Developing Robust Probiotic Consortia: A Methodological Optimization Approach.

Developing effective probiotic consortia requires a comprehensive understanding of strain interactions. While traditional methods focus on direct interactions of the participating microbes, the role of microbial metabolites remains largely unexplored. Present study introduces a novel approach of evaluating the impact of strains as well as their secondary metabolites on compatibility during co-culture by assessing the antagonistic and synergistic attributes for multi-strain probiotic formulation. Assessment of antagonistic activity by spot method indicated suppressive nature of PIG1FD and PIG1IR on other strain's growth, hence not appropriate for consortia formulation. Findings of synergistic attribute demonstrated growth promoting role of cell supernatants from isolates PIG6IR and PIG5CI significantly, as it accelerated the entry of all other isolates into the log phase by 5-6 h and 0-2 h, respectively. By employing this methodology, we identified PIG5CI and PIG6IR (isolates identified as Bacillus spizizenii BAB 7915 and Bacillus subtilis BAB 7918 by 16S RNA sequencing method) as promising candidates for consortium formation due to their ability to enhance the growth of other strains through metabolite production. By attempting to elucidate the microbial interactions and metabolite-mediated effects, this research contributes to a more comprehensive understanding of probiotic consortia dynamics and offers valuable insights for future translational studies.

Improved chickpea growth, physiology, nutrient assimilation and rhizoremediation of hydrocarbons by bacterial consortia.

BACKGROUND: Soil pollution by petroleum hydrocarbons (PHCs) reduces yield by changing the physico-chemical properties of soil and plants due to PHCs' biotoxicity and persistence. Thus, removing PHCs from the soil is crucial for ecological sustainability. Microbes-assisted phytoremediation is an economical and eco-friendly solution. The current work aimed to develop and use bacterial consortia (BC) for PHCs degradation and plant growth enhancement in hydrocarbon-contaminated soil. Initially, the enriched microbial cultures (that were prepared from PHCs-contaminated soils from five distinct regions) were obtained via screening through microcosm experiments. Afterward, two best microbial cultures were tested for PHCs degradation under various temperature and pH ranges. After culture optimization, isolation and characterization of bacterial strains were done to construct two BC. These constructed BC were tested in a pot experiment for hydrocarbons degradation and chickpea growth in PHCs contaminated soil. RESULTS: Findings revealed that PHCs exerted significant phytotoxic effects on chickpea growth and physiology when cultivated in PHCs contaminated soil, reducing agronomic and physiological traits by 13-29% and 12-43%, respectively. However, in the presence of BC, the phytotoxic impacts of PHCs on chickpea plants were reduced, resulting in up to 24 - 35% improvement in agronomic and physiological characteristics as compared to un-inoculated contaminated controls. Furthermore, the bacterial consortia boosted chickpea's nutritional absorption and antioxidant mechanism. Most importantly, chickpea plants phytoremediated 52% of the initial PHCs concentration; however, adding BC1 and BC2 with chickpea plants further increased this removal and remediated 74% and 80% of the initial PHCs concentration, respectively. CONCLUSION: In general, BC2 outperformed BC1 (with few exceptions) in promoting plant growth and PHCs elimination. Therefore, using multi-trait BC for PHCs degradation and plant growth improvement under PHCs stress may be an efficient and environmentally friendly strategy to deal with PHCs pollution and toxicity.

The Reverse Ecology-Based Approach to Design a Bacterial Consortium as Soybean Bioinoculant.

Bioinoculants traditionally rely on selecting efficient microbes from the soil with potential growth-enhancing traits for plants. However, such approaches often neglect microbe-microbe and microbe-plant interactions. In this study, we applied a reverse ecology framework to design and assess a bacterial consortium tailored for soybeans. Our analysis identified Paenibacillus polymyxa, Methylobacterium brachiatum, and Enterobacter sp. as key strains for their synergistic potential in promoting soybean growth. Computational analyses revealed that these selected strains exhibited low competitiveness and metabolic compatibility. Specifically, their complementary metabolic profiles suggested minimal competition for resources and potential for mutualistic interactions. In vitro experiments further supported these findings, demonstrating that the consortium maintained stable growth without inhibitory effects among strains. In addition, greenhouse validation experiments confirmed the efficacy of the microbial consortium in enhancing soybean growth such as root and shoot development and biomass production. Overall, this study underscores the potential of reverse ecology in optimizing microbial consortia design for bioinoculant applications.

