A feasible approach for azo-dye (methyl orange) degradation by textile effluent isolate Serratia marcescens ED1 strain for water sustainability: AST identification, degradation optimization and pathway hypothesis.

Methyl orange (MO) is a dye commonly used in the textile industry that harms aquatic life, soil and human health due to its potential as an environmental pollutant. The present study describes the dye degradation ability of Serratia marcescens strain ED1 isolated from textile effluent and characterized by 16S rRNA gene sequence analysis. The laccase property of bacterial isolate was confirmed qualitatively. The effects of various factors (pH, temperature, incubation time, and dye concentration) were evaluated using Response Surface Methodology (RSM). The maximum dye (MO) degradation was 81.02 % achieved at 37 °C temperature and 7.0 pH with 200 mg/L dye concentration after 48 h of incubation. The beef extract, ammonium nitrate and fructose supplementation showed better response during bioremediation among the different carbon and nitrogen sources. The degree of pathogenicity was confirmed through the simple plate-based method, and an antibiotic resistance profile was used to check the low-risk rate of antibiotic resistance. However, the fate and extinct of degraded MO products were analysed through UV-Vis spectroscopy, FT-IR, and GC-MS analysis to confirm the biodegradation potential of the bacterial strain ED1 and intermediate metabolites were identified to propose metabolic pathway. The phytotoxicity study on Vigna radiata L. seeds confirmed nontoxic effect of degraded MO metabolites and indicates promising degradation potential of S. marcescens strain ED1 to successfully remediate MO dye ecologically sustainably.

Copolymers as a turning point for large scale polyhydroxyalkanoates applications.

Traditional plastics reshaped the society thanks to their brilliant properties and cut-price manufacturing costs. However, their protracted durability and limited recycling threaten the environment. Worthy alternatives seem to be polyhydroxyalkanoates, compostable biopolymers produced by several microbes. The most common 3-hydroxybutyrate homopolymer has limited applications calling for copolymers biosynthesis to enhance material properties. As a growing number of researches assess the discovery of novel comonomers, great endeavors are dedicated as well to copolymers production scale-up, where the choice of the microbial carbon source significantly affects the overall economic feasibility. Diving into novel metabolic pathways, engineered strains, and cutting-edge bioprocess strategies, this review aims to survey up-to-date publications about copolymers production, focusing primarily on precursors origins. Specifically, in the core of the review, copolymers precursors have been divided into three categories based on their economic value: the costliest structurally related ones, the structurally unrelated ones, and finally various low-cost waste streams. The combination of cheap biomasses, efficient pretreatment strategies, and robust microorganisms paths the way towards the development of versatile and circular polymers. Conceived to researchers and industries interested in tackling polyhydroxyalkanoates production, this review explores an angle often underestimated yet of prime importance: if PHAs copolymers offer advanced properties and sustainable end-of-life, the feedstock choice for their upstream becomes a major factor in the development of plastic substitutes.

Effects of bagasse as a carbon source on biofloc formation, water quality, and microbial community structure in shrimp culture system.

As a widely available, low-cost agricultural byproduct, bagasse is a potential solid carbon source and provides microbial attachment as a biofilm carrier. In this study, the effects of bagasse as a carbon source on biofloc formation, water quality, microbial community structure, and nitrogen conversion in a shrimp culture system were explored, and the performance of bagasse bioflocs was assessed. No bagasse was added to the control group (CK), and three bagasse addition groups were set up, with the floc content of the water maintained at 5 mL/L (BF5 group), 10 mL/L (BF10 group), and 15 mL/L (BF15 group). The results showed that bagasse bioflocs formed in the fourth week when bagasse was placed in the culture water, and the surface of bagasse was covered with thick biofilm at that time. The DOC content of the BF15 group was significantly greater than that of the CK group, from 30.31 to 105.06% (P < 0.05), and the DOC increased with increasing bagasse biofloc content. The BF group rapidly converted TAN to NO2--N and then to NO3--N because the accumulation of nitrite nitrogen in the BF15 group occurred 1 week earlier than in the other groups; at the 8th week, the nitrite nitrogen conversion rate of each BF group was close to 100%, which was significantly greater than that of the CK group (P < 0.05). The relative abundances of genes encoding microbial glutamate dehydrogenase and glutamate synthase increased in the bagasse biofloc groups (P < 0.05). The relative abundances of genes from Rhodobacterales and Hyphomicrobiales in each group were greater, but bagasse bioflocs increased the proportion of Hyphomicrobiale. In summary, adding bagasse to the shrimp culture system can form a biofloc system, resulting in the formation of a rich bacterial biofilm on its surface. Bagasse addition not only affects the composition of microbial communities but also accelerates the nitrification process in water. As a result, ammonia and nitrite are converted into nitrate, which is essential for maintaining the stability of the ecosystem balance in aquaculture water.

