Vitreoscilla hemoglobin enhances the catalytic performance of industrial oxidases in vitro.

Oxidases are a group of oxidoreductases and need molecular oxygen in the catalytic process. Vitreoscilla hemoglobin (VHb) can improve the growth and productivity of host cells under hypoxic conditions, rendering it attractive for industrial application. In this work, we demonstrated the addition of immobilized VHb increased the catalytic activity of immobilized D-amino acid oxidase of Trigonopsis variabilis by two-fold when catalyzing cephalosporin C under oxygen-limited conditions. A similar increase of activities was observed in glucose oxidase, alcohol oxidase, and p-hydroxymandelate synthase by adding free VHb or immobilized VHb under hypoxic conditions. When L-glutamate oxidase was used to catalyze L-glutamate to produce α-ketoglutarate, the yield increased from 80.6 to 96.9% by fusing VHb with L-glutamate oxidase. Results demonstrated that the addition of free VHb, immobilized VHb, or fused VHb could increase the catalytic efficiency of oxidases, which was considered by increasing the concentration of the microenvironmental oxygen. Thus, VHb may become a potential additive agent to promote the efficiency of oxidases on industrial scale . KEY POINTS: • First time confirmation of facilitation of VHb on several industrial oxidases in vitro • VHb functions under hypoxic conditions rather than oxygen-enriched conditions • VHb functions in vitro in the form of free, immobilized protein and fusion enzyme.

Efficient production of GlcNAc in an aqueous-organic system with a Chitinolyticbacter meiyuanensis SYBC-H1 mutant.

OBJECTIVES: Shellfish waste is a primary source for making N-acetyl-D-glucosamine. Thus, establishing a high-efficiency and low-cost bioconversion method to produce N-acetyl-D-glucosamine directly from shellfish waste was promising. RESULTS: A mutant C81 was obtained from Chitinolyticbacter meiyuanensis SYBC-H1 via 60Co-γ irradiation. This mutant C81 showed the highest chitinase activity of 9.8 U/mL that was 85% higher than the parent strain. The mutant C81 exhibted improved antioxidant activities, including total antioxidant capacity, superoxide radical ability, and hydroxyl radical scavenging ability, compared to that of the parent strain. Four out of nine organic solvents increased the chitinase activity by 1.9%, 6.8%, 11.7%, and 15.8%, corresponding to methylbenzene, n-heptane, petroleum ether, and n-hexane, respectively. The biphase system composed of aqueous and hexane presented a five-fold reduction of cell viability compared to the control. Using a continuous fermentation bioconversion process, 4.2 g/L GlcNAc was produced from crayfish shell powder with a yield of 80% of the chitin content. CONCLUSIONS: This study demonstrated that the mutant C81 is suitable for converting crayfish shell powder into GlcNAc in an aqueous-organic system.

Porous carbon aerogel derived from bacterial cellulose with prominent potential for efficient removal of antibiotics from the aquatic matrix.

The development of adsorption methods for the remediation of antibiotics pollution in water is hindered by the lack of high-performance sorbents. In this study, a nanofiber carbon aerogel was prepared using bacterial cellulose and its adsorption performances for three common antibiotics (norfloxacin, sulfamethoxazole, and chloramphenicol) in water were evaluated. The as-prepared nanofiber carbon aerogel showed a higher adsorption capacity toward target antibiotics compared to other adsorbents reported in the literature. The maximum adsorption capacities for norfloxacin, sulfamethoxazole, and chloramphenicol were 1,926, 1,264, and 525 mg/g, respectively at 298 K. Notably, the nanofiber carbon aerogel was able to adsorb 80% of the equilibrium adsorption capacity within 1 min and reach equilibrium within 15 min. After five regeneration cycles, the adsorption capacity still reached 1,166, 847, and 428 mg/g for norfloxacin, sulfamethoxazole, and chloramphenicol, respectively. The characterization results showed that the carbon aerogel exhibited a high specific surface area (1,505 m2/g) and a layered porous network structure. Furthermore, the mechanistic study reveals that the enhanced antibiotic adsorption by the as-prepared nanofiber carbon aerogel was attributed to the pore filling effect, hydrogen bonding, hydrophobic effect, electrostatic interaction, and π-π interactions. Overall, these results imply that low-cost and green nanofiber carbon aerogels may be promising adsorbents for the remediation of antibiotic-contaminated wastewater. The materials prepared from natural and readily available bacterial cellulose can adsorb antibiotics efficiently, which provides a reference for the development of adsorbent materials using natural substances.

