CRISPR/Cas12a-Mediated Genome Editing in Thioalkalivibrio versutus.

Haloalkaliphilic Thioalkalivibrio versutus, a dominant species for sulfide removal, has attracted increasing attention. However, research on T. versutus is limited by the lack of genetic manipulation tools. In this work, we developed a CRISPR/AsCas12a-mediated system in T. versutus for an efficient and implementable genome editing workflow. Compared to the CRISPR/Cas9-mediated system, the CRISPR/AsCas12a system exhibited enhanced editing efficiency. Additionally, as Cas12a is capable of processing the crRNA maturation independently, the CRISPR/AsCas12a system allowed multiplex gene editing and large-fragment DNA knockout by expressing more than one crRNA under the control of one promoter. Using the CRISPR/AsCas12a system, five key genes of the elemental sulfur oxidation pathway were knocked out. Simultaneous deletion of the rhd and tusA genes disrupted the ability of T. versutus to metabolize elemental sulfur, resulting in a 24.7% increase in elemental sulfur generation and a 15.2% reduction in sulfate production. This genome engineering strategy significantly improved our understanding of sulfur metabolism in Thioalkalivibrio spp.

Organic layer characteristics and microbial utilization of the biosulfur globules produced by haloalkaliphilic Thioalkalivibrio versutus D301 during biological desulfurization.

The haloalkaliphilic genus Thioalkalivibrio, widely used in bio-desulfurization, can oxidize H2S to So, which is excreted outside cells in the form of biosulfur globules. As by-product of bio-desulfurization, information on biosulfur globules is still very scant, which limits its high-value utilization. In this paper, the characteristics of biosulfur globules produced by Thioalkalivibrio versutus D301 and the possibility of cultivating sulfur-oxidizing bacteria as a high biological-activity sulfur source were studied. The sulfur element in the biosulfur globules existed in the form α-S8, which was similar to chemical sulfur. The biosulfur globule was wrapped with an organic layer composed of polysaccharides and proteins. The composition of this organic layer could change. In the formation stage of biosulfur globules, the organic layer was dominated by polysaccharides, and in later stage, proteins became the main component. We speculated that the organic layer was mainly formed by the passive adsorption of organic matter secreted by cells. The existence of organic layer endowed biosulfur with better bioavailability. Compared with those found using chemical sulfur, the growth rates of Acidithiobacillus thiooxidans ATCC 19377T, Thiomicrospira microaerophila BDL05 and Thioalkalibacter halophilus BDH06 using biosulfur increased several folds to an order of magnitude, indicating that biosulfur was a good sulfur source for cultivating sulfur-oxidizing bacteria.

Enhanced Biodesulfurization with a Microbubble Strategy in an Airlift Bioreactor with Haloalkaliphilic Bacterium Thioalkalivibrio versutus D306.

Biodesulfurization under haloalkaline conditions requires limiting oxygen and additional energy in the system to deliver high mixing quality control. This study considers biodesulfurization in an airlift bioreactor with uniform microbubbles generated by a fluidic oscillation aeration system to enhance the biological desulfurization process and its hydrodynamics. Fluidic oscillation aeration in an airlift bioreactor requires minimal energy input for microbubble generation. This aeration system produced 81.87% smaller average microbubble size than the direct aeration system in a bubble column bioreactor. The biodesulfurization phase achieved a yield of 94.94% biological sulfur, 84.91% biological sulfur selectivity, and 5.06% sulfur oxidation performance in the airlift bioreactor with the microbubble strategy. The biodesulfurization conditions of thiosulfate via Thioalkalivibrio versutus D306 are revealed in this study. The biodesulfurization conditions in the airlift bioreactor with the fluidic oscillation aeration system resulted in the complete conversion of thiosulfate with 27.64% less sulfate production and 10.34% more biological sulfur production than in the bubble column bioreactor. Therefore, pleasant hydrodynamics via an airlift bioreactor mechanism with microbubbles is favored for biodesulfurization under haloalkaline conditions.