Mixotrophic denitrification of waste activated sludge fermentation liquid as an alternative carbon source for nitrogen removal: Reducing N2O emissions and costs.

Heterotrophic-sulfur autotrophic denitrification (HAD) has been proposed to be a prospective nitrogen removal process. In this work, the potential of fermentation liquid (FL) from waste-activated sludge (WAS) as the electron donor for denitrification in the HAD system was explored and compared with other conventional carbon sources. Results showed that when FL was used as a carbon source, over 99% of NO3--N was removed and its removal rate exceeded 14.00 mg N/g MLSS/h, which was significantly higher than that of methanol and propionic acid. The produced sulfate was below the limit value and the emission of N2O was low (1.38% of the NO3--N). Microbial community analysis showed that autotrophic denitrifiers were predominated in the HAD system, in which Thiobacillus (16.4%) was the dominant genus. The economic analysis showed the cost of the FL was 0.062 €/m3, which was 30% lower than that in the group dosed with methanol. Our results demonstrated the FL was a promising carbon source for the HAD system, which could reduce carbon emission and cost, and offer a creative approach for waste-activated sludge resource reuse.

Engineering carbon source division of labor for efficient α-carotene production in Corynebacterium glutamicum.

Effective utilization of glucose, xylose, and acetate, common carbon sources in lignocellulose hydrolysate, can boost biomanufacturing economics. However, carbon leaks into biomass biosynthesis pathways instead of the intended target product remain to be optimized. This study aimed to enhance α-carotene production by optimizing glucose, xylose, and acetate utilization in a high-efficiency Corynebacterium glutamicum cell factory. Heterologous xylose pathway expression in C. glutamicum resulted in strain m4, exhibiting a two-fold increase in α-carotene production from xylose compared to glucose. Xylose utilization was found to boost the biosynthesis of pyruvate and acetyl-CoA, essential precursors for carotenoid biosynthesis. Additionally, metabolic engineering including pck, pyc, ppc, and aceE deletion, completely disrupted the metabolic connection between glycolysis and the TCA cycle, further enhancing α-carotene production. This strategic intervention directed glucose and xylose primarily towards target chemical production, while acetate supplied essential metabolites for cell growth recovery. The engineered strain C. glutamicum m8 achieved 30 mg/g α-carotene, 67% higher than strain m4. In fed-batch fermentation, strain m8 produced 1802 mg/L of α-carotene, marking the highest titer reported to date in microbial fermentation. Moreover, it exhibited excellent performance in authentic lignocellulosic hydrolysate, producing 216 mg/L α-carotene, 1.45 times higher than the initial strain (m4). These labor-division strategies significantly contribute to the development of clean processes for producing various valuable chemicals from lignocellulosic resources.

Metabolic engineering of Pseudomonas bharatica CSV86T to degrade Carbaryl (1-naphthyl-N-methylcarbamate) via the salicylate-catechol route.

