Metataxonomic Studies to Evaluate the Beneficial Effect of Enzymatic Pretreatment on the Anaerobic Digestion of Waste Generated in Turkey Farming.

Turkey litter waste is lignocellulosic and keratinous, requiring prior enzymatic treatment to facilitate fiber hydrolysis and utilization by microorganisms in anaerobic digestion (AD) process. The understanding of the performance of microorganisms in AD can be facilitated through molecular biology and bioinformatics tools. This study aimed to determine the taxonomic profile and functional prediction of microbial communities in the AD of turkey litter waste subjected to enzymatic pretreatment and correlate it with operational parameters. The tests involved the use of turkey litter (T) at 25 g L-1 of volatile solids, a granular inoculum (S) (10% m/v), and the addition of cellulase (C), and pectinase (P) enzymes at four concentrations. The use of enzymes increased methane production by 19% (turkey litter, inoculum, and cellulase-TSC4) and 15% (turkey litter, inoculum, and enzymatic pectinase-TSP4) compared to the control (turkey litter and inoculum-TS), being more effective in TSC4 (667.52 mLCH4), where there was consumption of acetic, butyric, and propionic acids. The pectinase assay (TSP4) showed a methane production of 648 mLCH4 and there was the accumulation of metabolites. Cellulolytic microorganisms Bacteroides, Ruminofilibacter, Lachnospiraceae, Ruminococcaceae, and Methanosaeta were favored in TSC4. In TSP4, the predominant genus was Macellibacteroides and Methanosarcina, and genes involved in methylotrophic methanogenesis were also found (mtaB, mtmB, and mtbB). Enzymes involved in hydrogenotrophic methanogenesis were identified in both assays (TSC4 and TSP4). Molecular tools helped to understand the metabolic routes involved in AD with enzymatic treatment, allowing the elaboration of strategies to improve the sustainable degradation of turkey litter waste.

The functions of laccase gene GhLAC15 in fiber colouration and development in brown-colored cotton.

The monotonicity of color type in naturally colored cottons (NCCs) has become the main limiting factor to their widespread use, simultaneously coexisting with poor fiber quality. The synchronous improvement of fiber quality and color become more urgent and crucial as the demand for sustainable development increases. The homologous gene of wild cotton Gossypium stocksii LAC15 in G. hirsutum, GhLAC15, was also dominantly expressed in the developing fibers of brown cotton XC20 from 5 DPA (day post anthesis) to 25 DPA, especially at the secondary cell wall thickening stage (20 DPA and 25 DPA). In XC20 plants with downregulated GhLAC15 (GhLAC15i), a remarkable reduction in proanthocyanidins (PAs) and lignin contents was observed. Some of the key genes in the phenylpropane and flavonoid biosynthesis pathway were down-regulated in GhLAC15i plants. Notably, the fiber length of GhLAC15i plants showed an obvious increase and the fiber color was lightened. Moreover, we found that the thickness of cotton fiber cell wall was decreased in GhLAC15i plants and the fiber surface became smoother compared to that of WT. Taken together, this study revealed that GhLAC15 played an important role in PAs and lignin biosynthesis in naturally colored cotton fibers. It might mediate fiber color and fiber quality by catalyzing PAs oxidation and lignin polymerization, ultimately regulating fiber colouration and development.

Investigating the lignocellulolytic gut microbiome of huhu grubs (Prionoplus reticularis) using defined diets and dietary switch.

The huhu beetle (Prionoplus reticularis) is the largest endemic beetle found throughout Aotearoa New Zealand, and is characterised by feeding on wood during its larval stage. It has been hypothesised that its gut microbiome plays a fundamental role in the degradation of wood. To explore this idea we examined the fungal and bacterial community composition of huhu grubs' frass, using amplicon sequencing. Grubs were reared on an exclusive diet of either a predominantly cellulose source (cotton) or lignocellulose source (pine) for 4 months; subsequently a diet switch was performed and the grubs were grown for another 4 months. The fungal community of cellulose-reared huhu grubs was abundant in potential cellulose degraders, contrasting with the community of lignocellulose-reared grubs, which showed abundant potential soft rot fungi, yeasts, and hemicellulose and cellulose degraders. Cellulose-reared grubs showed a less diverse fungal community, however, diet switch from cellulose to lignocellulose resulted in a change in community composition that showed grubs were still capable of utilising this substrate. Conversely, diet seemed to have a limited influence on huhu grub gut bacterial communities.