Composition and key-influencing factors of bacterial communities active in sulfur cycling of soda lake sediments.

Bacteria are important participants in sulfur cycle of the extremely haloalkaline environment, e.g. soda lake. The effects of physicochemical factors on the composition of sulfide-oxidizing bacteria (SOB) and sulfate-reducing bacteria (SRB) in soda lake have remained elusive. Here, we surveyed the community structure of total bacteria, SOB and SRB based on 16S rRNA, soxB and dsrB gene sequencing, respectively, in five soda lakes with different physicochemical factors. The results showed that the dominant bacteria belonged to the phyla Proteobacteria, Bacteroidetes, Halanaerobiaeota, Firmicutes and Actinobacteria. SOB and SRB were widely distributed in lakes with different physicochemical characteristics, and the community composition were different. In general, salinity and inorganic nitrogen sources (NH4+-N, NO3--N) were the most significant factors. Specifically, the communities of SOB, mainly including Thioalkalivibrio, Burkholderia, Paracoccus, Bradyrhizobium, and Hydrogenophaga genera, were remarkably influenced by the levels of NH4+-N and salinity. Yet, for SRB communities, including Desulfurivibrio, Candidatus Electrothrix, Desulfonatronospira, Desulfonatronum, Desulfonatronovibrio, Desulfonatronobacter and so on, the most significant determinants were salinity and NO3--N. Besides, Rhodoplanes played a significant role in the interaction between SOB and SRB. From our results, the knowledge regarding the community structures of SOB and SRB in extremely haloalkaline environment was extended.

Recent advances in microbial capture of hydrogen sulfide from sour gas via sulfur-oxidizing bacteria.

Biological desulfurization offers several remarkably environmental advantages of operation at ambient temperature and atmospheric pressure, no demand of toxic chemicals as well as the formation of biologically re-usable sulfur (S0), which has attracted increasing attention compared to conventionally physicochemical approaches in removing hydrogen sulfide from sour gas. However, the low biomass of SOB, the acidification of process solution, the recovery of SOB, and the selectivity of bio-S0 limit its industrial application. Therefore, more efforts should be made in the improvement of the BDS process for its industrial application via different research perspectives. This review summarized the recent research advances in the microbial capture of hydrogen sulfide from sour gas based on strain modification, absorption enhancement, and bioreactor modification. Several efficient solutions to limitations for the BDS process were proposed, which paved the way for the future development of BDS industrialization.

Revealing sulfate role in empowering the sulfur-oxidizing capacity of Thioalkalivibrio versutus D301 for an enhanced desulfurization process.

Haloalkaliphilic Thioalkalivibrio, a dominant genus for sulfide removal, has attracted growing interest. However, the bacterial biological response to this process's final product, sulfate, has not been well-studied. Here, thiosulfate oxidation and sulfur formation by T. versutus D301 were being enhanced with increasing sulfate supply. With the addition of 0.73 M sulfate, the thiosulfate utilization rate and sulfur production were improved by 68.1% and 120.1% compared with carbonate-grown control at the same salinity (1.8 M). For sulfate-grown cells, based on metabolic analysis, the downregulation of central carbon metabolism indicated that sulfate triggered a decrease in energy conservation efficiency. Additionally, the gene expression analysis further revealed that sulfate induced the inhibition of sulfur to sulfate oxidation, causing the upregulation of thiosulfate to sulfur oxidation for providing cells with additional energy. This study enhances researchers' understanding regarding the sulfate effect on the bio-desulfurization process and presents a new perspective of optimizing the biotechniques.

[Inhibition of biological desulfurization by organosulfur: a review].

