgae to recover bioresources from toxic wastewater and reveals the critical underlying mechanisms.

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