Efficient conversion of hemicellulose into high-value product and electric power by enzyme-engineered bacterial consortia.

As an abundant agricultural and forestry biomass resource, hemicelluloses are hard to be effectively degraded and utilized by microorganisms due to the constraints of membrane and metabolic regulations. Herein, we report a synthetic extracellular metabolic pathway with hemicellulose-degrading-enzymes controllably displayed on Escherichia coli surface as engineered bacterial consortia members for efficient utilization of xylan, the most abundant component in hemicellulose. Further, we develop a hemicellulose/O2 microbial fuel cell (MFC) configuring of enzyme-engineered bacterial consortia based bioanode and bacterial-displayed laccase based biocathode. The optimized MFC exhibited an open-circuit voltage of 0.71 V and a maximum power density (Pmax) of 174.33 ± 4.56 µW cm-2. Meanwhile, 46.6% (w/w) α-ketoglutarate was produced in this hemicellulose fed-MFC. Besides, the MFC retained over 95% of the Pmax during 6 days' operation. Therefore, this work establishes an effective and sustainable one-pot process for catalyzing renewable biomass into high-value products and electricity in an environmentally-friendly way.

Omic-driven strategies to unveil microbiome potential for biodegradation of plastics: a review.

Plastic waste accumulation has lately been identified as the leading and pervasive environmental concern, harming all living beings, natural habitats, and the global market. Given this issue, developing ecologically friendly solutions, such as biodegradation instead of standard disposal, is critical. To effectively address and develop better strategies, it is critical to understand the inter-relationship between microorganisms and plastic, the role of genes and enzymes involved in this process. However, the complex nature of microbial communities and the diverse mechanisms involved in plastic biodegradation have hindered the development of efficient plastic waste degradation strategies. Omics-driven approaches, encompassing genomics, transcriptomics and proteomics have revolutionized our understanding of microbial ecology and biotechnology. Therefore, this review explores the application of omics technologies in plastic degradation studies and discusses the key findings, challenges, and future prospects of omics-based approaches in identifying novel plastic-degrading microorganisms, enzymes, and metabolic pathways. The integration of omics technologies with advanced molecular technologies such as the recombinant DNA technology and synthetic biology would guide in the optimization of microbial consortia and engineering the microbial systems for enhanced plastic biodegradation under various environmental conditions.

Species- and subspecies-level characterization of health-associated bacterial consortia that colonize the human gut during infancy.

BACKGROUND: The human gut microbiome develops rapidly during infancy, a key window of development coinciding with the maturation of the adaptive immune system. However, little is known about the microbiome growth dynamics over the first few months of life and whether there are any generalizable patterns across human populations. We performed metagenomic sequencing on stool samples (n = 94) from a cohort of infants (n = 15) at monthly intervals in the first 6 months of life, augmenting our dataset with seven published studies for a total of 4,441 metagenomes from 1,162 infants. RESULTS: Strain-level de novo analysis was used to identify 592 of the most abundant organisms in the infant gut microbiome. Previously unrecognized consortia were identified which exhibited highly correlated abundances across samples and were composed of diverse species spanning multiple genera. Analysis of a published cohort of infants with cystic fibrosis identified one such novel consortium of diverse Enterobacterales which was positively correlated with weight gain. While all studies showed an increased community stability during the first year of life, microbial dynamics varied widely in the first few months of life, both by study and by individual. CONCLUSION: By augmenting published metagenomic datasets with data from a newly established cohort, we were able to identify novel groups of organisms that are correlated with measures of robust human development. We hypothesize that the presence of these groups may impact human health in aggregate in ways that individual species may not in isolation.

Mixed and membrane-separated culturing of synthetic cyanobacteria-yeast consortia reveals metabolic cross-talk mimicking natural cyanolichens.