Integrated Multi-Omics Analysis to Reveal the Molecular Mechanisms of Inflorescence Elongation in Medicago sativa.

The morphological architecture of inflorescence influences seed production. The regulatory mechanisms underlying alfalfa (Medicago sativa) inflorescence elongation remain unclear. Therefore, in this study, we conducted a comparative analysis of the transcriptome, proteome, and metabolome of two extreme materials at three developmental stages to explore the mechanisms underlying inflorescence elongation in alfalfa. We observed the developmental processes of long and short inflorescences and found that the elongation capacity of alfalfa with long inflorescence was stronger than that of alfalfa with short inflorescences. Furthermore, integrative analysis of the transcriptome and proteome indicated that the phenylpropanoid biosynthesis pathway was closely correlated with the structural formation of the inflorescence. Additionally, we identified key genes and proteins associated with lignin biosynthesis based on the differential expressed genes and proteins (DEGs and DEPs) involved in phenylpropanoid biosynthesis. Moreover, targeted hormone metabolome analysis revealed that IAA, GA, and CK play an important role in the peduncle elongation of alfalfa inflorescences. Based on omics analysis, we detected key genes and proteins related to plant hormone biosynthesis and signal transduction. From the WGCNA and WPCNA results, we furthermore screened 28 candidate genes and six key proteins that were correlated with lignin biosynthesis, plant hormone biosynthesis, and signaling pathways. In addition, 19 crucial transcription factors were discovered using correlation analysis that might play a role in regulating candidate genes. This study provides insight into the molecular mechanism of inflorescence elongation in alfalfa and establishes a theoretical foundation for improving alfalfa seed production.

Sugar transport in thermophiles: Bridging lignocellulose deconstruction and bioconversion.

Biomass degrading thermophiles play an indispensable role in building lignocellulose-based supply chains. They operate at high temperatures to improve process efficiencies and minimize mesophilic contamination, can overcome lignocellulose recalcitrance through their native carbohydrate-active enzyme (CAZyme) inventory, and can utilize a wide range of sugar substrates. However, sugar transport in thermophiles is poorly understood and investigated, as compared to enzymatic lignocellulose deconstruction and metabolic conversion of sugars to value-added chemicals. Here, we review the general modes of sugar transport in thermophilic bacteria and archaea, covering the structural, molecular, and biophysical basis of their high-affinity sugar uptake. We also discuss recent genetic studies on sugar transporter function. With this understanding of sugar transport, we discuss strategies for how sugar transport can be engineered in thermophiles, with the potential to enhance the conversion of lignocellulosic biomass into renewable products. ONE-SENTENCE SUMMARY: Sugar transport is the understudied link between extracellular biomass deconstruction and intracellular sugar metabolism in thermophilic lignocellulose bioprocessing.

Biocalorimetry-aided monitoring of fungal pretreatment of lignocellulosic agricultural residues.

The present study aimed to investigate whether and how non-invasive biocalorimetric measurements could serve for process monitoring of fungal pretreatment during solid-state fermentation (SSF) of lignocellulosic agricultural residues such as wheat straw. Seven filamentous fungi representing different lignocellulose decay types were employed. Water-soluble sugars being immediately available after fungal pretreatment and those becoming water-extractable after enzymatic digestion of pretreated wheat straw with hydrolysing (hemi)cellulases were considered to constitute the total bioaccessible sugar fraction. The latter was used to indicate the success of pretreatments and linked to corresponding species-specific metabolic heat yield coefficients (YQ/X) derived from metabolic heat flux measurements during fungal wheat straw colonisation. An YQ/X range of about 120 to 140 kJ/g was seemingly optimal for pretreatment upon consideration of all investigated fungi and application of a non-linear Gaussian fitting model. Upon exclusion from analysis of the brown-rot basidiomycete Gloeophyllum trabeum, which differs from all other here investigated fungi in employing extracellular Fenton chemistry for lignocellulose decomposition, a linear relationship where amounts of total bioaccessible sugars were suggested to increase with increasing YQ/X values was obtained. It remains to be elucidated whether an YQ/X range being optimal for fungal pretreatment could firmly be established, or if the sugar accessibility for post-treatment generally increases with increasing YQ/X values as long as "conventional" enzymatic, i.e. (hemi)cellulase-based, lignocellulose decomposition mechanisms are operative. In any case, metabolic heat measurement-derived parameters such as YQ/X values may become very valuable tools supporting the assessment of the suitability of different fungal species for pretreatment of lignocellulosic substrates. KEY POINTS: • Biocalorimetry was used to monitor wheat straw pretreatment with seven filamentous fungi. • Metabolic heat yield coefficients (YQ/X) seem to indicate pretreatment success. • YQ/X values may support the selection of suitable fungal strains for pretreatment.

