Biofuneling lignin-derived compounds into lipids using a newly isolated Citricoccus sp. P2.

Lignin-derived compounds (LDCs) bioconversion into lipids is a promising yet challenging task. This study focuses on the isolation of the ligninolytic bacterium Citricoccus sp. P2 and investigates its mechanism for producing lipids from LDCs. Although strain P2 exhibits a relatively low lignin degradation rate of 44.63%, it efficiently degrades various concentrations of LDCs. The highest degradation rate is observed when incubated with 0.6 g/L vanillic acid, 0.6 g/L syringic acid, 0.8 g/L p-coumaric acid, and 0.4 g/L phenol, resulting in respective lipid yields of 0.16 g/L, 0.13 g/L, 0.24 g/L, and 0.13 g/L. The genome of strain P2 provides insights into LDCs bioconversion into lipids and stress tolerance. Moreover, Citricoccus sp. P2 has been successfully developed a non-sterilized lipid production using its native alkali-halophilic characteristics, which significantly enhances the lipid yield. This study presents a promising platform for lipids production from LDCs and has potential to promote valorization of lignin.

Valorization of lignin-derived compounds into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by engineered Halomonas sp. Y3.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a biopolyester with great potential, but its high production cost via the propionate-dependent pathway has hindered its development. Herein, we engineer Halomonas sp. Y3 to achieve efficient conversion of various LDCs into PHBV without propionate supplement. Initially, we successfully achieve PHBV production without propionate supplement by overexpressing threonine synthesis. The resulting biopolyester exhibits a 3 HV proportion of up to 7.89 mol%, comparable to commercial PHBV (8 mol%) available from Sigma Aldrich (403105). To further enhance PHBV production, we rationally design the reconstruction of aromatic compound catabolism. The engineered strain Y3_18 efficiently assimilates all LDCs containing syringyl (S), guaiacyl (G), and p-hydroxyphenyl-type (H) units. From 1 g/L of S-, G-, and H-type LDCs, Y3_18 produces PHBV at levels of 449 mg/L, 488 mg/L, and 716 mg/L, respectively, with yields of 44.9 % (g/g), 48.8 % (g/g), and 71.6 % (g/g). Moreover, to improve PHBV yield from lignin, we integrate laccase-secretion and PHBV production modules. This integration leads to the accumulation of 425.84 mg/L of PHBV with a yield of 21.29 % (g/g) and a 3 HV proportion of 6.38 mol%. By harnessing the capabilities of Halomonas sp. Y3, we demonstrate an efficient and sustainable approach for PHBV production from a variety of LDCs.

Ti4+-dopamine/sodium alginate multicomponent complex derived N-doped TiO2@carbon nanocomposites for efficient removal of methylene blue.

A one-pot route for the preparation of TiO2@carbon nanocomposite from Ti4+/polysaccharide coordination complex has been developed and shown advantages in operation, cost, environment, etc. However, the photodegradation rate of methylene blue (MB) needs to be improved. N-doping has been proven as an efficient means to enhance photodegradation performance. Thus, the present study upgraded the TiO2@carbon nanocomposite to N-doped TiO2@carbon nanocomposite (N-TiO2@C) from Ti4+-dopamine/sodium alginate multicomponent complex. The composites were characterized by FT-IR, XRD, XPS, UV-vis DRS, TG-DTA, and SEM-EDS. The obtained TiO2 was a typical rutile phase, and the carboxyl groups existed on N-TiO2@C. The photocatalyst consequently showed high removal efficiency of MB. The cycling experiment additionally indicated the high stability of N-TiO2@C. The present work provided a novel route for preparing N-TiO2@C. Moreover, it can be extended to prepare N-doped polyvalent metal oxides@carbon composites from all water-soluble polysaccharides such as cellulose derivatives, starch, and guar gum.

Non-sterilized conversion of whole lignocellulosic components into polyhydroxybutyrate by Halomonas sp. Y3 with a dual anti-microbial contamination system.

