Establishment of low-cost production platforms of polyhydroxyalkanoate bioplastics from Halomonas cupida J9.

Microbial production of polyhydroxyalkanoate (PHA) is greatly restricted by high production cost arising from high-temperature sterilization and expensive carbon sources. In this study, a low-cost PHA production platform was established from Halomonas cupida J9. First, a marker-less genome-editing system was developed in H. cupida J9. Subsequently, H. cupida J9 was engineered to efficiently utilize xylose for PHA biosynthesis by introducing a new xylose metabolism module and blocking xylonate production. The engineered strain J9UΔxylD-P8xylA has the highest PHA yield (2.81 g/L) obtained by Halomonas with xylose as the sole carbon source so far. This is the first report on the production of short- and medium-chain-length (SCL-co-MCL) PHA from xylose by Halomonas. Interestingly, J9UΔxylD-P8xylA was capable of efficiently utilizing glucose and xylose as co-carbon sources for PHA production. Furthermore, fed-batch fermentation of J9UΔxylD-P8xylA coupled to a glucose/xylose co-feeding strategy reached up to 12.57 g/L PHA in a 5-L bioreactor under open and unsterile condition. Utilization of corn straw hydrolysate as the carbon source by J9UΔxylD-P8xylA reached 7.0 g/L cell dry weight (CDW) and 2.45 g/L PHA in an open fermentation. In summary, unsterile production in combination with inexpensive feedstock highlights the potential of the engineered strain for the low-cost production of PHA from lignocellulose-rich agriculture waste.

Self-Buffering system for Cost-Effective production of lactic acid from glucose and xylose using Acid-Tolerant Issatchenkia orientalis.

This study presents a cost-effective strategy for producing organic acids from glucose and xylose using the acid-tolerant yeast, Issatchenkia orientalis. I. orientalis was engineered to produce lactic acid from xylose, and the resulting strain, SD108XL, successfully converted sorghum hydrolysates into lactic acid. In order to enable low-pH fermentation, a self-buffering strategy, where the lactic acid generated by the SD108XL strain during fermentation served as a buffer, was developed. As a result, the SD108 strain produced 67 g/L of lactic acid from 73 g/L of glucose and 40 g/L of xylose, simulating a sugar composition of sorghum biomass hydrolysates. Moreover, techno-economic analysis underscored the efficiency of the self-buffering strategy in streamlining the downstream process, thereby reducing production costs. These results demonstrate the potential of I. orientalis as a platform strain for the cost-effective production of organic acids from cellulosic hydrolysates.

Construction of an economical xylose-utilizing Saccharomyces cerevisiae and its ethanol fermentation.

Traditional industrial Saccharomyces cerevisiae could not metabolize xylose due to the lack of a specific enzyme system for the reaction from xylose to xylulose. This study aims to metabolically remould industrial S. cerevisiae for the purpose of utilizing both glucose and xylose with high efficiency. Heterologous gene xylA from Piromyces and homologous genes related to xylose utilization were selected to construct expression cassettes and integrated into genome. The engineered strain was domesticated with industrial material under optimizing conditions subsequently to further improve xylose utilization rates. The resulting S. cerevisiae strain ABX0928-0630 exhibits a rapid growth rate and possesses near 100% xylose utilization efficiency to produce ethanol with industrial material. Pilot-scale fermentation indicated the predominant feature of ABX0928-0630 for industrial application, with ethanol yield of 0.48 g/g sugars after 48 hours and volumetric xylose consumption rate of 0.87 g/l/h during the first 24 hours. Transcriptome analysis during the modification and domestication process revealed a significant increase in the expression level of pathways associated with sugar metabolism and sugar sensing. Meanwhile, genes related to glycerol lipid metabolism exhibited a pattern of initial increase followed by a subsequent decrease, providing a valuable reference for the construction of efficient xylose-fermenting strains.

Efficient and economical production of polyhydroxyalkanoate from sustainable rubber wood hydrolysate and xylose as co-substrate by mixed microbial cultures.