Effect of Oak Powdery Mildew on Ascorbate-Glutathione Cycle and Other Antioxidants in Plant-Erysiphe alphitoides Interaction.

Erysiphe alphitoides is a species of powdery mildew responsible for the major foliar disease of oak trees, including Quercus robur. Infection with E. alphitoides leads to a reduction in the growth of the trees and in their ability to survive. This paper reports on the biochemical changes characteristic of defence responses in oak leaves with different infection area sizes, collected in July, August, and September during three growing seasons. The study highlights the effect of E. alphitoides infection on changes in the ascorbate-glutathione cycle, phenolic compound profile, and metal content (mineral distribution). Visible symptoms of pathogen infection appeared gradually in July, but the most intense biochemical plant responses in oak leaves were detected mainly in August and September. These responses included increased ascorbate-glutathione enzyme activities, phenolic compounds, and metal contents. In addition, microscopic analyses revealed a strong fluorescence signal of lignin in the epidermis of pathogen-infected leaves. The involvement of the studied compounds in the basic defence mechanisms of oak against E. alphitoides infection is discussed in the paper.

Machine learning-aided engineering of a cytochrome P450 for optimal bioconversion of lignin fragments.

Using machine learning, molecular dynamics simulations, and density functional theory calculations we gain insight into the selectivity patterns of substrate activation by the cytochromes P450. In nature, the reactions catalyzed by the P450s lead to the biodegradation of xenobiotics, but recent work has shown that fungi utilize P450s for the activation of lignin fragments, such as monomer and dimer units. These fragments often are the building blocks of valuable materials, including drug molecules and fragrances, hence a highly selective biocatalyst that can produce these compounds in good yield with high selectivity would be an important step in biotechnology. In this work a detailed computational study is reported on two reaction channels of two P450 isozymes, namely the O-deethylation of guaethol by CYP255A and the O-demethylation versus aromatic hydroxylation of p-anisic acid by CYP199A4. The studies show that the second-coordination sphere plays a major role in substrate binding and positioning, heme access, and in the selectivity patterns. Moreover, the local environment affects the kinetics of the reaction through lowering or raising barrier heights. Furthermore, we predict a site-selective mutation for highly specific reaction channels for CYP199A4.

OsWRKY12 negatively regulates the drought-stress tolerance and secondary cell wall biosynthesis by targeting different downstream transcription factor genes in rice.

With the increasing occurrence of global warming, drought is becoming a major constraint for plant growth and crop yield. Plant cell walls experience continuous changes during the growth, development, and in responding to stressful conditions. The plant WRKYs play pivotal roles in regulating the secondary cell wall (SCW) biosynthesis and helping plant defend against abiotic stresses. qRT-PCR evidence showed that OsWRKY12 was affected by drought and ABA treatments. Over-expression of OsWRKY12 decreased the drought tolerance of the rice transgenics at the germination stage and the seedling stage. The transcription levels of drought-stress-associated genes as well as those genes participating in the ABA biosynthesis and signaling were significantly different compared to the wild type (WT). Our results also showed that less lignin and cellulose were deposited in the OsWRKY12-overexpressors, and heterogenous expression of OsWRKY12 in atwrky12 could lower the increased lignin and cellulose contents, as well as the improved PEG-stress tolerance, to a similar level as the WT. qRT-PCR results indicated that the transcription levels of all the genes related to lignin and cellulose biosynthesis were significantly decreased in the rice transgenics than the WT. Further evidence from yeast one-hybrid assay and the dual-luciferase reporter system suggested that OsWRKY12 could bind to promoters of OsABI5 (the critical component of the ABA signaling pathway) and OsSWN3/OsSWN7 (the key positive regulators in the rice SCW thickening), and hence repressing their expression. In conclusion, OsWRKY12 mediates the crosstalk between SCW biosynthesis and plant stress tolerance by binding to the promoters of different downstream genes.