Chromosomal mutations in Escherichia coli that improve tolerance to nonvolatile side-products from dilute acid treatment of sugarcane bagasse.

Lignocellulosic biomass provides attractive nonfood carbohydrates for the production of ethanol, and dilute acid pretreatment is a biomass-independent process for access to these carbohydrates. However, this pretreatment also releases volatile and nonvolatile inhibitors of fermenting microorganisms. To identify unique gene products contributing to sensitivity/tolerance to nonvolatile inhibitors, ethanologenic Escherichia coli strain LY180 was adapted for growth in vacuum-treated sugarcane bagasse acid hydrolysate (VBHz) lacking furfural and other volatile inhibitors. A mutant, strain AQ15, obtained after approximately 500 generations of growth in VBHz, grew and fermented the sugars in a medium with 50% VBHz. Comparative genome sequence analysis of strains AQ15 and LY180 revealed 95 mutations in strain AQ15. Six of these mutations were also found in strain SL112, an independent inhibitor-tolerant derivative of strain LY180. Among these six mutations, null mutations in mdh and bacA were identified as contributing factors to VBHz tolerance in strain AQ15, based on the genetic and physiological analysis. The deletion of either gene in strain LY180 increased tolerance to VBHz from approximately 30-50% (vol/vol). Considering the location and physiological role of the two enzymes in the cell, it is likely that the two enzymes contribute to the VBHz sensitivity of ethanologenic E. coli by different mechanisms.

Plasmidic Expression of nemA and yafC* Increased Resistance of Ethanologenic Escherichia coli LY180 to Nonvolatile Side Products from Dilute Acid Treatment of Sugarcane Bagasse and Artificial Hydrolysate.

Hydrolysate-resistant Escherichia coli SL100 was previously isolated from ethanologenic LY180 after sequential transfers in AM1 medium containing a dilute acid hydrolysate of sugarcane bagasse and was used as a source of resistance genes. Many genes that affect tolerance to furfural, the most abundant inhibitor, have been described previously. To identify genes associated with inhibitors other than furfural, plasmid clones were selected in an artificial hydrolysate that had been treated with a vacuum to remove furfural. Two new resistance genes were discovered from Sau3A1 libraries of SL100 genomic DNA: nemA (N-ethylmaleimide reductase) and a putative regulatory gene containing a mutation in the coding region, yafC*. The presence of these mutations in SL100 was confirmed by sequencing. A single mutation was found in the upstream regulatory region of nemR (nemRA operon) in SL100. This mutation increased nemA activity 20-fold over that of the parent organism (LY180) in AM1 medium without hydrolysate and increased nemA mRNA levels >200-fold. Addition of hydrolysates induced nemA expression (mRNA and activity), in agreement with transcriptional control. NemA activity was stable in cell extracts (9 h, 37°C), eliminating a role for proteinase in regulation. LY180 with a plasmid expressing nemA or yafC* was more resistant to a vacuum-treated sugarcane bagasse hydrolysate and to a vacuum-treated artificial hydrolysate than LY180 with an empty-vector control. Neither gene affected furfural tolerance. The vacuum-treated hydrolysates inhibited the reduction of N-ethylmaleimide by NemA while also serving as substrates. Expression of the nemA or yafC* plasmid in LY180 doubled the rate of ethanol production from the vacuum-treated sugarcane bagasse hydrolysate.

Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals.

Pretreatments such as dilute acid at elevated temperature are effective for the hydrolysis of pentose polymers in hemicellulose and also increase the access of enzymes to cellulose fibers. However, the fermentation of resulting syrups is hindered by minor reaction products such as furfural from pentose dehydration. To mitigate this problem, four genetic traits have been identified that increase furfural tolerance in ethanol-producing Escherichia coli LY180 (strain W derivative): increased expression of fucO, ucpA, or pntAB and deletion of yqhD. Plasmids and integrated strains were used to characterize epistatic interactions among traits and to identify the most effective combinations. Furfural resistance traits were subsequently integrated into the chromosome of LY180 to construct strain XW129 (LY180 ΔyqhD ackA::PyadC'fucO-ucpA) for ethanol. This same combination of traits was also constructed in succinate biocatalysts (Escherichia coli strain C derivatives) and found to increase furfural tolerance. Strains engineered for resistance to furfural were also more resistant to the mixture of inhibitors in hemicellulose hydrolysates, confirming the importance of furfural as an inhibitory component. With resistant biocatalysts, product yields (ethanol and succinate) from hemicellulose syrups were equal to control fermentations in laboratory media without inhibitors. The combination of genetic traits identified for the production of ethanol (strain W derivative) and succinate (strain C derivative) may prove useful for other renewable chemicals from lignocellulosic sugars.