As a green and economic emerging technology, biological desulfurization is popular. However, biological desulfurization is inhibited by organosulfur in the treatment gases which cannot be ignored. This article summarizes relevant studies on the influence of organosulfur on biological desulfurization in recent years, including the types and physicochemical characteristics of organosulfur, the influence of organosulfur on the desulfurization process, the reaction mechanism of organosulfur, the interplay between organosulfur and some operating conditions, and species of microorganisms that are tolerant to organosulfur. Methods for mitigating the effect of organosulfur on the desulfurization process are discussed, to provide references for the stable and efficient operation of biological desulfurization.

Transcriptional response of Thialkalivibrio versutus D301 to different sulfur sources and identification of the sulfur oxidation pathways.

The genus Thialkalivibrio plays an essential role in the biological desulfurization system. However, to date, the sulfur oxidation pathways of Thialkalivibrio are not clearly understood. Here, we performed transcriptomic analysis on Thialkalivibrio versutus D301 with either thiosulfate or chemical sulfur as the sulfur source to understand it. The results show that T. versutus D301 has a higher growth rate and sulfur oxidation activity when thiosulfate is utilized. The use of chemical sulfur as sulfur source leads to decreased expression of genes involved in carbon metabolism, ribosome synthesis and oxidative phosphorylation in T. versutus D301. Potentially due to the adsorption to sulfur particles, the genes related to flagellum assembly and motivation are significantly induced in T. versutus D301 in the presence of chemical sulfur. In the periplasm, both thiosulfate and polysulfide from the chemical sulfur are oxidized to sulfate via the similar truncated Sox system (SoxAXYZB). Then, part of polysulfide reached to cytoplasm through an unidentified route is oxidized to sulfite by the Dsr-like system. The sulfite in the cytoplasm is further catalyzed to sulfate by SoxB or SoeABC. Overall, the difference in the oxidation rates of D301 can be mainly attributed to the bioavailability of the two sulfur sources, not the sulfur oxidation pathways.

[Determination of sulfur compounds in biological desulfurization system by high performance liquid chromatography].

Biological desulfurization is a process in which sulfur compounds are removed from gas and oil using microorganisms. It is a simple process that has mild operating conditions, high desulfurization efficiency, low energy consumption and less environmental pollution. However, there is still a lack of simple and efficient analytical methods for quantitatively analyzing the sulfur compounds in the biological desulfurization process. In order to solve this problem, the analytical method for the simultaneous determination of sulfite, thiosulfate and sulfide in biological desulfurization solutions by pre-column fluorescence derivation using high performance liquid chromatography (HPLC) was developed. The standard curves of sulfur species in this analytical method had good linear relationships with correlation coefficients of 0.999 5, 0.999 7, and 0.999 7 for sulfite, thiosulfate and sulfide, respectively. The detection limits of these sulfur compounds were 0.000 6, 0.000 7 and 0.001 1 µmol/L; the range of recovery rates were 98.17 to 101.9%, 100.9 to 102.6%, and 101.1 to 104.2%; which had good repeatability and stability. The analytical method was simple, efficient and accurate, and could be used to simultaneously determine the sulfur compounds in different biological desulfurization systems.

Recent advances in biocatalysts engineering for polyethylene terephthalate plastic waste green recycling.

The massive waste of poly(ethylene terephthalate) (PET) that ends up in the landfills and oceans and needs hundreds of years for degradation has attracted global concern. The poor stability and productivity of the available PET biocatalysts hinder their industrial applications. Active PET biocatalysts can provide a promising avenue for PET bioconversion and recycling. Therefore, there is an urgent need to develop new strategies that could enhance the stability, catalytic activity, solubility, productivity, and re-usability of these PET biocatalysts under harsh conditions such as high temperatures, pH, and salinity. This has raised great attention in using bioengineering strategies to improve PET biocatalysts' robustness and catalytic behavior. Herein, historical and forecasting data of plastic production and disposal were critically reviewed. Challenges facing the PET degradation process and available strategies that could be used to solve them were critically highlighted and summarized. In this review, we also discussed the recent progress in enzyme bioengineering approaches used for discovering new PET biocatalysts, elucidating the degradation mechanism, and improving the catalytic performance, solubility, and productivity, critically assess their strength and weakness and highlighting the gaps of the available data. Discovery of more potential PET hydrolases and studying their molecular mechanism extensively via solving their crystal structure will widen this research area to move forward the industrial application. A deeper knowledge of PET molecular and degradation mechanisms will give great insight into the future identification of related enzymes. The reported bioengineering strategies during this review could be used to reduce PET crystallinity and to increase the operational temperature of PET hydrolyzing enzymes.