Whole-Cell Bioconversion Systems for Efficient Synthesis of Monolignols from L-Tyrosine in Escherichia coli.

Monolignols and their derivatives exhibit various pharmaceutical and physiological characteristics, such as antioxidant and anti-inflammatory properties. However, they remain difficult to synthesize. In this study, we engineered several whole-cell bioconversion systems with carboxylate reductase (CAR)-mediated pathways for efficient synthesis of p-coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine in Escherichia coli BL21 (DE3). By overexpressing the l-tyrosine ammonia lyase from Flavobacterium johnsoniae (FjTAL), carboxylate reductase from Segniliparus rugosus (SruCAR), alcohol dehydrogenase YqhD and hydroxylase HpaBC from E. coli, and caffeate 3-O-methyltransferase (COMT) from Arabidopsis thaliana, three enzyme cascades FjTAL-SruCAR-YqhD, FjTAL-SruCAR-YqhD-HpaBC, and FjTAL-SruCAR-YqhD-HpaBC-COMT were constructed to produce 1028.5 mg/L p-coumaryl alcohol, 1015.3 mg/L caffeyl alcohol, and 411.4 mg/L coniferyl alcohol from 1500, 1500, and 1000 mg/L l-tyrosine, with productivities of 257.1, 203.1, and 82.3 mg/L/h, respectively. This work provides an efficient strategy for the biosynthesis of p-coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine.

The RALF2-FERONIA-MYB63 module orchestrates growth and defense in tomato roots.

Plant secreted peptides RAPID ALKALINISATION FACTORs (RALFs), which act through the receptor FERONIA (FER), play important roles in plant growth. However, it remains unclear whether and how RALF-FER contributes to the trade-off of plant growth-defense. Here, we used a variety of techniques such as CRISPR/Cas9, protein-protein interaction and transcriptional regulation methods to investigate the role of RALF2 and its receptor FER in regulating lignin deposition, root growth, and defense against Fusarium oxysporum f. sp. lycopersici (Fol) in tomato (Solanum lycopersicum). The ralf2 and fer mutants show reduced primary root length, elevated lignin accumulation, and enhanced resistance against Fol than the wild-type. FER interacts with and phosphorylates MYB63 to promote its degradation. MYB63 serves as an activator of lignin deposition by regulating the transcription of dirigent protein gene DIR19. Mutation of DIR19 suppresses lignin accumulation, and reverses the short root phenotype and Fol resistance in ralf2 or fer mutant. Collectively, our results demonstrate that the RALF2-FER-MYB63 module fine-tunes root growth and resistance against Fol through regulating the deposition of lignin in tomato roots. The study sheds new light on how plants maintain the growth-defense balance via RALF-FER.

MicroRNA257 promotes secondary growth in hybrid poplar.

Populus, a significant fast-growing tree species with global afforestation and energy potential, holds considerable economic value. The abundant production of secondary xylem by trees, which serves as a vital resource for industrial purposes and human sustenance, necessitates the orchestration of various regulatory mechanisms, encompassing transcriptional regulators and microRNAs (miRNAs). Nevertheless, the investigation of microRNA-mediated regulation of poplar secondary growth remains limited. In this study, we successfully isolated a novel microRNA (Pag-miR257) from 84 K poplar and subsequently integrated it into the 35 S overexpression vector. The overexpression of Pag-miR257 resulted in notable increases in plant height, stem diameter, and fresh weight. Additionally, the overexpression of Pag-miR257 demonstrated a significant enhancement in net photosynthetic rate. The findings from the examination of cell wall autofluorescence indicated a substantial increase in both xylem area and the number of vessels in poplar plants overexpressing Pag-miR257. Furthermore, the cell wall of the Pag-miR257 overexpressing plants exhibited thickening as observed through transmission electron microscopy. Moreover, the Fourier Transforms Infrared (FTIR) analysis and phloroglucinol-HCl staining revealed an elevation in lignin content in Pag-miR257 overexpressing poplar plants. The findings of this study suggest that microRNA257 may play a role in the control of secondary growth in poplar stems, thereby potentially enhancing the development of wood engineering techniques for improved material and energy production.