Using co-substrate to accumulate polyhydroxyalkanoate (PHA) is an efficient approach to reduce production cost and improve yield of PHA. In the study, PHA was biosynthesized under full aerobic mode by using rubber wood hydrolysate and xylose co-substrate as the carbon source. The effects of co-substrate on PHA production, microbial community and carbon conversion were explored. The results showed that proper addition of xylose was beneficial for the synthesis of PHA and monomer 3-hydroxyvalerate (3HV). Higher conversion yield of substrate-to-PHA (YPHA/S) of 0.933 g COD PHA/g COD S and PHA content of 43.6 g PHA/100 g VSS were gained at co-substrate ratio of 1:1. Likewise, under this condition, PHA production also reached the highest value of 1849 mg COD/L (1088 mg/L). Moreover, the addition of xylose created a favorable screening of PHA dominant strains, improved the conversion of carbon source, and saved 72.3% of feedstock consumption.

Xylitol production by Pseudomonas gessardii VXlt-16 from sugarcane bagasse hydrolysate and cost analysis.

Xylitol is a well-known sugar alcohol with exponentially rising market demand due to its diverse industrial applications. Organic agro-industrial residues (OAIR) are economic alternative for the cost-effective production of commodity products along with addressing environmental pollution. The present study aimed to design a process for xylitol production from OAIR via microbial fermentation with Pseudomonas gessardii VXlt-16. Parametric analysis with Taguchi orthogonal array approach resulted in a conversion factor of 0.64 g xylitol/g xylose available in untreated sugarcane bagasse hydrolysate (SBH). At bench scale, the product yield increased to 71.98/100 g (0.66 g/L h). 48.49 g of xylitol crystals of high purity (94.56%) were recovered after detoxification with 2% activated carbon. Cost analysis identified downstream operations as one of the cost-intensive parts that can be countered by adsorbent recycling. Spent carbon, regenerated with acetic acid washing can be reused for six cycles effectively and reduced downstream cost by about ≈32%. The strategy would become useful in the cost-effective production of several biomass-dependent products like proteins, enzymes, organic acids, as well.

A cost-practical cell-recycling process for xylonic acid bioproduction from acidic lignocellulosic hydrolysate with whole-cell catalysis of Gluconobacter oxydans.

Xylonic acid (XA), as a bio-based platform chemical, is of considerable interest for xylose bioconversion. The whole-cell catalysis of Gluconobacter oxydans presents a promising application potential, while the hard works of cell culture still severely hinder XA business from the crude toxics-containing lignocellulosic hydrolysate. Hence, the bacterial cells should be recycled to reduce commercial production cost. The implementation of diatomite detoxification not only absorbs most of the degraded inhibitors in hydrolysate, but also confines the sugar contents loss with 10% and allows the bacterial cells to maintain 90% bioconversion performance during cell-recycling operation. Additionally, a scale-up of XA bioproduction was achieved in a sealed oxygen supply fermenter. Finally, 210 g XA was produced from 1000 g corncob originated hydrolysate within 24 h of whole-cell catalysis. Diatomite treatment provides an efficient and cost-practical approach for the commercial bioproduction of biochemicals like XA from lignocellulosic biomass.

Assessment of different Bacillus coagulans strains for l-lactic acid production from defined media and gardening hydrolysates: Effect of lignocellulosic inhibitors.

Cellulose valorisation has been successfully addressed for years. However, the use of hemicellulosic hydrolysates is limited due to the presence of C5-sugars and inhibitors formed during pretreatment. Bacillus coagulans is one of the few bacteria able to utilize both C6- and C5-sugars to produce l-lactic acid, but its susceptibility to the lignocellulosic inhibitors needs further investigation. For such a purpose, the tolerance of different B. coagulans strains to increasing concentrations of inhibitors is studied. The isolated A162 strain reached the highest l-lactic acid productivity in all cases (up to 2.4 g L-1  h-1), even in presence of 5 g L-1 of furans and phenols. Remarkably, most of furans and phenolic aldehydes were removed from defined media and hemicellulosic gardening hydrolysate after fermentation with A162. Considering the high productivities and the biodetoxifying effect attained, A162 could be pointed out as a great candidate for valorisation of mixed sugars from hemicellulosic hydrolysates with high inhibitors concentration, promoting the implementation of lignocellulosic biorefineries.