Polyhydroxybutyrate (PHB) production from lignocellulosic biomass is challenging due to the need for whole components and energy-effective conversion. Herein, Halomonas sp. Y3, a ligninolytic bacterium with the capacity to produce PHB from lignin and cellulose- and hemicellulose-derived sugars, is employed to explore its feasibility. This strain shows high sugar tolerance up to 200 g/L of glucose and 120 g/L of xylose. A dual anti-microbial contamination system (DACS) containing alkali-halophilic system (AHS) and phosphite-urea system (PUS) is presented, successfully achieving a completely aseptic effect and resulting in a total of 8.2 g of PHB production from 100 g bamboo biomass. We further develop a stage-fed-batch fermentation to promote the complete utilization of xylose. Approximately 69.99 g of dry cell weight (DCW) and 46.45 g of PHB with 66.35 % are obtained from a total of 296.58 g of sugars and 5.70 g of lignin, showing a significant advancement for LCB bioconversion. We then delete the native phosphate transporters, rendering the strain unable to grow on phosphate-loaded media, effectively improving the strain biosafety without compromising its ability to produce PHB. Overall, our findings demonstrate the potential of Y3 as a classic bacterium strain for PHB production with potential uses in industry.

Highly efficient polyhydroxyalkanoate production from lignin using genetically engineered Halomonas sp. Y3.

Lignin degradation represents a significant challenge in biological valorization, but it is suffering from insufficiency, putting barriers to efficient lignin conversion. Herein, the study first develops a highly efficient laccase secretion apparatus, enabling high enzyme activity of 184 U/mL, complementing the biochemical limits on lignin depolymerization well in Halomonas sp. Y3. Further engineering of PHA biosynthesis produces a significantly high PHA titer of 286, 742, and 868 mg/L from alkaline lignin, catechol, and protocatechuate, respectively. The integration of laccase-secretion and PHA production modules enables a record titer of 693 and 1209 mg/L in converting lignin and lignin-containing stream to PHA, respectively. The titer is improved furtherly to 740 and 1314 mg/L by developing a non-sterilized fermentation. This study advances a cheaper and greener production of valuable chemicals from lignin by constructing a biosynthetic platform for PHA production and provides novel insight into the lignin conversion by extremophilic microbes.

Efficiently unsterile polyhydroxyalkanoate production from lignocellulose by using alkali-halophilic Halomonas alkalicola M2.

The alkali-halophilic Halomonas alkalicola M2 was isolated and developed for an open unsterile polyhydroxyalkanoate (PHA) fermentation from lignocellulose at pH 10.0 and NaCl 70 g/L. The alkaline pretreatment liquid (APL) was converted into PHA by the strain, which was significantly affected by the cultural conditions, including pH, NaCl concentration, nitrogen source, and APL concentration. The extracted PHA was composed of three monomers and similar in physicochemical properties to conventional short chain-length PHA. A record yield of 2.1 and 5.9 g of PHA was accumulated from 100 g dry bamboo powder (BP) by using APL and APL combined with hydrolysate during a 48-h open unsterile fermentation process, respectively. In summary, the alkali-halophilic H. alkalicola M2 achieved the open unsterile fermentation for lignocellulose efficient bioconversion into PHA under high alkalinity and salinity conditions and would be an ideal producer in the field.

Comparative genomic and secretomic characterisation of endophytic Bacillus velezensis LC1 producing bioethanol from bamboo lignocellulose.

Bacillus is an excellent organic matter degrader, and it has exhibited various abilities required for lignocellulose degradation. Several B. velezensis strains encode lignocellulosases, however their ability to efficiently transform biomass has not been appreciated. In the present study, through the comparative genomic analysis of the whole genome sequences of 21 B. velezensis strains, CAZyome related to lignocellulose degradation was identified and their similarities and differences were compared. Subsequently, the secretome of B. velezensis LC1 by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were identified and confirmed that a considerable number of proteins were involved in lignocellulose degradation. Moreover, after 6-day treatment, the degradation efficiency of the B. velezensis LC1 toward cellulose, hemicellulose and lignin were 59.90%, 75.44% and 23.41%, respectively, the hydrolysate was subjected to ethanol fermentation with Saccharomyces cerevisiae and Escherichia coli KO11, yielded 10.44 g/L ethanol after 96 h. These results indicate that B. velezensis LC1 has the ability to effectively degrade bamboo lignocellulose and has the potential to be used in bioethanol production.

Genome sequencing of gut symbiotic Bacillus velezensis LC1 for bioethanol production from bamboo shoots.