Metabolite exchange mediates crucial interactions in microbial communities, significantly impacting global carbon and nitrogen cycling. Understanding these chemically-mediated interactions is essential for elucidating natural community functions and developing engineered synthetic communities. This study investigated membrane-separated bioreactors (mBRs) as a novel tool to identify transient metabolites and their producers/consumers in mixed microbial communities. We compared three co-culture methods (direct mixed, 2-chamber mBR, and 3-chamber mBR) to grow a synthetic binary community of the cyanobacterium Synechococcus elongatus PCC 7942 and the fungus Rhodotorula toruloides NBRC 0880, as well as axenic S. elongatus. Despite not being natural lichen constituents, these organisms exhibited interactions resembling those in cyanolichens. S. elongatus fixed CO2 into sugars as the primary shared metabolite, while R. toruloides secreted various biochemicals, predominantly sugar alcohols, mirroring the metabolite exchange observed in natural lichens. The mBR systems successfully captured metabolite gradients and revealed rapidly consumed compounds, including TCA cycle intermediates and amino acids. Our approach demonstrated that the 2-chamber mBR optimally balanced metabolite exchange and growth dynamics. This study provides insights into cross-species metabolic interactions and presents a valuable tool for investigating and engineering synthetic microbial communities with potential applications in biotechnology and environmental science.

Microbial inoculation accelerates rice straw decomposition by reshaping structure and function of lignocellulose-degrading microbial consortia in paddy fields.

Inoculating lignocellulose-degrading microorganisms can accelerate straw decomposition in paddy field; however, the relationship between indigenous and inoculated microorganisms remains unclear. This study explored the effects of microbial inoculation on straw decomposition, microbial community, lignocellulose-degrading consortia, and associated functional genes. After inoculation, straw degradation rate increased by up to 4.9 %, and the rice yield increased by 790 kg/ha. Microbial inoculation restructured soil microbial community, influencing key taxa and interactions within the microbial network. A lignocellulose-degrading consortia consisting 37 genera was established, with a notable increase in the relative abundance of lignocellulose-degrading bacteria following inoculation. Among them, Pseudarthrobacter, with high lignin-degrading enzyme activity, emerged as a key genus after inoculation. Additionally, the abundance of lignin-degrading enzyme genes also increased significantly after inoculation. These findings offer new insights into how microbial inoculation accelerates the in situ decomposition of rice straw by reshaping the structure and function of lignocellulose-degrading consortia within the soil ecosystem.

Tetracycline removal by immobilized indigenous bacterial consortium using biochar and biomass: Removal performance and mechanisms.

The significant influx of antibiotics into the environment represents ecological risks and threatens human health. Microbial degradation stands as a highly effective method for reducing antibiotic pollution. This study explored the potential of immobilized microbial consortia to efficiently degrade tetracycline. Concurrently, the suitability of different immobilization materials were assessed, with reed charcoal-immobilized consortia exhibiting the highest efficiency in removing tetracycline (92%). Similarly, wheat-bran-loaded bacterial consortia displayed a remarkable 11.43-fold increase in tetracycline removal compared with free consortia. Moreover, adding the carriers increased the nutrients, while the activities of both intracellular and extracellular catalases increased significantly post-immobilization, thus highlighting this enzyme's crucial role in tetracycline degradation. Finally, analysis of the microbial communities revealed the prevalence of Achromobacter and Parapedobacter, signifying their potential as key degraders. Overall, the immobilized consortia not only hold promise for application in the bioremediation of tetracycline-contaminated environment but also provide theoretical underpinnings for environmental remediation by microorganisms.

Predatory protists impact plant performance by promoting plant growth-promoting rhizobacterial consortia.

Plant performance is impacted by rhizosphere bacteria. These bacteria are subjected to both bottom-up control by root exudates as well as top-down control by predators, particularly protists. Protists stimulate plant growth-promoting microbes resulting in improved plant performance. However, knowledge of the mechanisms that determine the interconnections within such tripartite protist-bacteria-plant interactions remains limited. We conducted experiments examining the effects of different densities of the predatory protist Cercomonas lenta on rhizosphere bacterial communities, specifically zooming on interactions between Cercomonas lenta and key bacterial taxa, as well as interactions among key bacterial taxa. We tracked rhizosphere bacterial community composition, potential microbial interactions, and plant performance. We found that Cercomonas lenta inoculation led to an average increase in plant biomass of 92.0%. This effect was linked to an increase in plant growth-promoting rhizobacteria (Pseudomonas and Sphingomonas) and a decrease in bacteria (Chitinophaga) that negatively impact on plant growth-promoting rhizobacteria. We also found evidence for cooperative enhancements in biofilm formation within the plant growth-promoting rhizobacterial consortium. Cercomonas lenta enhanced a plant growth-promoting rhizobacterial consortium colonization by promoting its cooperative biofilm formation in the rhizosphere, leading to a 14.5% increase in phosphate solubilization that benefits plant growth. Taken together, we provide mechanistic insights into how the predatory protist Cercomonas lenta impacts plant growth, namely by stimulating plant beneficial microbes and enhancing their interactive activities such as biofilm formation. Predatory protists may therefore represent promising biological agents that can contribute to sustainable agricultural practices by promoting interactions between the plant and its microbiome.

