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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems have been widely applied for gene or genome editing. Adequate checking is important to screen mutants after CRISPR-mediated editing events. Here, we report gene escape cases after the knockout by Type I-F native CRISPR system in Zymomonas mobilis. Through amplifying both the gene of interest and its flanking homologous arms, followed by curing the editing plasmid, we found different destinies for gene-editing events. Some genes were readily knocked out and followed by the easy plasmid curing. In some other cases, however, the editing plasmid was difficult to remove from the cell, or the deleted genes were transferred into the editing plasmid. For example, the targeted region of fur can be integrated into the editing plasmid after the knockout, resulting in a spurious editing event. We supposed that the transfer of the gene may be attributed to bacterial insertion sequences. Searching for literatures on the gene knockout using CRISPR in bacteria reveals that the escape event is likely underestimated due to inadequate validation in other microbes. Hence, several strategies are proposed to enhance gene knockout and plasmid curing.

Lowering cost will prompt the sustainable development of sugarcane‐based ethanol industry. In this work, we developed a low‐cost process for ethanol production from sugarcane by a genetically engineered Zymomonas mobilis. Fermentation media were first optimized, resulting in a 15.54% increase in ethanol fermentation efficiency as compared to control media. To further reduce the byproduct levan formation, a levansucrase‐encoding gene of Z. mobilis, sacB, was deleted through the type I‐F CRISPR‐Cas system, which resulted in a further elevation of both ethanol conversion ratio and productivity comparing with the starting strain ZMS912 (87.50% vs. 76.77%, 1.95 g/L/h vs. 1.71 g/L/h). Moreover, we conducted fed‐batch fermentation for ethanol production using sugarcane juice in 5 L bioreactors and employing the optimized media and engineered strain. The results showed that maximum ethanol titer of 81.59 g/L and productivity of 5.83 g/L/h were achieved. Finally, preliminary techno‐economic assessment demonstrated that our efforts to modify media and strain could reduce the processing cost of ethanol production from sugarcane juice, which provides the feasibility for economic ethanol production in the future.

Microbial biomass and lipid production with mixed-culture of Rhodotorula glutinis and Chlorella vulgaris using acetate as sole carbon source was investigated. Synergistic effect of mixed-culture using 20 g/L acetate significantly promoted cell growth and acetate utilization efficiency. Increasing the proportion of algae in co-culture was beneficial for biomass and lipid accumulation and the optimal ratio of yeast/algae was 1:2. Light exposure further enhanced biomass and lipid titer with 6.9 g/L biomass and 2.6 g/L lipid (38.3% lipid content) obtained in a 5L bioreactor. The results of lipid classes and fatty acid profiles moreover indicated that more neutral lipids and linolenic acid were synthesized in mixed-culture under light exposure condition, suggesting the great potential in applications of biofuels production. This study provided new insight and strategy for economical microbial biomass and lipid production by light-exposed mixed-culture using inexpensive acetate as carbon source.

Pennisetum hydridum has several beneficial characteristics, including the ability to grow quickly, a highly developed root system, the ability to produce high yields, strong resistance to environmental stressors, and the ability to adapt to different growth environments. Recent studies have shown that Pennisetum hydridum has the potential to play an important role in the sectors of bioenergy, environmental remediation, and livestock fodder. The objective of this paper is to review the current research on Pennisetum hydridum in China and discuss its potential for phytoremediation, biofuel development, and feed utilization. Pennisetum hydridum is resistant to a variety of stressors and can adapt to different growth environments. It can be used for the remediation of heavy metal-contaminated land, restoration of saline-alkali land, and nutrient removal of constructed wetlands, as well as soil and water conservation. Pennisetum hydridum exhibits a higher biomass yield, caloric value, and cellulose content than other bioenergy crops, suggesting it has superior potential as a second-generation biofuel resource. As a type of forage grass, Pennisetum hydridum also has an appealing taste and high feeding value, in addition to its high nutrition value. A comprehensive utilization framework for Pennisetum hydridum has been proposed in order to promote high-efficiency utilization of this multipurpose crop and fully realize its maximum potential. Research on Pennisetum hydridum as a bioenergy resource, however, is still in its infancy. Additional work is needed to better understand its breeding and genetic transformation processes, its energy conversion mechanism, and the biological pathways that support its growth and adaptability, so that this valuable resource may be utilized in the most productive way possible.