Polyamine transporters and polyamines increase furfural tolerance during xylose fermentation with ethanologenic Escherichia coli strain LY180.

Expression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenic Escherichia coli LY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression of yqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion of potE in EMFR9 lowered furfural tolerance to that of the parent. Deletion of potE and puuP in LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural.

Manure enriched with nitrogen derived from high-protein food waste in a large dining facility.

Food waste (FW) from large dining facility has been a pressing environmental challenge in China recently. This study developed an innovative species-specific feeding strategy for producing pigeon meat and excellent manure from FW. Adding FW to the feed of pigeons significantly increased their feed intake and promoted their growth although the pigeons showed a strong aversion to the FW. We produced a "super manure" with exceptionally high nitrogen (N) content (mean = 10.77 % on a dry basis, 8.04-12.57 %, n = 264) by feeding slowly-growing pigeon species (Columba livia vs. and Caoge Huzhou 11) with protein-high commercial feed and FW. A significant negative relationship between the N and carbon (C) contents in the pigeon manure was found, with C depletion higher than N depletion. Furthermore, the N content in the anaerobic composting (AnC) manure was 29.16 % higher than that in the FW. Fourier transform infrared (FT-IR) analysis and stable isotopes δ13C and δ15N in the manure clearly identified the transformations of nutrients during pigeon feeding and the AnC process. This study opens a path for producing N-high manure using protein-high food waste.

Study on the biodegradation characteristics and mechanism of tetracycline by Serratia entomophila TC-1.

Microbial degradation is an important solution for antibiotic pollution in livestock and poultry farming wastes. This study reports the isolation and identification of the novel bacterial strain Serratia entomophila TC-1, which can degrade 87.8 % of 200 mg/L tetracycline (TC) at 35 °C, pH 6.0, and an inoculation amount of 1 % (v/v). Based on the intermediate products, a possible biological transformation pathway was proposed, including dehydration, oxidation ring opening, decarbonylation, and deamination. Using Escherichia coli and Bacillus subtilis as biological indicators, TC degraded metabolites have shown low toxicity. Whole-genome sequencing showed that the TC-1 strain contained tet (d) and tet (34), which resist TC through multiple mechanisms. In addition, upon TC exposure, TC-1 participated in catalytic and energy supply activities by regulating gene expression, thereby playing a role in TC detoxification. We found that TC-1 showed less interference with changes in the bacterial community in swine wastewater. Thus, TC-1 provided new insights into the mechanisms responsible for TC biodegradation and can be used for TC pollution treatment.

Improving Escherichia coli FucO for furfural tolerance by saturation mutagenesis of individual amino acid positions.

Furfural is an inhibitory side product formed during the depolymerization of hemicellulose with mineral acids. In Escherichia coli, furfural tolerance can be increased by expressing the native fucO gene (encoding lactaldehyde oxidoreductase). This enzyme also catalyzes the NADH-dependent reduction of furfural to the less toxic alcohol. Saturation mutagenesis was combined with growth-based selection to isolate a mutated form of fucO that confers increased furfural tolerance. The mutation responsible, L7F, is located within the interfacial region of FucO homodimers, replacing the most abundant codon for leucine with the most abundant codon for phenylalanine. Plasmid expression of the mutant gene increased FucO activity by more than 10-fold compared to the wild-type fucO gene and doubled the rate of furfural metabolism during fermentation. No inclusion bodies were evident with either the native or the mutated gene. mRNA abundance for the wild-type and mutant fucO genes differed by less than 2-fold. The Km (furfural) for the mutant enzyme was 3-fold lower than that for the native enzyme, increasing efficiency at low substrate concentrations. The L7F mutation is located near the FucO N terminus, within the ribosomal binding region associated with translational initiation. Free-energy calculations for mRNA folding in this region (nucleotides -7 to +37) were weak for the native gene (-4.1 kcal mol(-1)) but weaker still for the fucO mutant (-1.0 to -0.1 kcal mol(-1)). The beneficial L7F mutation in FucO is proposed to increase furfural tolerance by improving gene expression and increasing enzyme effectiveness at low substrate levels.