Thermophilic Hemicellulases Secreted by Microbial Consortia Selected from an Anaerobic Digester.

The rise of agro-industrial activities over recent decades has exponentially increased lignocellulose biomasses (LCB) production. LCB serves as a cost-effective source for fermentable sugars and other renewable chemicals. This study explores the use of microbial consortia, particularly thermophilic consortia, for LCB deconstruction. Thermophiles produce stable enzymes that retain activity under industrial conditions, presenting a promising approach for LCB conversion. This research focused on two microbial consortia (i.e., microbiomes) that were analyzed for enzyme production using a cheap medium, i.e., a mixture of spent mushroom substrate (SMS) and digestate. The secreted xylanolytic enzymes were characterized in terms of temperature and pH optima, thermal stability, and hydrolysis products from LCB-derived polysaccharides. These enzymes showed optimal activity aligning with common biorefinery conditions and outperformed a formulated enzyme mixture in thermostability tests in the digestate. Phylogenetic and genomic analyses highlighted the genetic diversity and metabolic potential of these microbiomes. Bacillus licheniformis was identified as a key species, with two distinct strains contributing to enzyme production. The presence of specific glycoside hydrolases involved in the cellulose and hemicellulose degradation underscores these consortia's capacity for efficient LCB conversion. These findings highlight the potential of thermophilic microbiomes, isolated from an industrial environment, as a robust source of robust enzymes, paving the way for more sustainable and cost-effective bioconversion processes in biofuel and biochemical production and other biotechnological applications.

Leveraging microbial synergy: Predicting the optimal consortium to enhance the performance of microbial fuel cell using Subspace-kNN.

Microbial Fuel Cells (MFCs) are a sophisticated and advanced system that uses exoelectrogenic microorganisms to generate bioenergy. Predicting performance outcomes under experimental settings is challenging due to the intricate interactions that occur in mixed-species bioelectrochemical reactors like MFCs. One of the key factors that limit the MFC's performance is the presence of a microbial consortium. Traditionally, multiple microbial consortia are implemented in MFCs to determine the best consortium. This approach is laborious, inefficient, and wasteful of time and resources. The increase in the availability of soft computational techniques has allowed for the development of alternative strategies like artificial intelligence (AI) despite the fact that a direct correlation between microbial strain, microbial consortium, and MFC performance has yet to be established. In this work, a novel generic AI model based on subspace k-Nearest Neighbour (SS-kNN) is developed to identify and forecast the best microbial consortium from the constituent microbes. The SS-kNN model is trained with thirty-five different microbial consortia sharing different effluent properties. Chemical oxygen demand (COD) reduction, voltage generation, exopolysaccharide (EPS) production, and standard deviation (SD) of voltage generation are used as input features to train the SS-kNN model. The proposed SS-kNN model offers an accuracy of 100% during training period and 85.71% when it is tested with the data obtained from existing literature. The implementation of selected consortium (as predicted by SS-kNN model) improves the COD reduction capability of MFC by 15.67% than that of its constituent microbes which is experimentally verified. In addition, to prevent the effects of climate change and mitigate water pollution, the implementation of MFC technology ensures clean and green electricity. Consequently, achieving sustainable development goals (SDG) 6, 7, and 13.

Adaptation of a microbial consortium to pelagic Sargassum modifies its taxonomic and functional profile that improves biomethane potential.