Background d-Lactic acid played an important role in the establishment of PLA as a substitute for petrochemical plastics. But, so far, the d-lactic acid production was limited in only pilot scale, which was definitely unable to meet the fast growing market demand. To achieve industrial scale d-lactic acid production, the cost-associated problems such as high-cost feedstock, expensive nutrient sources and fermentation technology need to be resolved to establish an economical fermentation process. Results In the present study, the combined effect of B vitamin supplementation and membrane integrated continuous fermentation on d-lactic acid production from agricultural lignocellulosic biomass by Lactobacillus delbrueckii was investigated. The results indicated the specific addition of vitamins B1, B2, B3 and B5 (VB1, VB2, VB3 and VB5) could reduce the yeast extract (YE) addition from 10 to 3 g/l without obvious influence on fermentation efficiency. By employing cell recycling system in 350 h continuous fermentation with B vitamin supplementation, YE addition was further reduced to 0.5 g/l, which resulted in nutrient source cost reduction of 86%. A maximum d-lactate productivity of 18.56 g/l/h and optical purity of 99.5% were achieved and higher than most recent reports. Conclusion These findings suggested the novel fermentation strategy proposed could effectively reduce the production cost and improve fermentation efficiency, thus exhibiting great potential in promoting industrial scale d-lactic acid production from lignocellulosic biomass. Graphical Abstract

BackgroundAcid pretreatment is a common strategy used to break down the hemicellulose component of the lignocellulosic biomass to release pentoses, and a subsequent enzymatic hydrolysis step is usually applied to release hexoses from the cellulose. The hydrolysate after pretreatment and enzymatic hydrolysis containing both hexoses and pentoses can then be used as substrates for biochemical production. However, the acid-pretreated liquor can also be directly used as the substrate for microbial fermentation, which has an acidic pH and contains inhibitory compounds generated during pretreatment. Although the natural ethanologenic bacteriumZymomonas mobiliscan grow in a broad range of pH 3.5 ~ 7.5, cell growth and ethanol fermentation are still affected under acidic-pH conditions below pH 4.0.ResultsIn this study, adaptive laboratory evolution (ALE) strategy was applied to adaptZ. mobilisunder acidic-pH conditions. Two mutant strains named 3.6M and 3.5M with enhanced acidic pH tolerance were selected and confirmed, of which 3.5M grew better than ZM4 but worse than 3.6M in acidic-pH conditions that is served as a reference strain between 3.6M and ZM4 to help unravel the acidic-pH tolerance mechanism. Mutant strains 3.5M and 3.6M exhibited 50 ~ 130% enhancement on growth rate, 4 ~ 9 h reduction on fermentation time to consume glucose, and 20 ~ 63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole-genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic-pH tolerance mechanism of mutant strains. WGR result indicated that compared to wild-type ZM4, 3.5M and 3.6M have seven and five single nucleotide polymorphisms (SNPs), respectively, among which four are shared in common. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of flagellar and mobility related genes would help generate and redistribute cellular energy to resist acidic pH while keeping normal biological processes inZ. mobilis. Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under the acidic-pH condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for acidic-pH resistance. Furthermore, our results demonstrated that in mutant 3.6M, genes encoding F1F0ATPase to pump excess protons out of cells were upregulated under pH 3.8 compared to pH 6.2. This difference might help mutant 3.6M manage acidic conditions better than ZM4 and 3.5M. A few gene targets were then selected for genetics study to explore their role in acidic pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids,ZMO0956–ZMO0958encoding cytochrome bc1 complex andZMO1428–ZMO1432encoding RND efflux pump, could helpZ. mobilistolerate acidic-pH conditions.ConclusionAn acidic-pH-tolerant mutant 3.6M obtained through this study can be used for commercial bioethanol production under acidic fermentation conditions. In addition, the molecular mechanism of acidic pH tolerance ofZ. mobiliswas further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic-pH conditions. Moreover, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq, and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.