Low-Cost Cellulase-Hemicellulase Mixture Secreted by Trichoderma harzianum EM0925 with Complete Saccharification Efficacy of Lignocellulose.

Fermentable sugars are important intermediate products in the conversion of lignocellulosic biomass to biofuels and other value-added bio-products. The main bottlenecks limiting the production of fermentable sugars from lignocellulosic biomass are the high cost and the low saccharification efficiency of degradation enzymes. Herein, we report the secretome of Trichoderma harzianum EM0925 under induction of lignocellulose. Numerously and quantitatively balanced cellulases and hemicellulases, especially high levels of glycosidases, could be secreted by T. harzianum EM0925. Compared with the commercial enzyme preparations, the T. harzianum EM0925 enzyme cocktail presented significantly higher lignocellulolytic enzyme activities and hydrolysis efficiency against lignocellulosic biomass. Moreover, 100% yields of glucose and xylose were obtained simultaneously from ultrafine grinding and alkali pretreated corn stover. These findings demonstrate a natural cellulases and hemicellulases mixture for complete conversion of biomass polysaccharide, suggesting T. harzianum EM0925 enzymes have great potential for industrial applications.

Uncertainty reduction in biochemical kinetic models: Enforcing desired model properties.

A persistent obstacle for constructing kinetic models of metabolism is uncertainty in the kinetic properties of enzymes. Currently, available methods for building kinetic models can cope indirectly with uncertainties by integrating data from different biological levels and origins into models. In this study, we use the recently proposed computational approach iSCHRUNK (in Silico Approach to Characterization and Reduction of Uncertainty in the Kinetic Models), which combines Monte Carlo parameter sampling methods and machine learning techniques, in the context of Bayesian inference. Monte Carlo parameter sampling methods allow us to exploit synergies between different data sources and generate a population of kinetic models that are consistent with the available data and physicochemical laws. The machine learning allows us to data-mine the a priori generated kinetic parameters together with the integrated datasets and derive posterior distributions of kinetic parameters consistent with the observed physiology. In this work, we used iSCHRUNK to address a design question: can we identify which are the kinetic parameters and what are their values that give rise to a desired metabolic behavior? Such information is important for a wide variety of studies ranging from biotechnology to medicine. To illustrate the proposed methodology, we performed Metabolic Control Analysis, computed the flux control coefficients of the xylose uptake (XTR), and identified parameters that ensure a rate improvement of XTR in a glucose-xylose co-utilizing S. cerevisiae strain. Our results indicate that only three kinetic parameters need to be accurately characterized to describe the studied physiology, and ultimately to design and control the desired responses of the metabolism. This framework paves the way for a new generation of methods that will systematically integrate the wealth of available omics data and efficiently extract the information necessary for metabolic engineering and synthetic biology decisions.

Biosynthetic strategies to produce xylitol: an economical venture.

Xylitol is a natural five-carbon sugar alcohol with potential for use in food and pharmaceutical industries owing to its insulin-independent metabolic regulation, tooth rehardening, anti-carcinogenic, and anti-inflammatory, as well as osteoporosis and ear infections preventing activities. Chemical and biosynthetic routes using D-xylose, glucose, or biomass hydrolysate as raw materials can produce xylitol. Among these methods, microbial production of xylitol has received significant attention due to its wide substrate availability, easy to operate, and eco-friendly nature, in contrast with high-energy consuming and environmental-polluting chemical method. Though great advances have been made in recent years for the biosynthesis of xylitol from xylose, glucose, and biomass hydrolysate, and the yield and productivity of xylitol are substantially improved by metabolic engineering and optimizing key metabolic pathway parameters, it is still far away from industrial-scale biosynthesis of xylitol. In contrary, the chemical synthesis of xylitol from xylose remains the dominant route. Economic and highly efficient xylitol biosynthetic strategies from an abundantly available raw material (i.e., glucose) by engineered microorganisms are on the hard way to forwarding. However, synthetic biology appears as a novel and promising approach to develop a super yeast strain for industrial production of xylitol from glucose. After a brief overview of chemical-based xylitol production, we critically analyzed and comprehensively summarized the major metabolic strategies used for the enhanced biosynthesis of xylitol in this review. Towards the end, the study is wrapped up with current challenges, concluding remarks, and future prospects for designing an industrial yeast strain for xylitol biosynthesis from glucose.