BACKGROUND: Bamboo, a lignocellulosic feedstock, is considered as a potentially excellent raw material and evaluated for lignocellulose degradation and bioethanol production, with a focus on using physical and chemical pre-treatment. However, studies reporting the biodegradation of bamboo lignocellulose using microbes such as bacteria and fungi are scarce. RESULTS: In the present study, Bacillus velezensis LC1 was isolated from Cyrtotrachelus buqueti, in which the symbiotic bacteria exhibited lignocellulose degradation ability and cellulase activities. We performed genome sequencing of B. velezensis LC1, which has a 3929,782-bp ring chromosome and 46.5% GC content. The total gene length was 3,502,596 bp using gene prediction, and the GC contents were 47.29% and 40.04% in the gene and intergene regions, respectively. The genome contains 4018 coding DNA sequences, and all have been assigned predicted functions. Carbohydrate-active enzyme annotation identified 136 genes annotated to CAZy families, including GH, GTs, CEs, PLs, AAs and CBMs. Genes involved in lignocellulose degradation were identified. After a 6-day treatment, the bamboo shoot cellulose degradation efficiency reached 39.32%, and the hydrolysate was subjected to ethanol fermentation with Saccharomyces cerevisiae and Escherichia coli KO11, yielding 7.2 g/L of ethanol at 96 h. CONCLUSIONS: These findings provide an insight for B. velezensis strains in converting lignocellulose into ethanol. B. velezensis LC1, a symbiotic bacteria, can potentially degrade bamboo lignocellulose components and further transformation to ethanol, and expand the bamboo lignocellulosic bioethanol production.

Bamboo lignocellulose degradation by gut symbiotic microbiota of the bamboo snout beetle Cyrtotrachelus buqueti.

BACKGROUND: Gut symbiotic microbiota plays a critical role in nutrient supply, digestion, and absorption. The bamboo snout beetle, Cyrtotrachelus buqueti, a common pest of several bamboo species, exhibits high lignocellulolytic enzyme activity and contains various CAZyme genes. However, to date, no studies have evaluated the role of gut symbiotic microbiota of the snout beetle on bamboo lignocellulose degradation. Therefore, the present study investigated the role of gut symbiotic microbiota of C. buqueti on bamboo lignocellulose degradation. RESULTS: Gut symbiotic microbiota of female (CCJ), male (XCJ), and larvae (YCJ) beetles was used to treat bamboo shoot particles (BSPs) in vitro for 6 days. Scanning electron microscopy (SEM) revealed significant destruction of the lignocellulose structure after treatment, which was consistent with the degradation efficiencies of CCJ, XCJ, and YCJ for cellulose (21.11%, 17.58% and 18.74%, respectively); hemicellulose (22.22%, 27.18% and 34.20%, respectively); and lignin (19.83%, 24.30% and 32.97%, respectively). Gut symbiotic microbiota of adult and larvae beetles was then identified using 16sRNA sequencing, which revealed that four microbes: Lactococcus, Serratia, Dysgonomonas and Enterococcus, comprise approximately 84% to 94% of the microbiota. Moreover, the genomes of 45 Lactococcus, 72 Serratia, 86 Enterococcus and 4 Dysgonomonas microbes were used to analyse resident CAZyme genes. These results indicated that gut symbiotic microbiota of adult and larvae C. buqueti is involved in the lignocellulose degradation traits shown by the host. CONCLUSIONS: This study shows that the gut symbiotic microbiota of C. buqueti participates in bamboo lignocellulose degradation, providing innovative findings for bamboo lignocellulose bioconversion. Furthermore, the results of this study will allow us to further isolate lignocellulose-degrading microbiota for use in bamboo lignocellulose bioconversion.

Degradation of bamboo lignocellulose by bamboo snout beetle Cyrtotrachelus buqueti in vivo and vitro: efficiency and mechanism.