Background Pretreatment of lignocellulosic biomass generates different types of inhibitors (e.g., furfural and acetic acid), which could remarkably inhibit subsequent ethanol fermentation. Here, biochar as an additive in the fermentation broth was first applied to enhance ethanol production by Z. mobilis wild-type strain ZM4 in the presence of typical inhibitors. Results This study showed that the biochar-mediated tolerance to furfural and acetic acid for the strain Z. mobilis ZM4 was the highest reported level, resulting in much higher ethanol productivity under stress conditions than that in non-treated conditions. Further analysis showed that adsorptive detoxification was not the controlling factor for enhanced ethanol production under stress conditions, attributed to its low removal of furfural (Z. mobilis immobilized on biochar was also observed. Thus, biochar extracts in the fermentation broth and cell immobilization on biochar might be the controlling factors for enhanced ethanol production under stress conditions. Conclusions These results indicate that biochar-mediated enhanced ethanol fermentation (BMEEF) might be a promising strategy for ethanol production from lignocellulosic biomass.

Zymomonas mobilis (Z. mobilis) is a potential candidate for consolidated bioprocessing (CBP) strain in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion affected by cell wall peptidoglycan is also not well understood. Here we constructed several Penicillin Binding Proteins (PBPs)-deficient strains derivated from Z. mobilis S192 to perturb the cell wall peptidoglycan network and investigated the effects of peptidoglycan on the endoglucanase secretion. Results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBPs mutant strains, notably, △1089/0959 (4.09-fold) and △0959 (5.76-fold) in comparison to parent strains. Besides, for PBPs-deficient strains, the growth performance was not significantly inhibited but with enhanced antibiotic sensitivity and reduced inhibitor tolerance, otherwise, cell morphology was altered obviously. The concentration of intracellular soluble peptidoglycan was increased, especially for single gene deletion. Outer membrane permeability of PBPs-deficient strains was also improved, notably, △1089/0959 (1.14-fold) and △0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our finding indicated that PBPs-deficient Z. mobilis is capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness, and also acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacterias. Herein, we perturb the peptidoglycan synthesis network via knocking out PBPs (ZMO0197, ZMO0959, ZMO1089) in order to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study can not only lay the foundation for understanding the regulatory network of cell wall synthesis but also provide guidance for the construction of CBP strains in Z. mobilis.

Background: D-lactic acid played an important role in the establishment of PLA as a substitute for petrochemical plastics. But, so far, the D-lactic acid production was limited in only pilot scale, which was definitely unable to meet the fast growing market demand. To achieve industrial scale D-lactic acid production, the cost-associated problems such as high-cost feedstock, expensive nutrient sources and fermentation technology need to be resolved to establish an economical fermentation process.Results: In the present study, the combined effect of B vitamin supplementation and membrane integrated continuous fermentation on D-lactic acid production from agricultural lignocellulosic biomass by Lactobacillus delbrueckii was investigated. The results indicated the specific addition of vitamins B1, B2, B3 and B5 could reduce the yeast extract (YE) addition from 10 g/l to 3 g/l without obvious influence on fermentation efficiency. By employing cell recycling system in 350 h continuous fermentation with B vitamin supplementation, YE addition was further reduced to 0.5 g/l, which resulted in nutrient source cost reduction of 86%. A maximum D-lactate productivity of 18.56 g/l/h and optical purity of 99.5% were achieved and higher than most recent reports. Conclusion: These findings suggested the novel fermentation strategy proposed could effectively reduce the production cost and improve fermentation efficiency, thus exhibiting great potential in promoting industrial scale D-lactic acid production from lignocellulosic biomass.

Pediococcus acidilactici is commonly used for pediocin production and lactic acid fermentation. However, a high-efficiency genome editing tool is unavailable for this species. In this study, we constructed endogenous subtype II-A CRISPR-Cas system-based genome interference plasmids which carried a "repeat-spacer-repeat" cassette in the pMG36e shuttle vector. These plasmids exhibited self-interference activities in P. acidilactici LA412. Then, the genome-editing plasmids were constructed by cloning the upstream/downstream donor DNA into the corresponding interference plasmids to exert high-efficiency markerless gene deletion, gene integration, and point mutation in P. acidilactici LA412. We found that endogenous CRISPR-mediated depletion of the native plasmids enhanced the cell growth and that integration of an l-lactate dehydrogenase gene into the chromosome enhanced both cell growth and lactic acid production. IMPORTANCE A rapid and precise genome editing tool will promote the practical application of Pediococcus acidilactici, one type of lactic acid bacterium with excellent stress tolerance and probiotic characteristics. This study established a high-efficiency endogenous CRISPR-Cas system-based genome editing tool for P. acidilactici and achieved different genetic manipulations, including gene deletion, gene insertion, mononucleotide mutation, and endogenous plasmid depletion. The engineered strain edited by this tool showed significant advantages in cell growth and lactic acid fermentation. Therefore, our tool can satisfy the requirements for genetic manipulations of P. acidilactici, thus making it a sophisticated chassis species for synthetic biology and bioindustry.