Harnessing xylose pathways for biofuels production.

Energy security, environmental pollution, and economic development drive the development of alternatives to fossil fuels as an urgent global priority. Lignocellulosic biomass has the potential to contribute to meeting the demand for biofuel production via hydrolysis and fermentation of released sugars, such as glucose, xylose, and arabinose. Construction of robust cell factories requires introducing and rewiring of their metabolism to efficiently use all these sugars. Here, we review recent advances in re-constructing pathways for metabolism of pentoses, with special focus on xylose metabolism in the most widely used cell factories Saccharomyces cerevisiae and Escherichia coli. We also highlight engineering advanced biofuels-synthesis pathways and describes progress toward overcoming the challenges facing adoption of large-scale biofuel production.

Lactic acid production from glucose and xylose using the lactogenic Escherichia coli strain JU15: Experiments and techno-economic results.

In this work, d-lactic acid production was evaluated from a simulated hydrolysate of corn stover (32 g/L xylose, 42 g/L glucose) with the metabolically engineered Escherichia coli strain JU15. Based on the experimental results, a technical and economic analysis of the entire process was performed using the Aspen Plus software. As a result, it was possible to show that the strain can efficiently produce lactic acid from both sugars, reaching a final concentration of 40 g/L and a yield of 0.6 g lactic acid/g sugars. The process is economically viable at higher scales of 1000 tons/day. The cost distribution is influenced by the scale of the process; on a larger scale, the cost of raw materials represents a higher percentage of total cost than it does on smaller scales. The use of a metabolically engineered strain allows a better use of the sugars obtained from agroindustrial residues.

Dietary D-xylose effects on growth performance, portal nutrient fluxes, and energy expenditure in growing pigs.

Xylanase is commonly added to pig diets rich in arabinoxylans to promote nutrient utilization and growth. However, high doses of xylanase could release high amounts of xylose in the upper gut, which could have negative nutritional and metabolic implications. However, the amount of xylose to elicit such adverse effects is not clear. Thus, two experiments were conducted to investigate the effects of dietary xylose on the growth performance and portal-drained viscera (PDV) fluxes of glucose (GLU), urea-N (BUN), insulin production, and O2 consumption in growing pigs. In Exp. 1, 64 pigs (21.4 ± 0.1 kg BW), housed as either two barrows or gilts per pen (eight pens per diet) were used to determine the effects of increasing levels of D-xylose (0, 5, 15, and 25%) in a corn-soybean meal-cornstarch-based diet on pig growth performance in a 28-d trial. Cornstarch was substituted for D-xylose (wt/wt) in the control diet. BW and feed intake were monitored weekly. D-xylose linearly reduced (P < 0.05) final BW, ADG, and G:F but not ADFI. However, final BW, ADG, and G:F of pigs fed 15% D-xylose did not differ from pigs fed 0% D-xylose. Thus, the results suggested that pigs could tolerate up to 15% dietary D-xylose. In Exp. 2, six gilts (22.8 ± 1.6 kg BW), fitted with permanent catheters in the portal vein, ileal vein, and carotid artery, were fed the 0% and 15% D-xylose diets at 4% of their BW once daily at 0900 h for 7 d in a cross-over design (six pigs per diet). On d 7, pigs were placed in indirect calorimeters to measure whole-animal O2 consumption and sample blood simultaneously for 6 h from the portal vein and carotid artery after feeding to assay GLU, O2, BUN, and insulin concentrations. Net portal nutrients and insulin production were calculated as porto-arterial concentration differences × portal blood flow (PBF) rate, whereas PDV O2 consumption was calculated as arterial-portal O2 differences × PBF. Diet had no effect on postprandial PBF, insulin production, and portal BUN flux and O2 consumption. Pigs fed 0% D-xylose had greater (P < 0.05) postprandial portal and arterial BUN concentrations, and portal GLU concentration and flux than pigs fed 15% D-xylose diet. In conclusion, feeding growing pigs a diet containing 15% D-xylose did not reduce pig performance or affect PDV energetic demand but reduced GLU fluxes.