Improving confirmed nanometric sulfur bioproduction using engineered Thioalkalivibrio versutus.

Complicated production procedures and superior characteristics of nano-sized sulfur elevate its price to 25-40 fold higher than micrograde kind. Also, natural gas hydrogen sulfide levels are restricted because of its toxic environmental consequences. Thioalkalivibrio versutus is a polyextremophilic industrial autotroph with high natural gas desulfurization capability. Here, nanometric (>50 nm) sulfur bioproduction using T. versutus while desulfurizing natural gas was validated. Also, this production was enhanced by 166.7% via lowering sulfate production by 55.1%. A specially-developed CRISPR system, with 42% editing efficiency, simplified the genome editing workflow scheme for this challenging bacterium. In parallel, sulfur metabolism was uncovered using proteins mining and transcriptome studies for defining sulfate-producing key genes (heterodisulfide reductase-like complex, sulfur dioxygenase, sulfite dehydrogenase and sulfite oxidase). This study provided cost-effective nanometric sulfur production and improved this production using a novel CRISPR strategy, which could be suitable for industrial polyextremophiles, after uncovering sulfur pathways in T. versutus.

Deep and high-efficiency removal of sulfate through a coupling system with sulfate-reducing and sulfur-oxidizing capacity under haloalkaliphilic condition.

Sulfide from anaerobic treatment of high-sulfate wastewater would always have some adverse effects on downstream processes. In this study, a coupling anaerobic/aerobic system was developed and operated under haloalkaliphilic condition to realize deep and high-efficiency removal of sulfate without production of sulfide. A haloalkaliphilic sulfur-oxidizing strain, Thioalkalivibrio versutus SOB306, was responsible for oxidation of sulfide. The anaerobic part was first operated at optimum condition based on a previous study. Then, its effluent with an average sulfide concentration of 674 ± 33 mg·l-1 was further directly treated by a set of 1 l biofilter with SOB306 strain under aerobic condition. Finally, 100% removal rate of sulfide was achieved at aeration rate of 0.75 l·l-1·min-1, ORP of - 392 mV and HRT of 4 h. The average yield of elemental sulfur reached 79.1 ± 1.3% in the filter, and the CROS achieved a conversion rate of sulfate to sulfur beyond 54%. This study for the first time revealed the characteristics and performance of the haloalkaliphilic CROS in deep treatment of high-sulfate wastewater, which paved the way for the development and application of this method in the real world.

Effective production of succinic acid from coconut water (Cocos nucifera) by metabolically engineered Escherichia coli with overexpression of Bacillus subtilis pyruvate carboxylase.

Succinic acid is an important acid which is used in medicine and pharmaceutical companies. Metabolically engineered Escherichia coli strain was used for the effective production of succinic acid using Cocos nucifera water, which contained 5.00 ± 0.02 g/L glucose, 6.10 ± 0.01 g /L fructose and 6.70 ± 0.02 g /L sucrose. Fermentation of C. nucifera water with E. coli M6PM produced a final concentration of 11.78 ± 0.02 g/L succinic acid and yield of 1.23 ± 0.01 mol/mol, 0.66 ± 0.01 g/g total sugars after 72 h dual-phase fermentation in M9 medium while modeled sugar was 0.38 ± 0.02 mol/mol total sugars. It resulted in 72% of the maximum theoretical yield of succinic acid. Here we show that novel substrate of C. nucifera water resulted in effective production of succinic acid. These investigations unveil the importance of C. nucifera water as a substrate for the production of biochemicals.