In recent years, pelagic Sargassum has invaded the Caribbean coasts, and anaerobic digestion has been proposed as a sustainable management option. However, the complex composition of these macroalgae acts as a barrier to microbial degradation, thereby limiting methane production. Microbial adaptation is a promising strategy to improve substrate utilization and stress tolerance. This study aimed to investigate the adaptation of a microbial consortium to enhance methane production from the pelagic Sargassum. Microbial adaptation was performed in a fed-batch mode for 100 days by progressive feeding of Sargassum. The evolution of the microbial community was analyzed by high-throughput sequencing of 16S rRNA amplicons. Additionally, 16S rRNA data were used to predict functional profiles using the iVikodak platform. The results showed that, after adaptation, the consortium was dominated by the bacterial phyla Bacteroidota, Firmicutes, and Atribacterota, as well as methanogens of the families Methanotrichaceae and Methanoregulaceae. The abundance of predicted genes related to different metabolic functions was affected during the adaptation stage when Sargassum concentration was increased. At the end of the adaptation stage, the abundance of the predicted genes increased again. The adapted microbial consortium demonstrated a 60% increase in both biomethane potential and biodegradability index. This work offers valuable insights into the development of treatment technologies and the effective management of pelagic Sargassum in coastal regions, emphasizing the importance of microbial adaptation in this context.

Sustainable production of C50 carotenoid bacterioruberin from methane using soil-enriched microbial consortia.

Bacterioruberin is widely used in medicine, food, and cosmetics owing to its prominent characteristics of antioxidants and bioactivities. Bioconversion of methane into bacterioruberin is a promising way to address biomanufacturing substrate costs and greenhouse gas emissions but has not been achieved yet. Herein, this study aimed to upcycle methane to bacterioruberin by microbial consortia. The microbial consortia consist of Methylomonas and Methylophilus capable of synthesizing carotenoids from methane was firstly enriched from paddy soil. Through this microbial community, methane was successfully converted into C50 bacterioruberin for the first time. The bioconversion process was then optimized by the response surface methodology. Finally, the methane-derived bacterioruberin reached a record yield of 280.88 ± 2.94 µg/g dry cell weight. This study presents a cost-effective and eco-friendly approach for producing long-chain carotenoids from methane, offering a significant advancement in the direct conversion of greenhouse gases into value-added products.

Metabolic versatility of aerobic methane-oxidizing bacteria under anoxia in aquatic ecosystems.

The potential positive feedback between global aquatic deoxygenation and methane (CH4) emission emphasizes the importance of understanding CH4 cycling under O2-limited conditions. Increasing observations for aerobic CH4-oxidizing bacteria (MOB) under anoxia have updated the prevailing paradigm that MOB are O2-dependent; thus, clarification on the metabolic mechanisms of MOB under anoxia is critical and timely. Here, we mapped the global distribution of MOB under anoxic aquatic zones and summarized four underlying metabolic strategies for MOB under anoxia: (a) forming a consortium with oxygenic microorganisms; (b) self-generation/storage of O2 by MOB; (c) forming a consortium with non-oxygenic heterotrophic bacteria that use other electron acceptors; and (d) utilizing alternative electron acceptors other than O2. Finally, we proposed directions for future research. This study calls for improved understanding of MOB under anoxia, and underscores the importance of this overlooked CH4 sink amidst global aquatic deoxygenation.

Enhanced Dechlorination of the Herbicide Acetochlor by an Anaerobic Consortium via Sulfate Acclimation.

Acetochlor residues can contaminate anoxic habitats where anaerobic microbial transformation dominates. Herein, a highly efficient anaerobic acetochlor-degrading consortium ACT6 was enriched using sulfate and acetochlor as selection pressures. The acclimated consortium ACT6 showed an 8.7-fold increase in its ability to degrade acetochlor compared with the initial consortium ACT1. Two degradation pathways of acetochlor were found: reductive dechlorination and thiol-substitution dechlorination in the chloroacetyl group, in which the latter dominated. Acclimation enhanced the abundances of Desulfovibrio, Proteiniclasticum, and Lacrimispora from 0.7 to 28.0% (40-fold), 4.7 to 18.1% (4-fold), and 2.3 to 12.3% (5-fold), respectively, which were positively correlated with sulfate concentrations and acetochlor degradation ability. Three acetochlor-degrading anaerobes were isolated from the acclimated consortium ACT6, namely Cupidesulfovibrio sp. SRB-5, Proteiniclasticum sp. BAD-10, and Lacrimispora sp. BAD-7. This study provides new insights into the anaerobic catabolism of acetochlor and the anaerobic treatment of acetochlor in wastewater.