Pseudomonas bharatica CSV86T displays the unique property of preferential utilization of aromatic compounds over simple carbon sources like glucose and glycerol and their co-metabolism with organic acids. Well-characterized growth conditions, aromatic compound metabolic pathways and their regulation, genome sequence, and advantageous eco-physiological traits (indole acetic acid production, alginate production, fusaric acid resistance, organic sulfur utilization, and siderophore production) make it an ideal host for metabolic engineering. Strain CSV86T was engineered for Carbaryl (1-naphthyl-N-methylcarbamate) degradation via salicylate-catechol route by expression of a Carbaryl hydrolase (CH) and a 1-naphthol 2-hydroxylase (1NH). Additionally, the engineered strain exhibited faster growth on Carbaryl upon expression of the McbT protein (encoded by the mcbT gene, a part of Carbaryl degradation upper operon of Pseudomonas sp. C5pp). Bioinformatic analyses predict McbT to be an outer membrane protein, and Carbaryl-dependent expression suggests its probable role in Carbaryl uptake. Enzyme activity and protein analyses suggested periplasmic localization of CH (carrying transmembrane domain plus signal peptide sequence at the N-terminus) and 1NH, enabling compartmentalization of the pathway. Enzyme activity, whole-cell oxygen uptake, spent media analyses, and qPCR results suggest that the engineered strain preferentially utilizes Carbaryl over glucose. The plasmid-encoded degradation property was stable for 75-90 generations even in the absence of selection pressure (kanamycin or Carbaryl). These results indicate the utility of P. bharatica CSV86T as a potential host for engineering various aromatic compound degradation pathways.IMPORTANCEThe current study describes engineering of Carbaryl metabolic pathway in Pseudomonas bharatica CSV86T. Carbaryl, a naphthalene-derived carbamate pesticide, is known to act as an endocrine disruptor, mutagen, cytotoxin, and carcinogen. Removal of xenobiotics from the environment using bioremediation faces challenges, such as slow degradation rates, instability of the degradation phenotype, and presence of simple carbon sources in the environment. The engineered CSV86-MEC2 overcomes these disadvantages as Carbaryl was degraded preferentially over glucose. Furthermore, the plasmid-borne degradation phenotype is stable, and presence of glucose and organic acids does not repress Carbaryl metabolism in the strain. The study suggests the role of outer membrane protein McbT in Carbaryl transport. This work highlights the suitability of P. bharatica CSV86T as an ideal host for engineering aromatic pollutant degradation pathways.

PoSnf1 affects cellulose utilization through interaction with cellobiose transporter in Pleurotus ostreatus.

Pleurotus ostreatus is one of the most cultivated edible fungi worldwide, but its lignocellulose utilization efficiency is relatively low (<50 %), which eventually affects the biological efficiency of P. ostreatus. Improving cellulase production and activity will contribute to enhancing the lignocellulose-degrading capacity of P. ostreatus. AMP-activated/Snf1 protein kinase plays important roles in regulating carbon and energy metabolism. The Snf1 homolog (PoSnf1) in P. ostreatus was obtained and analyzed using bioinformatics. The cellulose response of PoSnf1, the effect of the phosphorylation level of PoSnf1 on the expression of cellulose degradation-related genes, the putative proteins that interact with the phosphorylated PoSnf1 (P-PoSnf1), the cellobiose transport function of two sugar transporters (STP1 and STP2), and the interactions between PoSnf1 and STP1/STP2 were studied in this research. We found that cellulose treatment improved the phosphorylation level of PoSnf1, which further affected cellulase activity and the expression of most cellulose degradation-related genes. A total of 1, 024 proteins putatively interacting with P-PoSnf1 were identified, and they were enriched mainly in the substances transport and metabolism. Most of the putative cellulose degradation-related protein-coding genes could respond to cellulose. Among the P-PoSnf1-interacting proteins, the functions of two sugar transporters (STP1 and STP2) were further studied, and the results showed that both could transport cellobiose and were indirectly regulated by P-PoSnf1, and that STP2 could directly interact with PoSnf1. The results of this study indicated that PoSnf1 plays an important role in regulating the expression of cellulose degradation genes possibly by affecting cellobiose transport.

Construction of an enzyme-constrained metabolic network model for Myceliophthora thermophila using machine learning-based kcat data.