Anaerobic Storage Completely Removes Suspected Fungal Pathogens but Increases Antibiotic Resistance Gene Levels in Swine Wastewater High in Sulfonamides.

Wastewater storage before reuse is regulated in some countries. Investigations of pathogens and antibiotic resistance genes (ARGs) during wastewater storage are necessary for lowering the risks for wastewater reuse but are still mostly lacking. This study aimed to investigate pathogens, including harmful plant pathogens, and ARGs during 180 d of swine wastewater (SWW) storage in an anaerobic storage experiment. The contents of total organic carbon and total nitrogen in SWW were found to consistently decrease with the extension of storage time. Bacterial abundance and fungal abundance significantly decreased with storage time, which may be mainly attributed to nutrient loss during storage and the long period of exposure to a high level (4653.2 µg/L) of sulfonamides in the SWW, which have an inhibitory effect. It was found that suspected bacterial pathogens (e.g., Escherichia-Shigella spp., Vibrio spp., Arcobacter spp., Clostridium_sensu_stricto_1 spp., and Pseudomonas spp.) and sulfonamide-resistant genes Sul1, Sul2, Sul3, and SulA tended to persist and even become enriched during SWW storage. Interestingly, some suspected plant fungal species (e.g., Fusarium spp., Ustilago spp. and Blumeria spp.) were detected in SWW. Fungi in the SWW, including threatening fungal pathogens, were completely removed after 60 d of anaerobic storage, indicating that storage could lower the risk of using SWW in crop production. The results clearly indicate that storage time is crucial for SWW properties, and long periods of anaerobic storage could lead to substantial nutrient loss and enrichment of bacterial pathogens and ARGs in SWW.

Application of oil-degrading agents consisted of thermophilic Bacillus subtilis and Bacillus glycinifermentans in food waste.

This work aims to investigate the effective removal of oil in food waste (FW). Two bacteria, Bacillus subtilis and Bacillus glycinifermentans, were obtained under high temperature conditions and named YZQ-2 and YZQ-5, respectively. The oil degradation rate of two bacteria was explored under different pH value, temperature, and NaCl concentration. In addition, the lipase and emulsifying activity were evaluated. The maximum oil degradation rate was 83.41 ± 0.86% and the maximum lipase activity reached 89.73 ± 20.89 U L-1 with YZQ-2. The fermentation broth of YZQ-2 displayed exceptional emulsification activity. Subsequently, YZQ-2 and YZQ-5 were added to aerobic FW composting. The moisture content of the compost treated with inoculated strains decreased at a faster rate during the first three days of composting. The microbial quantity increased rapidly in the first three days, and the oil degradation rate reached 39.96% after five days. Due to the excellent adaptability to high temperature and ability to degrade oil, strains YZQ-2 and YZQ-5 exhibit superior potential for various applications.

A high-yielding strain of indole-3-acetic acid isolated from food waste compost: metabolic pathways, optimization of fermentation conditions, and application.

Food waste is a potential resource to prepare microbial fertilizer. However, functional microorganisms derived from the food waste compost (FWC) are relatively lacking. We have isolated, identified, characterized and optimized a high-yielding indole-3-acetic acid (IAA) strain from FWC and further evaluated its growth promoting effect on plants. A IAA high-yielding strain, Providencia sp.Y, with an initial IAA yield of 139.98 mg L-1, was obtained through high-throughput screening, and identified by 16S rRNA gene sequence. The novel strain Y may simultaneously involve the following three pathways from L-tryptophan to IAA, which were identified using liquid chromatography-tandem mass spectrometry: (1) L-tryptophan-indole-3-ethanol-indole-3-acetaldehyde-indole-3-acetic acid; (2) L-tryptophan-1-hydroxy-indole-3-ethanol-indole-3-acetic acid; (3) L-tryptophan-indole-3-acetamide-indole-3-acetic acid. The most suitable comprehensive conditions for IAA production, which were optimized by single factor experiment, were: culture time 12 h, inoculation amount 2% (v/v), NaCl concentration 4% (w/v), culture temperature 25℃, initial pH = 5, and L-tryptophan concentration 3.0 g L-1. The yield of IAA after optimization was increased by 590.48%, from 139.98 mg L-1 (before optimization) to 966.54 mg L-1. Diluted 200-fold microbial suspension could significantly improve the growth of pakchoi seedlings. The seedling plant height, root length, leaf width, leaf length, and fresh weight with microbial suspension increased by 17.39%, 107.35%, 77.98%, 37.75%, and 215.38%, respectively, compared with those without microbial suspension. The increase was greater than that of commercial bacterial agents. In conclusion, this isolated strain can be used as an economical microbial inoculant and provides a new germplasm resource for developing microbial fertilizers.