BACKGROUND: As an important biomass raw material, the lignocellulose in bamboo is of significant value in energy conversion. The conversion of bamboo lignocellulose into fermentable reducing sugar, i.e. the degradation of bamboo lignocellulose, is an important step in lignocellulose conversion. However, little research has focussed on excavating the enzymes and microbes that are related to the degradation of bamboo lignocellulose, which is important for its utilisation. This study used Cyrtotrachelus buqueti (bamboo snout beetle) to evaluate the efficiency of bamboo lignocellulose degradation. RESULTS: RNA sequencing was conducted to sequence the transcriptome of the insect before and after feeding on bamboo shoots. The expression levels of genes encoding several carbohydrate-active enzymes, such as endoglucanase (evgtrinloc27093t1 and evgtrinloc16407t0) and laccase (evgtrinloc15173t0 and evgtrinloc11252t0), were found to be upregulated after feeding. Faecal component analysis showed that the degradation efficiencies of cellulose, hemicellulose and lignin were 61.82%, 87.65% and 69.05%, respectively. After 6 days of co-culture with crude enzymes in vitro, the degradation efficiencies of cellulose, hemicellulose and lignin in bamboo shoot particles (BSPs) were 24.98%, 37.52% and 26.67%, respectively. These results indicated that lignocellulosic enzymes and related enzymes within the insect itself co-degraded bamboo lignocellulose. These finding can potentially be used for the pre-treatment and enzymatic hydrolysis of bamboo lignocellulose. CONCLUSION: Our results showed that intestinal digestive enzymes from C. buqueti degraded bamboo shoot lignocellulose both in vivo and in vitro. In addition, the expression levels of many carbohydrate-active enzyme (CAZyme) genes were upregulated in the transcriptome, including those for cellulase, xylanase and ligninase genes. Therefore, we proposed a scheme for applying the lignocellulolytic enzymes from C. buqueti to degrade bamboo lignocellulose using genetic, enzymatic and fermentation engineering techniques to overexpress the lignocellulolytic enzymes genes in vitro and obtain large quantities of enzymes that could efficiently degrade bamboo lignocellulose and be used for lignocellulose bioconversion.

Picea wilsonii transcription factor NAC2 enhanced plant tolerance to abiotic stress and participated in RFCP1-regulated flowering time.

KEY MESSAGE: Picea wilsonii transcription factor PwNAC2 enhanced plant tolerance to salt and drought stress through multiple signaling pathway and interacted with PwRFCP1 to participate in flowering regulation. NAC is one of the largest transcription factor families in plants, however, its role is not yet fully understood. Here, we identified a transcription factor PwNAC2 in Picea wilsonii, which localized in nucleus with transcriptional activity in C-terminal region and can form homodimer by itself. Expression analysis by real-time PCR showed that PwNAC2 was induced by multiple abiotic stresses and phytohormones stimuli. PwRFCP1 (Resemble-FCA-contain-PAT1 domain), an interaction protein of PwNAC2 was screened via yeast two hybrid. Luciferase complementation assay confirmed the interaction in vivo and bimolecular fluorescence complementation assay showed the interaction in nucleus. PwNAC2 overexpression retarded Arabidopsis hypocotyls growth which is closely related to light, whereas promotion of hypocotyls growth by PwRFCP1 is independent on light. Under drought or salt treatment, overexpression of PwNAC2 in Arabidopsis showed more vigorous seed germination and significant tolerance for seedlings by ROS scavenging, reducing of membrane damage, slower water loss and increased stomatal closure. ABA or CBF-pathway marker genes were substantially higher in PwNAC2 transgenic Arabidopsis. Overexpression of PwRFCP1 promotes flowering in transgenic Arabidopsis, whereas PwNAC2 delayed flowering by altering the expression of FT, SOC1 and FLC. In addtioin, PwRFCP1 overexpression plants showed no higher tolerance to stress treatment than Col-0. Collectively, our results indicate that PwNAC2 enhanced plant tolerance to abiotic stress through multiple signaling pathways and participated in PwRFCP1-regulated flowering time.

De novo transcriptome assembly of the bamboo snout beetle Cyrtotrachelus buqueti reveals ability to degrade lignocellulose of bamboo feedstock.

BACKGROUND: The bamboo weevil Cyrtotrachelus buqueti, which is considered a pest species, damages bamboo shoots via its piercing-sucking mode of feeding. C. buqueti is well known for its ability to transform bamboo shoot biomass into nutrients and energy for growth, development and reproduction with high specificity and efficacy of bioconversion. Woody bamboo is a perennial grass that is a potential feedstock for lignocellulosic biomass because of its high growth rate and lignocellulose content. To verify our hypothesis that C. buqueti efficiently degrades bamboo lignocellulose, we assessed the bamboo lignocellulose-degrading ability of this insect through RNA sequencing for identifying a potential route for utilisation of bamboo biomass. RESULTS: Analysis of carbohydrate-active enzyme (CAZyme) family genes in the developmental transcriptome of C. buqueti revealed 1082 unigenes, including 55 glycoside hydrolases (GH) families containing 309 GHs, 51 glycosyltransferases (GT) families containing 329 GTs, 8 carbohydrate esterases (CE) families containing 174 CEs, 6 polysaccharide lyases (PL) families containing 11 PLs, 8 auxiliary activities (AA) families containing 131 enzymes with AAs and 17 carbohydrate-binding modules (CBM) families containing 128 CBMs. We used weighted gene co-expression network analysis to analyse developmental RNA sequencing data, and 19 unique modules were identified in the analysis. Of these modules, the expression of MEyellow module genes was unique and the module included numerous CAZyme family genes. CAZyme genes in this module were divided into two groups depending on whether gene expression was higher in the adult/larval stages or in the egg/pupal stages. Enzyme assays revealed that cellulase activity was highest in the midgut whereas lignin-degrading enzyme activity was highest in the hindgut, consistent with findings from intestinal gene expression studies. We also analysed the expression of CAZyme genes in the transcriptome of C. buqueti from two cities and found that several genes were also assigned to CAZyme families. The insect had genes and enzymes associated with lignocellulose degradation, the expression of which differed with developmental stage and intestinal region. CONCLUSION: Cyrtotrachelus buqueti exhibits lignocellulose degradation-related enzymes and genes, most notably CAZyme family genes. CAZyme family genes showed differences in expression at different developmental stages, with adults being more effective at cellulose degradation and larvae at lignin degradation, as well as at different regions of the intestine, with the midgut being more cellulolytic than the hindgut. This degradative system could be utilised for the bioconversion of bamboo lignocellulosic biomass.