Background: As one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. Two of the conditions that are needed to attain cost-efficient lignocellulosic ethanol production at an industrial scale are the simultaneous transformation of glucose and xylose to ethanol and a highly efficient ethanol fermentation process. Results: In this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, at the end of fermentation compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various concentrations of corn stover hydrolysates, in which the sum of glucose and xylose concentrations in the hydrolysates were 60, 90, and 120 g/L respectively, were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56% - 4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12% - 102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Conclusions: The fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. Thus, this strategy has potential for future lignocellulosic ethanol production.

Background As one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. However, the simultaneous transformation of glucose and xylose to ethanol is one of the key technologies for attaining cost-efficient lignocellulosic ethanol production at an industrial scale. Genetic modification of strains and constructing consortia were two approaches to resolve this issue. Compared with strain improvement, the synergistic interaction of consortia in metabolic pathways should be more useful than using each one separately. Results In this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in artificially simulated 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various corn stover hydrolysates with different sugar concentrations (glucose and xylose 60, 90, 120 g/L), were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56–4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12–102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Furthermore, qRT-PCR analysis of xylose/glucose transporter and other genes responsible for CCR explained the reason why the initial ratio inoculation of 1:3 in artificially simulated 80G40XRM had the best fermentation performance in the consortium. Conclusions The fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. This result showed that this strategy has potential for future lignocellulosic ethanol production.

Background Zymomonas mobilis is a natural ethanologen with many desirable characteristics, making it an ideal platform for future biorefineries. Recently, an endogenous CRISPR-based genome editing tool has been developed for this species. However, a simple and high-efficient genome editing method is still required. Results We developed a novel gene deletion tool based on the endogenous subtype I–F CRISPR-Cas system and the microhomology-mediated end joining (MMEJ) pathway. This tool only requires a self-interference plasmid carrying the mini-CRISPR (Repeat–Spacer–Repeat) expression cassette, where the spacer matches the target DNA. Transformation of the self-interference plasmid leads to target DNA damage and subsequently triggers the endogenous MMEJ pathway to repair the damaged DNA, leaving deletions normally smaller than 500 bp. Importantly, the MMEJ repair efficiency was increased by introducing mutations at the second repeat of the mini-CRISPR cassette expressing the guide RNA. Several genes have been successfully deleted via this method, and the phenotype of a σ28 deletion mutant generated in this study was characterized. Moreover, large fragment deletions were obtained by transformation of the self-interference plasmids expressing two guide RNAs in tandem. Conclusions Here, we report the establishment of an efficient gene deletion tool based on the endogenous subtype I–F CRISPR-Cas system and the MMEJ pathway in Zymomonas mobilis. We achieved single gene deletion and large-fragment knockout using this tool. In addition, we further promoted the editing efficiency by modifying the guide RNA expression cassette and selecting lower GC% target sites. Our study has provided an effective method for genetic manipulation in Z. mobilis.