BACKGROUND: Genome-scale metabolic models (GEMs) serve as effective tools for understanding cellular phenotypes and predicting engineering targets in the development of industrial strain. Enzyme-constrained genome-scale metabolic models (ecGEMs) have emerged as a valuable advancement, providing more accurate predictions and unveiling new engineering targets compared to models lacking enzyme constraints. In 2022, a stoichiometric GEM, iDL1450, was reconstructed for the industrially significant fungus Myceliophthora thermophila. To enhance the GEM's performance, an ecGEM was developed for M. thermophila in this study. RESULTS: Initially, the model iDL1450 underwent refinement and updates, resulting in a new version named iYW1475. These updates included adjustments to biomass components, correction of gene-protein-reaction (GPR) rules, and a consensus on metabolites. Subsequently, the first ecGEM for M. thermophila was constructed using machine learning-based kcat data predicted by TurNuP within the ECMpy framework. During the construction, three versions of ecGEMs were developed based on three distinct kcat collection methods, namely AutoPACMEN, DLKcat and TurNuP. After comparison, the ecGEM constructed using TurNuP-predicted kcat values performed better in several aspects and was selected as the definitive version of ecGEM for M. thermophila (ecMTM). Comparing ecMTM to iYW1475, the solution space was reduced and the growth simulation results more closely resembled realistic cellular phenotypes. Metabolic adjustment simulated by ecMTM revealed a trade-off between biomass yield and enzyme usage efficiency at varying glucose uptake rates. Notably, hierarchical utilization of five carbon sources derived from plant biomass hydrolysis was accurately captured and explained by ecMTM. Furthermore, based on enzyme cost considerations, ecMTM successfully predicted reported targets for metabolic engineering modification and introduced some new potential targets for chemicals produced in M. thermophila. CONCLUSIONS: In this study, the incorporation of enzyme constraint to iYW1475 not only improved prediction accuracy but also broadened the model's applicability. This research demonstrates the effectiveness of integrating of machine learning-based kcat data in the construction of ecGEMs especially in situations where there is limited measured enzyme kinetic parameters for a specific organism.

Research on the Influence of Carbon Sources and Buffer Layers on the Homogeneous Epitaxial Growth of 4H-SiC.

In this study, a 4H-SiC homoepitaxial layer was grown on a 150 mm 4° off-axis substrate using a horizontal hot wall chemical vapor deposition reactor. Comparing C3H8 and C2H4 as C sources, the sample grown with C2H4 exhibited a slower growth rate and lower doping concentration, but superior uniformity and surface roughness compared to the C3H8-grown sample. Hence, C2H4 is deemed more suitable for commercial epitaxial wafer growth. Increasing growth pressure led to decreased growth rate, worsened thickness uniformity, reduced doping concentration, deteriorated uniformity, and initially improved and then worsened surface roughness. Optimal growth quality was observed at a lower growth pressure of 40 Torr. Furthermore, the impact of buffer layer growth on epitaxial quality varied significantly based on different C/Si ratios, emphasizing the importance of selecting the appropriate conditions for subsequent device manufacturing.

Carbon slow-release and enhanced nitrogen removal performance of plant residue-based composite filler and ecological mechanisms in constructed wetland application.

Stable carbon release and coupled microbial efficacy of external carbon source solid fillers are the keys to enhanced nitrogen removal in constructed wetlands. The constructed wetland plant residue Acorus calamus was cross-linked with poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) to create composite solid carbon source fillers (Ac-BDPs). The study demonstrated the slow release of carbon sources from Ac-BDPs with 35.27 mg/g under an average release rate of 0.88 mg/(g·d). Excellent denitrification was also observed in constructed wetlands with Ac-BDPs. Moreover, the average removal rate of nitrate nitrogen (NO3--N) was increased by 1.94 and 3.85 times of the blank groups under initial NO3--N inputs of 5 and 15 mg/L, respectively. Furthermore, the relatively high abundances of nap, narG, nirKS, norB, qnorZ and nosZ guaranteed efficient denitrification performance in constructed wetlands with Ac-BDPs. The study introduced a reliable technique for biological nitrogen removal by using composite carbon source fillers in constructed wetlands.

Preparation and Performance Verification of a Solid Slow-Release Carbon Source Material for Deep Nitrogen Removal in Urban Tailwater.