Effect of combined bacteria on the flax dew degumming process: Substance degradation sequence and changes in functional bacteria taxa.

In this study, 16S rDNA high-throughput sequencing, Fourier transform infrared spectroscopy, and two-dimensional correlation spectroscopy techniques were used to analyze the mechanisms driving the sequence of degradation of gummy substances by the microbial community and hydrolytic enzymes during the flax dew degumming process. The results revealed that the inoculation of combined bacteria induced quorum sensing, modulated hydrolytic enzyme production, and reshaped the community structure. Lignin-degraded genera (Pseudomonas and Sphingobacterium) were enriched, and the relative abundances of pectin- and cellulose-degraded genera (Chryseobacterium) decreased in the early degumming stages. Hemicellulose-degraded genera (Brevundimonas) increased over the degumming time. Moreover, the abundance of lignin hydrolytic enzymes improved in the early stages, while the abundance of pectin hydrolytic enzymes increased at the end of degumming. Various types of functional bacteria taxa changed the sequence of substance degradation. Electron scanning microscopy and differential scanning calorimetry results indicated that the degumming, facilitated by the inoculation of combined bacteria, was nearly completed by 21 d. The fibers exhibited smoother and more intact properties, along with higher thermal stability, as indicated by a melting temperature of 71.54 °C. This study provides a reference for selecting precise degumming bacterial agents to enhance degumming efficiency.

Study on cultivation of aerobic granular sludge and its application in degrading lignin models in the sequencing batch biofilter granular reactor.

In this study, three sequencing batch biofilter granular reactors (SBBGRs) were employed to treat model lignin wastewater containing different lignin models (2,6-dimethoxyphenol, 4-methoxyphenol, and vanillin). After 40 days of cultivation, uniform-shaped aerobic granular sludge (AGS) was successfully developed through nutrient supplementation with synthetic wastewater. During the acclimation stage, the chemical oxygen demand (COD) reduction efficiencies of the three reactors showed a trend of initial decreasing (5-20%) and then recovering to a high reduction efficiency (exceeding 90%) in a short period of time. During the stable operation stage, all three reactors achieved COD reduction efficiencies exceeding 90%. These findings indicated the cultivated AGS's robust resistance to changes in lignin models in water. UV-Vis spectra analysis confirmed the effective degradation of the three lignin models. Microbiological analysis showed that Proteobacteria and Bacteroidetes were always the dominant phyla. At the genus level, while Acinetobacter (15.46%) dominated in the inoculation sludge, Kapabacteriales (7.93%), SBR1031 (11.77%), and Chlorobium (25.37%) were dominant in the three reactors (for 2,6-dimethoxyphenol, 4-methoxyphenol, and vanillin) after degradation, respectively. These findings demonstrate that AGS cultured with SBBGR effectively degrades lignin models, with different dominant strains observed for various lignin models.

Unraveling interspecies cross-feeding during anaerobic lignin degradation for bioenergy applications.

Lignin, a major component of plant biomass, remains underutilized for renewable biofuels due to its complex and heterogeneous structure. Although investigations into depolymerizing lignin using fungi are well-established, studies of microbial pathways that enable anaerobic lignin breakdown linked with methanogenesis are limited. Through an enrichment cultivation approach with inoculation of freshwater sediment, we enriched a microbial community capable of producing methane during anaerobic lignin degradation. We reconstructed the near-complete population genomes of key lignin degraders and methanogens using metagenome-assembled genomes finally selected in this study (MAGs; 92 bacterial and 4 archaeal MAGs affiliated into 45 and 2 taxonomic groups, respectively). This study provides genetic evidence of microbial interdependence in conversion of lignin to methane in a syntrophic community. Metagenomic analysis revealed metabolic linkages, with lignin-hydrolyzing and/or fermentative bacteria such as the genera Alkalibaculum and Propionispora transforming lignin breakdown products into compounds such as acetate to feed methanogens (two archaeal MAGs classified into the genus Methanosarcina or UBA6 of the family Methanomassiliicoccaceae). Understanding the synergistic relationships between microbes that convert lignin could inform strategies for producing renewable bioenergy and treating aromatic-contaminated environments through anaerobic biodegradation processes. Overall, this study offers fundamental insights into complex community-level anaerobic lignin metabolism, highlighting hitherto unknown players, interactions, and pathways in this biotechnologically valuable process.