Backgroud: Acetic acid and furfural are two major inhibitors during lignocellulosic ethanol production. In our previous study, we successfully constructed an engineered Zymomonas mobilis ZM532 strain tolerant these double inhibitors by genome shuffling, but the molecular mechanisms of tolerance to these inhibitors are still unknown. This study investigated the responses of ZM532 and wild-type ZM4 to acetic acid and furfural using genomics, transcriptomics and label free quantitative proteomics. Results: By Sanger sequencing technology we re-verified of previously identified 19 mutations in ZM532, but we found a total of 23 single nucleotide polymorphisms (SNPs) in the coding sequence (CDS; 4) and intergenic region (19) in ZM532. Six SNPs were novel in this study. We also identified a total of 1865 and 14 novel differentially expressed genes (DEGs) in ZM532 and wild-type ZM4. Among these, 352 DEGs were up-regulated; while and 393 were down-regulated in AF_ZM532 vs RM_532, respectively. However, 442 DEGs were up while 463 were down-regulated in AF_ZM4 vs RM_ZM4. Moreover, 2 up and 8 down-regulated genes were identified in AF_ZM532 vs AF_ZM4; while 7 up and 1 down-regulated genes were found in RM_ZM532 vs RM_ZM. We also identified 1,532 proteins among 107 up and 204 down-regulated proteins detected in ZM4_AF vs ZM4_RM, 123 up and 205 down regulated proteins were identified in ZM532_AF vs ZM532_RM, respectively. In addition, a total of 16 up and 5 down-regulated proteins were identified out of 1462 in ZM4_AF vs ZM532_AF, while 8 up and 5 down-regulated proteins were observed out of 1491 in ZM4_RM vs ZM532_RM. These proteins and genes are involved in amino acid biosynthesis, macromolecules repair, glycolysis, flagella assembly, ABC transporter, fermentation, and ATP synthesis pathways and stress response. These mentioned genes and proteins confirmed and help to unravel the acetic acid and furfural tolerance mechanism between ZM532 and wild-type ZM4. May be these proteins and genes play key roles in ZM532 regulation with strong expressions under acids stress conditions. Furthermore, we knocked-out and overexpressed two differentially expressed genes (DEGs), ZMO_RS02740 up-regulated and ZMO_RS06525 down-regulated to investigate their roles in acetic acid and furfural tolerance. Our knockout and complementary experiments revealed that up-regulated expression gene ZMO_RS02740 and the down-regulated expression gene ZMO_RS06525 play important roles in dealing with furfural and acetic acid stress. Conclusion: ZM532 can be used to substitute ZM4 as a biocatalyst for bioethanol under acetic acid and furfural condition, with a shorter fermentation time and higher productivity. Further studies may be required to clarify the relationship between the acid resistance and the genetic disparity of mutant strains.

The presence of inhibitory compounds as well as low sugar concentration was the main challenge in lignocellulosic hydrolysate based biofuels production. The objective of this study was to develop an effective membrane filtration process for simultaneously separating inhibitors and concentrating sugars to improve the lignocellulosic hydrolysate fermentability. The nanofiltration (NF) and reverse osmosis (RO) membranes were chosen to evaluate their sugar rejection and inhibitor removal performance using real lignocellulosic hydrolysate in batch recycling mode. The influence of soluble anionic polymer addition, anionic polymer concentration and pH on sugar rejection and inhibitor removal was examined. The results implied that application of sodium tripolyphosphate during NF/RO could improve the inhibitor separation in particular formic acid and acetic acid removal was significantly enhanced. Moreover, pH adjustment for hydrolysate prior to detoxification and concentration and fermentation could be avoided. The concentration experiment indicated that NF-RO hybrid membrane improved sugar rejection but did not cause the obvious increase in inhibitor concentration. Overall, the total sugar titer at the end of concentration mode was 145.6 g/L compared to the initial titer of 38.4 g/L at a volume concentration ratio (VCR) of 4. Furthermore, diafiltration with RO after NF-RO hybrid membrane filtration achieved maximum sugar recovery and inhibitor removal, consequently led to successful ethanol fermentation using detoxified retentate. After 134 h cultivation, the ethanol maximum concentration, productivity and yield of diafiltrated retentate reached 45.16 g/L, 0.34 g/L/h and 0.44 g/g, which were comparable to that of control. These observations indicated that the novel membrane filtration system proposed could achieve simultaneous lignocellulosic hydrolysate detoxification and concentration, which would greatly reduce the number of processing steps, operating cost and energy consumption, consequently improve the production efficiency. Therefore, it could be considered as promising strategy for large scale industrial lignocellulosic bioenergy production.