A dual bacterial alliance removed erythromycin residues by immobilizing on activated carbon.

Removing erythromycin from the environment is a major challenge. In this study, a dual microbial consortium (Delftia acidovorans ERY-6A and Chryseobacterium indologenes ERY-6B) capable of degrading erythromycin was isolated, and the erythromycin biodegradation products were studied. Coconut shell activated carbon was modified and its adsorption characteristics and erythromycin removal efficiency of the immobilized cells were studied. It was indicated that alkali-modified and water-modified coconut shell activated carbon and the dual bacterial system had excellent erythromycin removal ability. The dual bacterial system follows a new biodegradation pathway to degrade erythromycin. The immobilized cells removed 95% of erythromycin at a concentration of 100 mg L-1 within 24 h through pore adsorption, surface complexation, hydrogen bonding, and biodegradation. This study provides a new erythromycin removal agent and for the first time describes the genomic information of erythromycin-degrading bacteria, providing new clues regarding bacterial cooperation and efficient erythromycin removal.

Reuse of composted food waste from rural China as vermicomposting substrate: effects on earthworms, associated microorganisms, and economic benefits.

ABSTRACTAerobic composting of food waste (FW) from rural China using a composting device results in a substantial financial burden on the government. This study aimed to assess the feasibility of mitigating this cost using vermicomposting of composted FW. The specific aims were to elucidate the effects of composted FW on earthworm growth and reproduction, reveal the changes in the physical and chemical properties of earthworm casts during vermicomposting, identify the microbial community structure associated with vermicomposting, and perform a financial analysis based on the yield of earthworms and earthworm casts. Mixing composted FW and mature cow dung in an equal ratio achieved the highest earthworm reproduction rate, where 100 adult earthworms produced 567 juvenile earthworms and 252 cocoons in 40 d. Earthworms reduce salt content of vermicomposting substrates by assimilating Na+ and promoting humification by transforming humin into humic and fulvic acid, thus producing earthworm casts with a high generation index > 80%. When composted FW was added to a vermicomposting substrate, a distinctive microbial community structure with alkaliphilic, halophilic, and lignocellulolytic microorganisms dominated the microflora. The dominant bacterial species was Saccharopolyspora rectivirgula, and the dominant fungal species changed from Kernia nitida to Coprinopsis scobicola. Furthermore, microbial genes for refractory organic matter and fat degradation were observed in Vibrio cholerae, Kernia nitida, and Coprinopsis scobicola. Financial analysis showed that vermicomposting has the potential to reduce the cost associated with FW disposal from $ 57 to $ 18/t.

Evaluation of a data-driven intelligent waste classification system for scientific management of garbage recycling in a Chinese community.

Waste classification management is effective in addressing the increasing waste output and continuous deterioration of environmental conditions. The waste classification behaviour of resident is an important basis for managers to collect and allocate resources. Traditional analysis methods, such as questionnaire, have limitations considering the complexity of individual behaviour. An intelligent waste classification system (IWCS) was applied and studied in a community for 1 year. Time-based data analysis framework was constructed to describe the residents' waste sorting behaviour and evaluate the IWCS. The results showed that residents preferred to use face recognition than other modes of identification. The ratio of waste delivery frequency was 18.34% in the morning and 81.66% in the evening, respectively. The optimal time windows of disposing wastes were from 6:55 to 9:05 in the morning and from 18:05 to 20:55 in the evening which can avoid crowding. The percentage of accuracy of waste disposal increased gradually in a year. The amount of waste disposal was largest on every Sunday. The average accuracy was more than 94% based on monthly data, but the number of participating residents decreased gradually. Therefore, the study demonstrates that IWCS is a potential platform for increasing the accuracy and efficiency of waste disposal and can promote regulations implementation.

Construction and application of highly efficient waste cooking oil degrading bacteria consortium in oily wastewater.