Urban tailwater typically has a low carbon-to-nitrogen ratio and adding external carbon sources can effectively improve the denitrification performance of wastewater. However, it is difficult to determine the dosage of additional carbon sources, leading to insufficient or excessive addition. Therefore, it is necessary to prepare solid slow-release carbon source (SRC) materials to solve the difficulty in determining the dosage of carbon sources. This study selected two SRCs of slow-release carbon source 1 (SRC1) and slow-release carbon source 2 (SRC2), with good slow-release performance after static carbon release and batch experiments. The composition of SRC1 was: hydroxypropyl methylcellulose/disodium fumarate/polyhydroxy alkanoate (HPMC/DF/PHA) at a ratio of 3:2:4, with an Fe3O4 mass fraction of 3%. The composition of SRC2 was: HPMC/DF/PHA with a ratio of 1:1:1 and an Fe3O4 mass fraction of 3%. The fitted equations of carbon release curves of SRC1 and SRC2 were y = 61.91 + 7190.24e-0.37t and y = 47.92 + 8770.42e-0.43t, respectively. The surfaces of SRC1 and SRC2 had a loose and porous morphological structure, which could increase the specific surface area of materials and be more conducive to the adhesion and metabolism of microorganisms. The experimental nitrogen removal by denitrification with SRCs showed that when the initial total nitrogen concentration was 40.00 mg/L, the nitrate nitrogen (NO3--N) concentrations of the SRC1 and SRC2 groups on the 10th day were 2.57 and 2.66 mg/L, respectively. On the 20th day, the NO3--N concentrations of the SRC1 and SRC2 groups were 1.67 and 2.16 mg/L, respectively, corresponding to removal efficiencies of 95.83% and 94.60%, respectively. The experimental results indicated that SRCs had a good nitrogen removal effect. Developing these kinds of materials can provide a feasible way to overcome the difficulty in determining the dosage of carbon sources in the process of heterotrophic denitrification.

Insight into the microbial community of denitrification process using different solid carbon sources: Not only bacteria.

There is a lack of understanding about the bacterial, fungal and archaeal communities' composition of solid-phase denitrification (SPD) systems. We investigated four SPD systems with different carbon sources by analyzing microbial gene sequences based on operational taxonomic unit (OTU) and amplicon sequence variant (ASV). The results showed that the corncob-polyvinyl alcohol sodium alginate-polycaprolactone (CPSP, 0.86±0.04 mg NO3--N/(g·day)) and corncob (0.85±0.06 mg NO3--N/(g·day)) had better denitrification efficiency than polycaprolactone (PCL, 0.29±0.11 mg NO3--N/(g·day)) and polyvinyl alcohol-sodium alginate (PVA-SA, 0.24±0.07 mg NO3--N/(g·day)). The bacterial, fungal and archaeal microbial composition was significantly different among carbon source types such as Proteobacteria in PCL (OTU: 83.72%, ASV: 82.49%) and Rozellomycota in PVA-SA (OTU: 71.99%, ASV: 81.30%). ASV methods can read more microbial units than that of OTU and exhibit higher alpha diversity and classify some species that had not been identified by OTU such as Nanoarchaeota phylum, unclassified_ f_ Xanthobacteraceae genus, etc., indicating ASV may be more conducive to understand SPD microbial communities. The co-occurring network showed some correlation between the bacteria fungi and archaea species, indicating different species may collaborate in SPD systems. Similar KEGG function prediction results were obtained in two bioinformatic methods generally and some fungi and archaea functions should not be ignored in SPD systems. These results may be beneficial for understanding microbial communities in SPD systems.

Enhanced biomass production and harvesting efficiency of Chlamydomonas reinhardtii under high-ammonium conditions by powdered oyster shell.

Chlamydomonas reinhardtii prefers ammonium (NH4+) as a nitrogen source, but its late-stage growth under high-NH4+ concentrations (0.5 âˆ¼ 1 g/L) is retarded due to medium acidification. In this study, oyster shell powders were shown to increase the tolerance of C. reinhardtii to NH4+ supplementation at 0.7 g/L in TAP medium in 1-L bubble-column bioreactors, resulting in a 22.9 % increase in biomass production, 62.1 % rise in unsaturated fatty acid accumulation, and 19.2 % improvement in harvesting efficiency. Powdered oyster shell mitigated medium acidification (pH 7.2-7.8) and provided dissolved inorganic carbon up to 8.02 × 103 µmol/L, facilitating a 76.3 % NH4+ consumption, release of up to 189 mg/L of Ca2+, a 42.1 % reduction in ζ-potential and 27.7 % increase in flocculation activity of microalgae cells. This study highlights a promising approach to utilize powdered oyster shell as a liming agent, supplement carbon source, and bio-flocculant for enhancing biomass production and microalgae harvesting in NH4+-rich environments.

Improvement of biotic nitrate reduction in constructed photoautotrophic biofilm-soil microbial fuel cells.