Agricultural residues are among the most important and abundant biomass resources in the world. This study aimed to highlight the characterization of common agricultural residues in China. Six indicators, including biomass, cellulose, hemicelluloses, lignin, caloric value, and ash, were selected to evaluate agricultural residue for its potential energy utilization using grey relational analysis. The results showed that residues of grain crops, including corn (439.1 million tons, 46.6%), rice (207.1 million tons, 21.5%), and wheat (150.7 million tons, 16.0%) were the top three agricultural residues, accounting for 84.1% of total Chinese agricultural residues. Among the agricultural residue samples, rice, wheat, and corn exhibited relatively low ash content; and sugarcane, cotton, and rape residue exhibited low lignin content. Rape, corn, and hemp showed higher cellulose content; and rice, wheat, and corn exhibited higher hemicellulose content. Among the agricultural residues, hemp, corn, and sugarcane showed higher caloric value compared to other residues. According to the grey relational analysis, these combined properties have shown that corn, rice, and wheat can act as potential candidates for energy utilization. The evaluation results were essentially identical to the performance of agricultural residues. Therefore, the comprehensive evaluation of agricultural residues provides sound scientific evidence for their use in energy production.

Enhancement of biological H2 production efficiency with pre-ozonation process of palm oil mill effluent (POME) prior to thermophilic dark fermentation (55 °C) was investigated. H2 fermentation experiments were conducted using varying concentrations of raw and ozonated POME. Results revealed that H2 can be produced from both raw and ozonated POME under thermophilic fermentation. Maximum H2 production yield of 77 mL.g−1CODremoved was obtained from ozonated POME, which was higher than that of 51 mL·g−1 CODremoved obtained from raw POME at the highest concentration of 35,000 mg COD.L−1. Meanwhile, the specific H2 production rate (R'max) of 1.9 and 1.5 mL·h−1·g−1 TVS were observed in raw and ozonated POME at the concentration of 25,000 mg COD.L−1, respectively. The main metabolic products during POME fermentation were acetic and butyric acids and trace amount of valeric acid. Propionic acid and ethanol have contributed, which could be reduced H2 production in all batch experiments for both POME. The highest efficiency of total and soluble COD removal of 24 and 25% was obtained from the raw POME, and those of 19 and 25% was obtained from the ozonated POME. The present study demonstrates that the POME loading was greatly influenced on the H2 production yields and rates. The comparative results showed that the ozonated POME gave higher H2 yields than the raw POME. Thus, demonstrating that the ozonation process significantly improved the POME biodegradability, which is able to enhance H2 production yields. However, the ozone pre-treatment was not improved in the specific H2 production rates.

Mild traumatic brain injury (mTBI) is the most common form of brain trauma caused by physical impact. The subventricular zone (SVZ) is a neurogenetic niche that contributes to homeostasis and repair after brain injury. It is particularly challenging to fully elucidate the molecular alterations in the SVZ occurring in response to injury due to its cell diversity and the complex network. In this study, we aimed to address this issue using a novel transcriptomic technique- unbiased single-cell RNA sequencing. We resolved previous unknown cell subpopulations harbored in the niche, and uncovered cell type-specific alterations in gene expression, enriched pathways, and cell-cell crosstalk following mTBI. Notably, we also report novel lineage trajectories and molecular hallmarks that govern neurogenesis. This study dissects the delicate transcriptome changes of individual cell types as well as the reprogramming process of cells in the SVZ niche after mTBI, and our findings are expected to facilitate the development of therapeutic interventions or diagnostic tests for mTBI.

The bioavailability of heavy metal and growth of hyperaccumulator are key factors controlling the phytoextraction of heavy metal from soil. In this study, the efficacy and potential microbial mechanisms of digestate application in enhancing Cd extraction from soil by Pennisetum hybridum were investigated. The results showed that digestate application significantly promoted the height, tiller number, and biomass yield of Pennisetum hybridum. The application also increased the activities of urease, sucrase, dehydrogenase, available Cd contents of rhizosphere soils (from 2.21 to 2.46 mg kg−1), and the transfer factors of Cd from root to shoot and leaf. Assuming three annual harvests, digestate application would substantially reduce time needed for Pennisetum hybridum to completely absorb Cd from soil—from 15-16 yr–10 yr. Furthermore, the results of microbial community diversity analysis showed that digestate irrigation was more facilitated for the growth of the predominant bacteria, which were Actinobacteria and Chloroflexi at phylum level, and Sphingomonas and Nitrospiraat genus level, which mainly have the functions of promoted plant growth and metal resistance. The results suggested that the enhanced phytoextraction of Cd by Pennisetum hybridum with digestate application might mainly attributed to the increased Cd bio-availability and the enhanced plant growth, indicating that an approach combining digestate and Pennisetum hybridum could be a promising strategy for remediating Cd-contaminated soils.