Efficient methane production from agro-industrial residues using anaerobic fungal-rich consortia.

Anaerobic digestion (AD) emerges as a pivotal technique in climate change mitigation, transforming organic materials into biogas, a renewable energy form. This process significantly impacts energy production and waste management, influencing greenhouse gas emissions. Traditional research has largely focused on anaerobic bacteria and methanogens for methane production. However, the potential of anaerobic lignocellulolytic fungi for degrading lignocellulosic biomass remains less explored. In this study, buffalo rumen inocula were enriched and acclimatized to improve lignocellulolytic hydrolysis activity. Two consortia were established: the anaerobic fungi consortium (AFC), selectively enriched for fungi, and the anaerobic lignocellulolytic microbial consortium (ALMC). The consortia were utilized to create five distinct microbial cocktails-AF0, AF20, AF50, AF80, and AF100. These cocktails were formulated based on varying of AFC and ALMC by weights (w/w). Methane production from each cocktail of lignocellulosic biomasses (cassava pulp and oil palm residues) was evaluated. The highest methane yields of CP, EFB, and MFB were obtained at 337, 215, and 54 mL/g VS, respectively. Cocktails containing a mix of anaerobic fungi, hydrolytic bacteria (Sphingobacterium sp.), syntrophic bacteria (Sphaerochaeta sp.), and hydrogenotrophic methanogens produced 2.1-2.6 times higher methane in cassava pulp and 1.1-1.2 times in oil palm empty fruit bunch compared to AF0. All cocktails effectively produced methane from oil palm empty fruit bunch due to its lipid content. However, methane production ceased after 3 days when oil palm mesocarp fiber was used, due to long-chain fatty acid accumulation. Anaerobic fungi consortia showed effective lignocellulosic and starchy biomass degradation without inhibition due to organic acid accumulation. These findings underscore the potential of tailored microbial cocktails for enhancing methane production from diverse lignocellulosic substrates.

Transcriptomic and physiological analysis provide new insight into seed shattering mechanism in Pennisetum alopecuroides 'Liqiu'.

KEY MESSAGE: Through the histological, physiological, and transcriptome-level identification of the abscission zone of Pennisetum alopecuroides 'Liqiu', we explored the structure and the genes related to seed shattering, ultimately revealing the regulatory network of seed shattering in P. alopecuroides. Pennisetum alopecuroides is one of the most representative ornamental grass species of Pennisetum genus. It has unique inflorescence, elegant appearance, and strong stress tolerance. However, the shattering of seeds not only reduces the ornamental effect, but also hinders the seed production. In order to understand the potential mechanisms of seed shattering in P. alopecuroides, we conducted morphological, histological, physiological, and transcriptomic analyses on P. alopecuroides cv. 'Liqiu'. According to histological findings, the seed shattering of 'Liqiu' was determined by the abscission zone at the base of the pedicel. Correlation analysis showed that seed shattering was significantly correlated with cellulase, lignin, auxin, gibberellin, cytokinin and jasmonic acid. Through a combination of histological and physiological analyses, we observed the accumulation of cellulase and lignin during 'Liqiu' seed abscission. We used PacBio full-length transcriptome sequencing (SMRT) combined with next-generation sequencing (NGS) transcriptome technology to improve the transcriptome data of 'Liqiu'. Transcriptomics further identified many differential genes involved in cellulase, lignin and plant hormone-related pathways. This study will provide new insights into the research on the shattering mechanism of P. alopecuroides.

Peroxidase in plant defense: Novel insights for cadmium accumulation in rice (Oryza sativa L.).