Transcriptome analysis of Cyrtotrachelus buqueti in two cities in China.

In order to reduce the Cyrtotrachelus buqueti population, which is a serious pest in the bamboo industry, a range of approaches is required, which will be dependent on diverse gene expression influenced by environmental factors. In this study, samples from two regions of China, Muchuan in Sichuan Province and Chishui in Guizhou Province, were investigated through RNA-seq. Approximately 44 million high-quality reads were generated and 94.2% of the data was mapped to the transcriptome. A total of 15,641 out of the 29,406 identified genes were predicted. Moreover, 348 genes were differentially expressed between the two groups of imagoes (77 upregulated and 271 downregulated). The functional analysis showed that these genes were significantly enriched in the ribosome and metabolic pathway categories. The candidate genes contributing to the reduction in C. buqueti included 41 genes involved in the ribosome constitution category, five in the one­carbon pool pathway by folate category, and five heat shock protein genes. The downregulation of these candidate genes seems to have impaired metabolic processes, such as protein, DNA, RNA, and purine synthesis, as well as carbon and folate metabolism, subsequently resulting in the observed population reduction of C. buqueti. Furthermore, temperature, heavy metal content, and pH might influence the population by altering the expressions of genes involved in these metabolic processes.

Morphological observation and characterization of the Pseudoregma bambucicola with the scanning electron microscope.

Both leica microscopic camera system and scanning electron microscopy was used to observe and characterize the feet, back, abdomen, antennae and mouthparts of the Pseudoregma bambucicola from the bamboo, Bambusa multiplex. The possible functions of all the external morphological characteristics of the P. bambucicola were described and discussed in detail, which offers a basis for further enriching the biology, phylogeny and ecological niche of the P. bambucicola. Moreover, the morphological results should contribute to morphological identification and differentiation of the P. bambucicola from other aphids in the same family.

Overexpression of a NF-YB3 transcription factor from Picea wilsonii confers tolerance to salinity and drought stress in transformed Arabidopsis thaliana.

Nuclear factor Y (NF-Y) is a highly conserved transcription factor comprising NF-YA, NF-YB and NF-YC subunits. To date, the roles of NF-Y subunit in plant still remain elusive. In this study, a subunit NF-YB (PwNF-YB3), was isolated from Picea wilsonii Mast. and its role was studied. PwNF-YB3 transcript was detected in all vegetative and reproductive tissues with higher levels in stem and root and was greatly induced by salinity, heat and PEG but not by cold and ABA treatment. Over-expression of PwNF-YB3 in Arabidopsis showed a significant acceleration in the onset of flowering and resulted in more vigorous seed germination and significant tolerance for seedlings under salinity, drought and osmotic stress compared with wild type plants. Transcription levels of salinity-responsive gene (SOS3) and drought-induced gene (CDPK1) were substantially higher in transgenic Arabidopsis than in wild-type plants. Importantly, CBF pathway markers (COR15B, KIN1, LEA76), but not ABA pathway markers CBF4, were greatly induced under condition of drought. The nuclear localization showed that NF-YB3 acted as a transcription factor. Taken together, the data provide evidence that PwNF-YB3 positively confers significant tolerance to salt, osmotic and drought stress in transformed Arabidopsis plants probably through modulating gene regulation in CBF-dependent pathway.