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against mobile genetic elements (MGEs) through uptake of invader-derived spacers. De novo adaptation samples spacers from both invaders and hosts, whereas primed adaptation shows higher specificity to sample spacers from invaders in many model systems as well as in the subtype I-F system of Zymomonas mobilis. Self-derived spacers will lead to CRISPR self-interference. However, our in vivo study demonstrated that this species used the microhomology-mediated end joining (MMEJ) pathway to efficiently repair subtype I-F CRISPR-Cas system-mediated DNA breaks guided by the self-targeting spacers. MMEJ repair of DNA breaks requires direct microhomologous sequences flanking the protospacers and leads to DNA deletions covering the protospacers. Importantly, CRISPR-mediated genomic DNA breaks failed to be repaired via MMEJ pathway in presence of higher copies of short homologous DNA. Moreover, CRISPR-cleaved exogenous plasmid DNA was failed to be repaired through MMEJ pathway, probably due to the inhibition of MMEJ by the presence of higher copies of the plasmid DNA in Z. mobilis. Our results infer that MMEJ pathway discriminates DNA damages between in the host chromosome versus mobile genetic element (MGE) DNA, and maintains genome stability post CRISPR immunity in Z. mobilis.

Biological ethylene production is a promising sustainable alternative approach for fossil-based ethylene production. The high glucose utilization of Z. mobilis makes it as a promising bioethylene producer. In this study, Zymomonas mobilis has been engineered to produce ethylene through the introduction of the synthetic ethylene-forming enzyme (EFE). We also investigated the effect of systematically knocking out the competitive metabolic pathway of pyruvate in an effort to improve the availability of pyruvate for ethylene production in Z. mobilis expressing EFE. Guided by these results, we tested a number of conjectures that could improve the α-ketoglutarate supply. Optimization of these pathways and different substrate supplies resulted in a greater production of ethylene (from 1.36 to 12.83 nmol/OD600/mL), which may guide future engineering work on ethylene production using other organisms. Meanwhile, we achieved an ethylene production of 5.8 nmol/OD600/mL in the ZM532-efe strain using enzymatic straw hydrolysate of corn straw as the sole carbon source. As a preferred host in biorefinery technologies using lignocellulosic biomass as feedstock, heterologous expression of EFE in Z. mobilis converts the non-ethylene producing strain into an ethylene-producing one using a metabolic engineering approach, which is of great significance for the utilization of cellulosic biomass in the future. KEY POINTS: • Heterologous expression of EFE in Z. mobilis successfully converted the non-ethylene producing strain into an ethylene producer (1.36 nmol/OD600/mL). Targeted modifications of the central carbon metabolism can effectively improve ethylene production (peak production: 8.3 nmol/OD600/mL). • The addition of nutrients to the medium can further increase the production of ethylene (peak production: 12.8 nmol/OD600/mL). • The ZM532-efe strain achieved an ethylene production of 5.8 nmol/OD600/mL when enzymatic hydrolysate of corn straw was used as the sole carbon source.

Additional file 2: Fig. S1. The Venn diagrams of significantly differentially expressed genes of same strain under different pH conditions (A) and two different strains at acidic pH condition (B).

Additional file 4: Fig. S2. The transmembrane domain prediction of the inner membrane component of RND efflux system without mutation (A) and with mutation (B) using TMHMM. The arrow points the T11 domain, where the mutation located.

Additional file 7: Table S3: Primers used in this study.

Additional file 5: Fig. S3. Acetate production of Z. mobilis wild-type ZM4 and mutant strains of 3.5M and 3.6M at pH 3.8 and 6.2 when the glucose was completely consumed. At least two independent experiments were performed with similar results. Values are the mean of one representative experiment with three technical replicates. Error bars represent standard deviations. Statistics analysis was calculated using one-way ANOVA by GraphPad Prism 8.3.0. ** indicates adjusted p-value