Enhanced growth-driven stepwise inducible expression system development in haloalkaliphilic desulfurizing Thioalkalivibrio versutus.

Highly toxic and flammable H2S gas has become an environmental threat. Because of its ability to efficiently remove H2S by oxidation, Thioalkalivibrio versutus is gaining more attention. Haloalkaliphilic autotrophs, like the bio-desulfurizing T. versutus, grow weakly. Weak growth makes any trial for developing potent genetic tools required for genetic engineering far from achieved. In this study, the fed-batch strategy improved T. versutus growth by 1.6 fold in maximal growth rate, 9-fold in O.D600 values and about 3-fold in biomass and protein productions. The strategy also increased the favorable desulfurization product, sulfur, by 2.7 fold in percent yield and 1.5-fold in diameter. A tight iron-inducible expression system for T. versutus was successfully developed. The system was derived from fed-batch cultivation coupled with new design, build, test and validate (DPTV) approach. The inducible system was validated by toxin expression. Fed-batch cultivation coupled with DPTV approach could be applied to other autotrophs.

Enhanced production of succinic acid from methanol-organosolv pretreated Strophanthus preussii by recombinant Escherichia coli.

A biorefinery process for high yield production of succinic acid from biomass sugars was investigated using recombinant Escherichia coli. The major problem been addressed is utilization of waste biomass for the production of succinic acid using metabolic engineering strategy. Here, methanol extract of Strophanthus preussii was used for fermentation. The process parameters were optimized. Glucose (9 g/L), galactose (4 g/L), xylose (6 g/L) and arabinose (0.5 g/L) were the major sugars present in the methanol extract of S. preussii. E. coli K3OS with overexpression of soluble nucleotide pyridine transhydrogenase sthA and mutation of lactate dehydrogenase A (ldhA), phosphotransacetylase acetate kinase A (pta-ackA), pyruvate formate lyase B (pflB), pyruvate oxidase B (poxB), produced a final succinic acid concentration of 14.40 g/L and yield of 1.10 mol/mol total sugars after 72 h dual-phase fermentation in M9 medium. Here, we show that the maximum theoretical yield using methanol extracts of S. preussii was 64%. Hence, methanol extract of S. preussii could be used for the production of biochemicals such as succinate, malate and pyruvate.

Switch on a more efficient pyruvate synthesis pathway based on transcriptome analysis and metabolic evolution.

Due to the decrease of intracellular NADH availability, gluconate metabolism is more conducive to pyruvate production than glucose. Transcriptome analysis revealed that the Entner-Doudoroff (ED) pathway was activated by gluconate in Escherichia coli YP211 (MG1655 ΔldhA ΔpflB Δpta-ackA ΔpoxB Δppc ΔfrdBC). To construct a new pyruvate producing strain with glucose metabolism via ED pathway, the genes ppsA, ptsG, pgi and gnd were deleted sequentially to reduce the demand for PEP and block the Embden-Meyerhor-Parnas pathway and Pentose-Phosphate pathway. After nearly 1000 generations of growth-based selection, the evolved strain YP404 was isolated and the ED pathway was proved to be activated as the primary glycolytic pathway. Comparing with YP211, the pyruvate concentration and yield increased by 59% and 10.1%, respectively. In fed-batch fermentation, the pyruvate concentration reached 83.5 g l-1 with a volumetric productivity of 2.3 g l-1 h-1. This was the first time to produce pyruvate via ED pathway, and prove that this was a more effective way.

Desulfurization with Thialkalivibrio versutus immobilized on magnetic nanoparticles modified with 3-aminopropyltriethoxysilane.