Engineering NOG-pathway in Escherichia coli for poly-(3-hydroxybutyrate) production from low cost carbon sources.

Poly-(3-hydroxybutyrate) (P3HB) is a polyester with biodegradable and biocompatible characteristics suitable for bio-plastics and bio-medical use. In order to reduce the raw material cost, cheaper carbon sources such as xylose and glycerol were evaluated for P3HB production. We first conducted genome-scale metabolic network analysis to find the optimal pathways for P3HB production using xylose or glycerol respectively as the sole carbon sources. The results indicated that the non-oxidative glycolysis (NOG) pathway is important to improve the product yields. We then engineered this pathway into E. coli by introducing foreign phophoketolase enzymes. The results showed that the carbon yield improved from 0.19 to 0.24 for xylose and from 0.30 to 0.43 for glycerol. This further proved that the introduction of NOG pathway can be used as a general strategy to improve P3HB production.

Comparative assessment of fermentative capacity of different xylose-consuming yeasts.

BACKGROUND: Understanding the effects of oxygen levels on yeast xylose metabolism would benefit ethanol production. In this work, xylose fermentative capacity of Scheffersomyces stipitis, Spathaspora passalidarum, Spathaspora arborariae and Candida tenuis was systematically compared under aerobic, oxygen-limited and anaerobic conditions. RESULTS: Fermentative performances of the four yeasts were greatly influenced by oxygen availability. S. stipitis and S. passalidarum showed the highest ethanol yields (above 0.44 g g-1) under oxygen limitation. However, S. passalidarum produced 1.5 times more ethanol than S. stipitis under anaerobiosis. While C. tenuis showed the lowest xylose consumption rate and incapacity to produce ethanol, S. arborariae showed an intermediate fermentative performance among the yeasts. NAD(P)H xylose reductase (XR) activity in crude cell extracts correlated with xylose consumption rates and ethanol production. CONCLUSIONS: Overall, the present work demonstrates that the availability of oxygen influences the production of ethanol by yeasts and indicates that the NADH-dependent XR activity is a limiting step on the xylose metabolism. S. stipitis and S. passalidarum have the greatest potential for ethanol production from xylose. Both yeasts showed similar ethanol yields near theoretical under oxygen-limited condition. Besides that, S. passalidarum showed the best xylose consumption and ethanol production under anaerobiosis.

Fed-batch hydrolysate addition and cell separation by settling in high cell density lignocellulosic ethanol fermentations on AFEX™ corn stover in the Rapid Bioconversion with Integrated recycling Technology process.

The Rapid Bioconversion with Integrated recycling Technology (RaBIT) process uses enzyme and yeast recycling to improve cellulosic ethanol production economics. The previous versions of the RaBIT process exhibited decreased xylose consumption using cell recycle for a variety of different micro-organisms. Process changes were tested in an attempt to eliminate the xylose consumption decrease. Three different RaBIT process changes were evaluated in this work including (1) shortening the fermentation time, (2) fed-batch hydrolysate addition, and (3) selective cell recycling using a settling method. Shorting the RaBIT fermentation process to 11 h and introducing fed-batch hydrolysate addition eliminated any xylose consumption decrease over ten fermentation cycles; otherwise, decreased xylose consumption was apparent by the third cell recycle event. However, partial removal of yeast cells during recycle was not economical when compared to recycling all yeast cells.

Comparative techno-economic analysis of steam explosion, dilute sulfuric acid, ammonia fiber explosion and biological pretreatments of corn stover.

Pretreatment is required to destroy recalcitrant structure of lignocelluloses and then transform into fermentable sugars. This study assessed techno-economics of steam explosion, dilute sulfuric acid, ammonia fiber explosion and biological pretreatments, and identified bottlenecks and operational targets for process improvement. Techno-economic models of these pretreatment processes for a cellulosic biorefinery of 113.5 million liters butanol per year excluding fermentation and wastewater treatment sections were developed using a modelling software-SuperPro Designer. Experimental data of the selected pretreatment processes based on corn stover were gathered from recent publications, and used for this analysis. Estimated sugar production costs ($/kg) via steam explosion, dilute sulfuric acid, ammonia fiber explosion and biological methods were 0.43, 0.42, 0.65 and 1.41, respectively. The results suggest steam explosion and sulfuric acid pretreatment methods might be good alternatives at present state of technology and other pretreatment methods require research and development efforts to be competitive with these pretreatment methods.