Additional file 6: Fig. S4. Cell growth of recombination strains and wild type of Z. mobilis containing the control plasmid pEZ15A and plasmid constructs of pEZ-Tc1, pEZ-Tc2, pEZ-Tc4, and pEZ-Tc6, respectively, at pH 3.6, 4.0, 6.0 without tetracycline induction (A, C, E, G), or with the induction of 0.8 μg/mL tetracycline (B, D, F, H). In these graphs, red line represents pH 3.6, blue line represents pH 4.0, green line represents pH 6.0 (solid line represents pEZ15A and dotted line represents pEZ-Tc1, pEZ-Tc2, pEZ-Tc4 or pEZ-Tc6, respectively). pEZ-Tc1, plasmid construct expressing operon ZMO0142-ZMO0145 encoding ABC transporter related protein; pEZ-Tc2, plasmid construct expressing operon ZMO0798-ZMO0801 encoding multiple drug efflux related proteins; pEZ-Tc4, plasmid construct expressing operon ZMO0238 ~ ZMO0242 encoding ATP synthesis F1F0 submits; pEZ-Tc6, plasmid construct expressing operon ZMO2005, ZMO0667-ZMO0671 encoding ATP synthesis F1F0 submits. Experiments have been repeated at least three times with similar result, and results from one experiment with three triplicate technical repeats were presented.

Biorefinery of biomass-based biofuels and biochemicals by microorganisms is a competitive alternative of traditional petroleum refineries. Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for commercial production of desirable bioproducts through metabolic engineering. In this review, we summarize the metabolic engineering progress achieved in Z. mobilis to expand its substrate and product ranges as well as to enhance its robustness against stressful conditions such as inhibitory compounds within the lignocellulosic hydrolysates and slurries. We also discuss a few metabolic engineering strategies that can be applied in Z. mobilis to further develop it as a robust workhorse for economic lignocellulosic bioproducts. In addition, we briefly review the progress of metabolic engineering in Z. mobilis related to the classical synthetic biology cycle of “Design-Build-Test-Learn”, as well as the progress and potential to develop Z. mobilis as a model chassis for biorefinery practices in the synthetic biology era.

Background: Biological ethylene production via the ethylene-forming enzyme (EFE) can offer a promising sustainable alternative approach for fossil-based ethylene production. The high stress tolerance of Z. mobilis make it as promising bio-ethylene producer.Results: In this study, Heterologous expression of the efe gene in Z. mobilis successfully converted the non-ethylene producing strain into an ethylene producer. What’s more, we systematically performed the effect of knocking out the competitive metabolic pathway of pyruvate and the addition of nutrients to the medium to improve the ethylene production in Z. mobilis. These optimization pathways and different substrate supplies resulted in higher ethylene productivity (from 1.36 to 12.83 nmol/OD600/ml), which may guide future engineering work on ethylene production in other organisms to further improve ethylene productivity. Meanwhile, we obtained ethylene production of 5.8 nmol/OD600/ml in strain ZM532-efe by using enzymatic hydrolysate of corn straw as the sole carbon source. This is also the first report on the production of ethylene from cellulosic biomass.Conclusions: These results indicate that the engineered Z. mobilis show great potential for production of ethylene from cellulosic biomass in the future.

Background Furfural and acetic acid are the two major inhibitors generated during lignocellulose pretreatment and hydrolysis, would severely inhibit the cell growth, metabolism, and ethanol fermentation efficiency of Zymomonas mobilis. Effective genome shuffling mediated by protoplast electrofusion was developed and then applied to Z. mobilis. Results After two rounds of genome shuffling, 10 different mutants with improved cell growth and ethanol yield in the presence of 5.0 g/L acetic acid and 3.0 g/L furfural were obtained. The two most prominent genome-shuffled strains, 532 and 533, were further investigated along with parental strains in the presence of 7.0 g/L acetic acid and 3.0 g/L furfural. The results showed that mutants 532 and 533 were superior to the parental strain AQ8-1 in the presence of 7.0 g/L acetic acid, with a shorter fermentation time (30 h) and higher productivity than AQ8-1. Mutant 533 exhibited subtle differences from parental strain F34 in the presence of 3.0 g/L furfural. Mutations present in 10 genome-shuffled strains were identified via whole-genome resequencing, and the source of each mutation was identified as either de novo mutation or recombination of the parent genes. Conclusions These results indicate that genome shuffling is an efficient method for enhancing stress tolerance in Z. mobilis. The engineered strains generated in this study could be potential cellulosic ethanol producers in the future.