OBJECTIVE: Thialkalivibrio versutus D301 cells were immobilized on Fe3O4 nanoparticles (NPs) synthesized by an improved chemical coprecipitation method and modified with 3-aminopropyltriethoxysilane (APTES), then the immobilized cells were used in sulfur oxidation. RESULTS: The prepared Fe3O4-APTES NPs had a narrow size distribution (10 ± 2 nm) and were superparamagnetic, with a saturation magnetization of 60.69 emu/g. Immobilized cells had a saturation magnetization of 34.95 emu/g and retained superparamagnetism. The optimum conditions for cell immobilization were obtained at pH 9.5 and 1 M Na+. The immobilization capacity of Fe3O4-APTES NPs was 7.15 g DCW/g-NPs that was 2.3-fold higher than that of Fe3O4 NPs. The desulfurization efficiency of the immobilized cells was close to 100%, having the same sulfur oxidation capacity as free cells. Further, the immobilized cells could be reused at least eight times, retaining more than 85% of their desulfurization efficiency. CONCLUSION: Immobilization of cells with the modified magnetic NPs efficiently increased cell controllability, have no effect on their desulfurization activity and could be effectively used in large-scale industrial applications.

Improvement of desulfurizing activity of haloalkaliphilic Thialkalivibrio versutus SOB306 with the expression of Vitreoscilla hemoglobin gene.

OBJECTIVE: To construct efficient transformation and expression system and further improve desulfurizing activity of cells through expression of Vitreoscilla hemoglobin (VHb) in haloalkaliphilic Thialkalivibrio versutus SOB306. RESULTS: We transferred plasmids pKT230 and pBBR-smr into T. versutus SOB306 via a conjugation method. We identified four promoters from among several predicted promoters by scoring for streptomycin resistance, and finally selected tac and p3 based on the efficiency of expression of red fluorescent protein (RFP). Expression of RFP when regulated by tac was more than three times that of p3 in SOB306. Further, we expressed VHb under the control of tac promoter in SOB306. Expression of VHb was verified using CO-difference spectra. The results showed that VHb expression can boost sulfur metabolism, as evidenced by an increase of about 11.7 ± 1.8% in the average rate of thiosulfate removal in the presence of VHb. CONCLUSION: A conjugation transfer and an expression system for Thialkalivibrio, has been developed for the first time and used for expression of VHb to improve desulfurizing activity.

Efficient production of succinic acid from Palmaria palmata hydrolysate by metabolically engineered Escherichia coli.

Succinic acid, a C4 dicarboxylic acid is used in many fields such as food, agriculture, pharmaceutical and polymer industries. In this study, microbial production of succinic acid from Palmaria palmata was investigated for the first time. In engineered Escherichia coli KLPPP, lactate dehydrogenase, pyruvate formate lyase, phosphotransacetylase-acetate kinase and pyruvate oxidase genes were deleted while phosphoenolpyruvate carboxykinase was overexpressed. The recombinant exhibited higher molar yield of succinic acid on galactose (1.20±0.02mol/mol) than glucose (0.48±0.03mol/mol). The concentration and molar yield of succinic acid were 22.40±0.12g/L and 1.13±0.02mol/mol total sugar respectively after 72h dual phase fermentation from P. palmata hydrolysate which composed of glucose (12.57±0.17g/L) and galactose (18.03±0.10g/L). The results demonstrate that P. palmata red macroalgae biomass represents a novel and an economically alternative feedstock for biochemicals production.

Complete genome sequence of Thialkalivibrio versutus D301 isolated from Soda Lake in northern China, a typical strain with great ability to oxidize sulfide.

Thioalkalivibrio versutus D301 isolated from Soda Lake is a haloalkaliphilic and obligated chemolithoautotrophic Gram-negative bacterium. The strain has a good adaption to hyperhaline and highly alkaline environment and a powerful sulfur-oxidizing ability. Here, we present the complete genome sequence of T. versutus D301, providing insights into the genomic basis of its effects and facilitating its application in microbial desulfurization.