The biotic nitrate reduction rate in freshwater ecosystems is typically constrained by the scarcity of carbon sources. In this study, 'two-chambers' - 'two-electrodes' photoautotrophic biofilm-soil microbial fuel cells (P-SMFC) was developed to accelerate nitrate reduction by activating in situ electron donors that originated from the soil organic carbon (SOC). The nitrate reduction rate of P-SMFC (0.1341 d-1) improved by âˆ¼ 1.6 times on the 28th day compared to the control photoautotrophic biofilm. The relative abundance of electroactive bacterium increased in the P-SMFC and this bacterium contributed to obtain electrons from SOC. Biochar amendment decreased the resistivity of P-SMFC, increased the electron transferring efficiency, and mitigated anodic acidification, which continuously facilitated the thriving of putative electroactive bacterium and promoted current generation. The results from physiological and ecological tests revealed that the cathodic photoautotrophic biofilm produced more extracellular protein, increased the relative abundance of Lachnospiraceae, Magnetospirillaceae, Pseudomonadaceae, and Sphingomonadaceae, and improved the activity of nitrate reductase and ATPase. Correspondingly, P-SMFC in the presence of biochar achieved the highest reaction rate constant for nitrate reduction (kobs) (0.2092 d-1) which was 2.4 times higher than the control photoautotrophic biofilm. This study provided a new strategy to vitalize in situ carbon sources in paddy soil for nitrate reduction by the construction of P-SMFC.

How to develop a bio-based phosphorus mining strategy for eutrophic marine sediments: Unlocking native microbial processes for anaerobic phosphorus release.

This study examined the anaerobic release of phosphorus (P) from two different Baltic Sea sediments (B and F), focusing on the impact of initial concentration of externally introduced waste-derived volatile fatty acids (VFA) as the carbon source, temperature, pH, and mixing conditions. The first batch bioreactor set was operated to demonstrate the effect of VFA on anaerobic P release at different concentrations (1000-10000 mg/L as COD) at 20 °C. A notable P release of up to 15.85 mg/L PO4-P was observed for Sediment B at an initial carbon concentration of 10000 mg COD/L. However, VFA consumption in the bioreactors was minimal or no subsequent. The second batch bioreactor set was carried out to investigate the effect of temperature (20 °C-35 °C), pH (5.5, 7.0 and 8.5) and mixing conditions on P release by introducing lower initial carbon concentration (1000 mg COD/L) considering the potential risk for VFA accumulation in the bioreactors. Maximum P releases of 4.4 mg/L and 3.5 mg/L were for Sediment B and Sediment F, respectively. Two-way ANOVA tests revealed that the operation time and pH and their interactions were statistically significant (p < 0.05) for both sediments while the effect of mixing was not statistically significant. Most of the sulfate was reduced during batch bioreactor operation and Desulfomicobiaceae became dominant among other sulfate-reducing bacteria (SRB) possibly shows the importance of SRB in terms of anaerobic P release. This study gives an insight into future implementations of phosphorus mining from eutrophic environment under anaerobic conditions.

Acidification induce chemical and microbial variation in tea plantation soils and bacterial degradation of the key acidifying phenolic acids.

Camellia sinensis is an important economic plant grown in southern subtropical hilly areas, especially in China, mainly for the production of tea. Soil acidification is a significant cause of the reduction of yield and quality and continuous cropping obstacles in tea plants. Therefore, chemical and microbial properties of tea growing soils were investigated and phenolic acid-degrading bacteria were isolated from a tea plantation. Chemical and ICP-AES investigations showed that the soils tested were acidic, with pH values of 4.05-5.08, and the pH negatively correlated with K (p < 0.01), Al (p < 0.05), Fe and P. Aluminum was the highest (47-584 mg/kg) nonessential element. Based on high-throughput sequencing, a total of 34 phyla and 583 genera were identified in tea plantation soils. Proteobacteria and Acidobacteria were the main dominant phyla and the highest abundance of Acidobacteria was found in three soils, with nearly 22% for the genus Gp2. Based on the functional abundance values, general function predicts the highest abundance, while the abundance of amino acids and carbon transport and metabolism were higher in soils with pH less than 5. According to Biolog Eco Plate™ assay, the soil microorganisms utilized amino acids well, followed by polymers and phenolic acids. Three strains with good phenolic acid degradation rates were obtained, and they were identified as Bacillus thuringiensis B1, Bacillus amyloliquefaciens B2 and Bacillus subtilis B3, respectively. The three strains significantly relieved the inhibition of peanut germination and growth by ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, cinnamic acid, and mixed acids. Combination of the three isolates showed reduced relief of the four phenolic acids due to the antagonist of B2 against B1 and B3. The three phenolic acid degradation strains isolated from acidic soils display potential in improving the acidification and imbalance in soils of C. sinensis.