The treatment of cooking oil wastewater is an urgent issue need to be solved. We aimed to screen for efficient oil-degrading bacteria and develop a new microbial agent for degrading waste cooking oil in oily wastewater. Three extremely effective oil-degrading bacteria, known as YZQ-1, YZQ-3, and YZQ-4, were found by the enrichment and acclimation of samples from various sources and separation using oil degradation plates. The 16S rRNA sequencing analysis and phylogenetic tree construction showed that the three strains were Bacillus tropicus, Pseudomonas multiresinivorans, and Raoultella terrigena. Under optimal degradation conditions, the maximal degradation rates were 67.30 ± 3.69%, 89.65 ± 1.08%, and 79.60 ± 5.30%, respectively, for YZQ-1, YZQ-3, and YZQ-4. Lipase activity was highest for YZQ-3, reaching 94.82 ± 12.89 U/L. The best bacterial alliance was obtained by adding equal numbers of microbial cells from the three strains. Moreover, when this bacterial alliance was applied to oily wastewater, the degradation rate of waste cooking oil was 61.13 ± 7.30% (3.67% ± 2.13% in the control group), and COD removal was 62.4% ± 5.65% (55.60% ± 0.71% in the control group) in 72 h. Microbial community analysis results showed YZQ-1 and YZQ-3 were adaptable to wastewater and could coexist with local bacteria, whereas YZQ-4 could not survive in wastewater. Therefore, the combination of YZQ-1 and YZQ-3 can efficiently degrade oil and shows great potential for oily wastewater treatment.

Increase in furfural tolerance in ethanologenic Escherichia coli LY180 by plasmid-based expression of thyA.

Furfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilis YB886, Escherichia coli NC3, and Zymomonas mobilis CP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing the thyA gene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in the de novo pathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression of thyA was no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA in E. coli and to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA in E. coli would be expected to increase the cellular requirement for dTMP. Increased expression of thyA (E. coli, B. subtilis, or Z. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.

High-Energy-Density Organic Amendments Enhance Soil Health.

Soil microbial biomass (SMB) and soil microbial communities (SMCs) are the key factors in soil health and agricultural sustainability. We hypothesized that low bioavailable carbon (C) and energy were the key limiting factors influencing soil microbial growth and developed a new fertilization system to address this: the simultaneous application of mineral fertilizers and high-energy-density organic amendments (HED-OAs). A microcosm soil incubation experiment and a Brassica rapa subsp. chinensis pot culture experiment were used to test the effects of this new system. Compared to mineral fertilizer application alone, the simultaneous input of fertilizers and vegetable oil (SIFVO) achieved a bacterial abundance, fungal abundance, and fungal:bacterial ratio that were two orders of magnitude higher, significantly higher organic C and nitrogen (N) content, significantly lower N loss, and nearly net-zero N2O emissions. We proposed an energy and nutrient threshold theory to explain the observed bacterial and fungal growth characteristics, challenging the previously established C:N ratio determination theory. Furthermore, SIFVO led to microbial community improvements (an increased fungal:bacterial ratio, enriched rhizosphere bacteria and fungi, and reduced N-transformation bacteria) that were beneficial for agricultural sustainability. A low vegetable oil rate (5 g/kg) significantly promoted Brassica rapa subsp. chinensis growth and decreased the shoot N content by 35%, while a high rate caused severe N deficiency and significantly inhibited growth of the crop, confirming the exceptionally high microbial abundance and indicating severe microbe-crop competition for nutrients in the soil.

Efficient degradation of tylosin by Klebsiella oxytoca TYL-T1.

Tylosin is widely used in livestock; however, the release of tylosin through animal manure can cause serious environmental problems. In this study, a new tylosin-degrading strain, TYL-T1, was isolated. Its phylogenetic similarity to Klebsiella oxytoca was found to be 99.17 %. TYL-T1 maintained good growth at 40 °C over a broad pH range (4.0-10). TYL-T1 degraded 99.34 % of tylosin in 36 h under optimal conditions (tylosin initial concentration: 25 mg/L, pH: 7.0, and temperature: 35 °C). After LC-MS-MS analysis, a new degradation pathway for tylosin was proposed, including ester bond breaking of the macrolide lactone ring, redox reaction, and loss of mycinose and mycarose. Based on a transcriptome analysis, 164 genes essential for degradation were upregulated through hydrolysis and redox of tylosin. Among various transferases, lipopolysaccharide methyltransferase, glycogen glucosyltransferase, and fructotransferase were responsible for tylosin degradation. The present study revealed the degradation mechanism of tylosin and highlighted the potential of Klebsiella oxytoca TYL-T1 to remove tylosin from the environment.