Phenylpropanoid biosynthesis plays crucial roles in the adaptation to cadmium (Cd) stress. Nevertheless, few reports have dabbled in physiological mechanisms of such super pathway regulating Cd accumulation in plants. Herein, by integrating transcriptomic, histological and molecular biology approaches, the present study dedicated to clarify molecular mechanism on how rice adapt to Cd stress via phenylpropanoid biosynthesis. Our analysis identified that the enhancement of phenylpropanoid biosynthesis was as a key response to Cd stress. Intriguingly, POD occupied a significant part in this process, with the number of POD related genes accounted for 26/29 of all upregulated genes in phenylpropanoid biosynthesis. We further used SHAM (salicylhydroxamic acid, the POD inhibitor) to validate that POD exhibited a negative correlation with the Cd accumulation in rice tissues, and proposed two intrinsic molecular mechanisms on POD in contributing to Cd detoxification. One strategy was that POD promoted the formation of lignin and CSs both in endodermis and exodermis for intercepting Cd influx. In detail, inhibited POD induced by external addition of SHAM decreased the content of lignin by 50.98-66.65 % and delayed percentage of the DTIP-CS to root length by 39.17-104.51 %. The other strategy was expression of transporter genes involved in Cd uptake, including OsIRT1, OsIRT2, OsZIP1 and OsZIP, negatively regulated by POD. In a word, our findings firstly draws a direct link between POD activity and the Cd accumulation, which is imperative for the breeding of rice with low-Cd-accumulating capacity in the future.

Evaluating lignin degradation under limited oxygen conditions by bacterial isolates from forest soil.

Lignin, a heterogeneous aromatic polymer present in plant biomass, is intertwined with cellulose and hemicellulose fibrils, posing challenges to its effective utilization due to its phenolic nature and recalcitrance to degradation. In this study, three lignin utilizing bacteria, Klebsiella sp. LEA1, Pseudomonas sp. LEA2, and Burkholderia sp. LEA3, were isolated from deciduous forest soil samples in Nan province, Thailand. These isolates were capable of growing on alkali lignin and various lignin-associated monomers at 40 °C under microaerobic conditions. The presence of Cu2+ significantly enhanced guaiacol oxidation in Klebsiella sp. LEA1 and Pseudomonas sp. LEA2. Lignin-related monomers and intermediates such as 2,6-dimethoxyphenol, 4-vinyl guaiacol, 4-hydroxybenzoic acid, benzoic acid, catechol, and succinic acid were detected mostly during the late stage of incubation of Klebsiella sp. LEA1 and Pseudomonas sp. LEA2 in lignin minimal salt media via GC-MS analysis. The intermediates identified from Klebsiella sp. LEA1 degradation suggested that conversion and utilization occurred through the ß-ketoadipate (ortho-cleavage) pathway under limited oxygen conditions. The ability of these bacteria to thrive on alkaline lignin and produce various lignin-related intermediates under limited oxygen conditions suggests their potential utility in oxygen-limited processes and the production of renewable chemicals from plant biomass.

Efficient production of polyhydroxybutyrate using lignocellulosic biomass derived from oil palm trunks by the inhibitor-tolerant strain Burkholderia ambifaria E5-3.

Lignocellulosic biomass is a valuable, renewable substrate for the synthesis of polyhydroxybutyrate (PHB), an ecofriendly biopolymer. In this study, bacterial strain E5-3 was isolated from soil in Japan; it was identified as Burkholderia ambifaria strain E5-3 by 16 S rRNA gene sequencing. The strain showed optimal growth at 37 °C with an initial pH of 9. It demonstrated diverse metabolic ability, processing a broad range of carbon substrates, including xylose, glucose, sucrose, glycerol, cellobiose, and, notably, palm oil. Palm oil induced the highest cellular growth, with a PHB content of 65% wt. The strain exhibited inherent tolerance to potential fermentation inhibitors derived from lignocellulosic hydrolysate, withstanding 3 g/L 5-hydroxymethylfurfural and 1.25 g/L acetic acid. Employing a fed-batch fermentation strategy with a combination of glucose, xylose, and cellobiose resulted in PHB production 2.7-times that in traditional batch fermentation. The use of oil palm trunk hydrolysate, without inhibitor pretreatment, in a fed-batch fermentation setup led to significant cell growth with a PHB content of 45% wt, equivalent to 10 g/L. The physicochemical attributes of xylose-derived PHB produced by strain E5-3 included a molecular weight of 722 kDa, a number-average molecular weight of 191 kDa, and a polydispersity index of 3.78. The amorphous structure of this PHB displayed a glass transition temperature of 4.59 °C, while its crystalline counterpart had a melting point of 171.03 °C. This research highlights the potential of lignocellulosic feedstocks, especially oil palm trunk hydrolysate, for PHB production through fed-batch fermentation by B. ambifaria strain E5-3, which has high inhibitor tolerance.