Xylitol fermentation characteristics with a newly isolated yeast Wickerhamomyces anomalus WA.

Xylitol is an increasingly popular functional food additive, and the newly isolated yeast Wickerhamomyces anomalus WA has shown extensive substrate utilization capability, with the ability to grow on hexose (d-galactose, d-glucose, d-mannose, l-fructose, and d-sorbose) and pentose (d-xylose and l-arabinose) substrates, as well as high tolerance to xylose at concentrations of up to 300 g/L. Optimal xylitol fermentation conditions were achieved at 32 °C, 140 rpm, pH 5.0, and initial cell concentration OD600 of 2.0, with YP (yeast extract 10 g/L, peptone 20 g/L) as the optimal nitrogen source. Xylitol yield increased from 0.61 g/g to 0.91 g/g with an increase in initial substrate concentration from 20 g/L to 180 g/L. Additionally, 20 g/L glycerol was found to be the optimal co-substrate for xylitol fermentation, resulting in an increase in xylitol yield from 0.82 g/g to 0.94 g/g at 140 rpm, enabling complete conversion of xylose to xylitol.

Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection.

BACKGROUND: Most recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors. RESULTS: In micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct. CONCLUSIONS: For the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

Levan Production by Suhomyces kilbournensis Using Sugarcane Molasses as a Carbon Source in Submerged Fermentation.

The valorization of byproducts from the sugarcane industry represents a potential alternative method with a low energy cost for the production of metabolites that are of commercial and industrial interest. The production of exopolysaccharides (EPSs) was carried out using the yeast Suhomyces kilbournensis isolated from agro-industrial sugarcane, and the products and byproducts of this agro-industrial sugarcane were used as carbon sources for their recovery. The effect of pH, temperature, and carbon and nitrogen sources and their concentration in EPS production by submerged fermentation (SmF) was studied in 170 mL glass containers of uniform geometry at 30 °C with an initial pH of 6.5. The resulting EPSs were characterized with Fourier-transform infrared spectroscopy (FT-IR). The results showed that the highest EPS production yields were 4.26 and 44.33 g/L after 6 h of fermentation using sucrose and molasses as carbon sources, respectively. Finally, an FT-IR analysis of the EPSs produced by S. kilbournensis corresponded to levan, corroborating its origin. It is important to mention that this is the first work that reports the production of levan using this yeast. This is relevant because, currently, most studies are focused on the use of recombinant and genetically modified microorganisms; in this scenario, Suhomyces kilbournensis is a native yeast isolated from the sugar production process, giving it a great advantage in the incorporation of carbon sources into their metabolic processes in order to produce levan sucrose, which uses fructose to polymerize levan.

Optimal Carbon Storage During Drought.

Allocation of non-structural carbohydrates (NSC) to storage allows plants to maintain a carbon pool in anticipation of future stress. However, to do so, plants must forego use of the carbon for growth, creating a trade-off between storage and growth. It is possible that plants actively regulate the storage pool to maximise fitness in a stress-prone environment. Here, we attempt to identify the patterns of growth and storage that would result during drought stress under the hypothesis that plants actively regulate carbon storage. We use optimal control theory to calculate the optimal allocation to storage and utilisation of stored carbon over a single drought stress period. We examine two fitness objectives representing alternative life strategies: prioritisation of growth (MaxM) and prioritisation of storage (MaxS), as well as strategies in between these extremes. We find that optimal carbon storage consists of three discrete phases: 'growth', 'storage without growth', and the 'stress' phase where there is no carbon source. This trajectory can be defined by the time point when the plant switches from growth to storage. Growth-prioritising plants switch later and fully deplete their stored carbon over the stress period, while storage-prioritising plants either do not grow or switch early in the drought period. The switch time almost always occurs before soil water is depleted, meaning that growth stops before photosynthesis. We conclude that the common observation of increasing carbon storage during drought could be interpreted as an active process that optimises plant performance during stress.