Lipids of Rhodotorula mucilaginosa IIPL32 with biodiesel potential: Oil yield, fatty acid profile, fuel properties.

This study analyzes the single cell oil (SCO), fatty acid profile, and biodiesel fuel properties of the yeast Rhodotorula mucilaginosa IIPL32 grown on the pentose fraction of acid pre-treated sugarcane bagasse as a carbon source. The yeast biomass from nitrogen limiting culture conditions (15.3 g L-1 ) was able to give the SCO yield of 0.17 g g-1 of xylose consumed. Acid digestion, cryo-pulverization, direct in situ transesterification, and microwave assisted techniques were evaluated in comparison to the Soxhlet extraction for the total intracellular yeast lipid recovery. The significant differences were observed among the SCO yield of different methods and the in situ transesterification stood out most for effective yeast lipid recovery generating 97.23 mg lipid as FAME per gram dry biomass. The method was fast and consumed lesser solvent with greater FAME yield while accessing most cellular fatty acids present. The yeast lipids showed the major presence of monounsaturated fatty esters (35-55%; 18:1, 16:1) suitable for better ignition quality, oxidative stability, and cold-flow properties of the biodiesel. Analyzed fuel properties (density, kinematic viscosity, cetane number) of the yeast oil were in good agreement with international biodiesel standards. The sugarcane bagasse-derived xylose and the consolidated comparative assessment of lab scale SCO recovery methods highlight the necessity for careful substrate choice and validation of analytical method in yeast oil research. The use of less toxic co-solvents together with solvent recovery and recycling would help improve process economics for sustainable production of biodiesel from the hemicellulosic fraction of cheap renewable sources.

Direct bioethanol production from wheat straw using xylose/glucose co-fermentation by co-culture of two recombinant yeasts.

To achieve a cost-effective bioconversion of lignocellulosic materials, a novel xylose/glucose co-fermentation process by co-culture of cellulose-utilizing recombinant Saccharomyces cerevisiae (S. cerevisiae) and xylan-utilizing recombinant Pichia pastoris (P. pastoris) was developed, in which ethanol was produced directly from wheat straw without additional hydrolytic enzymes. Recombinant S. cerevisiae coexpressing three types of cellulase and recombinant P. pastoris coexpressing two types of xylanase were constructed, respectively. All cellulases and xylanases were successfully expressed and similar extracellular activity was demonstrated. The maximum ethanol concentration of 32.6 g L-1 with the yield 0.42 g g-1 was achieved from wheat straw corresponding to 100 g L-1 of total sugar after 80 h co-fermentation, which corresponds to 82.6% of the theoretical yield. These results demonstrate that the direct and efficient ethanol production from lignocellulosic materials is accomplished by simultaneous saccharification (cellulose and hemicellulose) and co-fermentation (glucose and xylose) with the co-culture of the two recombinant yeasts.

High-pressure carbon dioxide/water pre-treatment of sugarcane bagasse and elephant grass: Assessment of the effect of biomass composition on process efficiency.

The performance of two lignocellulosic biomasses was studied in high-pressure carbon dioxide/water pre-treatment. Sugarcane bagasse and elephant grass were used to produce C5-sugars from hemicellulose and, simultaneously, to promote cellulose digestibility for enzymatic saccharification. Different pre-treatment conditions, with combined severity factor ranging from -1.17 to -0.04, were evaluated and maximal total xylan to xylose yields of 59.2wt.% (34.4wt.% xylooligomers) and 46.4wt.% (34.9wt.% xylooligomers) were attained for sugarcane bagasse and elephant grass, respectively. Furthermore, pre-treated biomasses were highly digestible, with glucan to glucose yields of 77.2mol% and 72.4mol% for sugarcane bagasse and elephant grass, respectively. High-pressure carbon dioxide/water pre-treatment provides high total C5-sugars and glucose recovery from both lignocellulosic biomasses; however it is highly influenced by composition and intrinsic features of each biomass. The obtained results confirm this approach as an effective and greener alternative to conventional pre-treatment processes.