Mother's milk microbiota is associated with the developing gut microbial consortia in very-low-birth-weight infants.

Mother's milk contains diverse bacterial communities, although their impact on microbial colonization in very-low-birth-weight (VLBW, <1,500 g) infants remains unknown. Here, we examine relationships between the microbiota in preterm mother's milk and the VLBW infant gut across initial hospitalization (n = 94 mother-infant dyads, 422 milk-stool pairs). Shared zero-radius operational taxonomic units (zOTUs) between milk-stool pairs account for ∼30%-40% of zOTUs in the VLBW infant's gut. We show dose-response relationships between intakes of several genera from milk and their concentrations in the infant's gut. These relationships and those related to microbial sharing change temporally and are modified by in-hospital feeding practices (especially direct breastfeeding) and maternal-infant antibiotic use. Correlations also exist between milk and stool microbial consortia, suggesting that multiple milk microbes may influence overall gut communities together. These results highlight that the mother's milk microbiota may shape the gut colonization of VLBW infants by delivering specific bacteria and through intricate microbial interactions.

Enhancing nitrogen transformation and humification in cow manure composting through psychrophilic and thermophilic nitrifying bacterial consortium inoculation.

Excessive nitrogen release during composting poses significant challenges to both the environment and compost quality. Biological enhancement of humification and nitrogen conservation is an environmentally friendly and cost-effective approach to composting. The aim of this study was to develop a psychrophilic and thermophilic nitrifying bacterial consortium (CNB) and investigate its role in nitrogen transformation and humification during cow manure composting. Analysis revealed that CNB inoculation promoted microbial proliferation and metabolism, significantly increased the number of nitrifying bacteria (p < 0.05), and elevated the activity of nitrite oxidoreductase and nxrA gene abundance. Compared to the control, CNB inoculation promoted the formation of NO3--N (77.87-82.35 %), while reducing NH3 (48.89 %) and N2O (20.05 %) emissions, and increased humus content (16.22 %). Mantel analysis showed that the higher abundance of nitrifying bacteria and nxrA facilitated the nitrification of NH4+-N. The improvement in nitrite oxidoreductase activity promoted NO3--N formation, leading to increased humus content and enhanced compost safety.

Metabolic pathway analysis of methane from methanol as substrate in microbial consortium.

Simplified anaerobic digestion (SAD) of substrates facilitates microbial methanogenic pathways. In this study, a methane-producing microbial consortium from cow dung was enriched to determine the metabolism and metabolic pathway in the SAD of methanol. The results showed that methanol as a sole substrate produced 167 mL of methane at 10 days significantly higher than 58 mL, 17.6 mL, and 4 mL generated when methanol was combined with sodium formate, sodium formate alone, or sodium acetate. The relative abundance of Methanobacterium, Candidatus_Methanomethylophilus, Methanomassiliicoccus, and Methanosarcina was increased by 5.96 %, 3.77 %, 2.85 %, and 0.14 % in the methanol substrate of AD, respectively. Macrogenome sequencing indicates that methanol wasconverted into Methyl-CoM in the presence of Methanosarcina, which combines with Coenzyme B to produce methane. This study revealed that methanol is converted into methane by a simple pathway.

Consolidated bioprocessing of lignocellulosic wastes in Northwest China for D-glucaric acid production by an artificial microbial consortium.

D-glucaric acid is a platform chemical of great importance and the consolidated bioprocessing (CBP) of lignocellulose by the microbial consortium of Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 features prospects in biomanufacturing it. Here we compared some representative lignocelluloses in Northwest China including corn stover, wheat straw and switchgrass, and the leading pretreatments including steam explosion, subcritical water pretreatment, sodium hydroxide pretreatment, aqueous ammonia pretreatment, lime pretreatment, and diluted sulfuric acid pretreatment. It was found that sodium hydroxide pretreated switchgrass (SHPSG) was the best substrate for D-glucaric acid production, resulting in the highest D-glucaric acid titers, 11.69 ± 0.73 g/L in shake flask and 15.71 ± 0.80 g/L in 10L airlift fermenter, respectively. To the best of our knowledge, this is the highest D-glucaric acid production titer from lignocellulosic biomass. This work offers a paradigm of producing low-cost D-glucaric acid for low-carbon polyethylene 2,5-furandicarboxylate (PEF) and a reference on developing biorefinery in Northwest China.