Lignocellulosic Hydrolysates Research Articles

Microbial detoxification of lignocellulosic biomass hydrolysates: Biochemical and molecular aspects, challenges, exploits and future perspectives.

Valorization of lignocellulosic biomass (LB) has the potential to secure sustainable energy production without impacting food insecurity, whist relieving over reliance on finite fossil fuels. Agro-derived lignocellulosic residues such as wheat straw, switchgrass, rice bran, and miscanthus have gained relevance as feedstocks for the production of biofuels and chemicals. However, the microorganisms employed in fermentative conversion of carbohydrates to fuels and chemicals are unable to efficiently utilize the sugars derived from LB due to co-production of lignocellulose-derived microbial inhibitory compounds (LDMICs) during LB pretreatment. LDMICs impact microbial growth by inhibition of specific enzymes, cause DNA and cell membrane damage, and elicit cellular redox imbalance. Over the past decade, success has been achieved with the removal of LDMICs prior to fermentation. However, LDMICs removal by chemical processes is often accompanied by sugar losses, which negatively impacts the overall production cost. Hence, in situ removal of LDMICs by fermentative organisms during the fermentation process has garnered considerable attention as the "go-to" approach for economical LDMICs detoxification and bio-chemicals production. In situ removal of LDMICs has been pursued by either engineering more robust biocatalysts or isolating novel microbial strains with the inherent capacity to mineralize or detoxify LDMICs to less toxic compounds. While some success has been made along this line, efficient detoxification and robust production of target bio-chemicals in lignocellulosic hydrolysates (LHs) under largely anaerobic fermentative conditions remains a lingering challenge. Consequently, LB remains an underutilized substrate for bio-chemicals production. In this review, the impact of microbial LH detoxification on overall target molecule production is discussed. Further, the biochemical pathways and mechanisms employed for in situ microbial detoxification of furanic LDMICs [e.g., furfural and 5-hydroxymethylfurfural (HMF)] and phenolic LDMICs (e.g., syringaldehyde, p-coumaric acid, 4-hydroxybenzaldehyde, vanillin, and ferulic acid) are discussed. More importantly, metabolic engineering strategies for the development of LDMIC-tolerant and bio-chemicals overproducing strains and processes are highlighted.

Overexpression of PtoMYB115 improves lignocellulose recalcitrance to enhance biomass digestibility and bioethanol yield by specifically regulating lignin biosynthesis in transgenic poplar

BackgroundWoody plants provide the most abundant biomass resource that is convertible for biofuels. Since lignin is a crucial recalcitrant factor against lignocellulose hydrolysis, genetic engineering of lignin biosynthesis is considered as a promising solution. Many MYB transcription factors have been identified to involve in the regulation of cell wall formation or phenylpropanoid pathway. In a previous study, we identified that PtoMYB115 contributes to the regulation of proanthocyanidin pathway, however, little is known about its role in lignocellulose biosynthesis and biomass saccharification in poplar.ResultsHere, we detected the changes of cell wall features and examined biomass enzymatic saccharification for bioethanol production under various chemical pretreatments in PtoMYB115 transgenic plants. We reported that PtoMYB115 might specifically regulate lignin biosynthesis to affect xylem development. Overexpression of PtoMYB115 altered lignin biosynthetic gene expression, resulting in reduced lignin deposition, raised S/G and beta-O-4 linkage, resulting in a significant reduction in cellulase adsorption with lignin and an increment in cellulose accessibility. These alterations consequently improved lignocellulose recalcitrance for significantly enhanced biomass saccharification and bioethanol yield in the PtoMYB115-OE transgenic lines. In contrast, the knockout of PtoMYB115 by CRISPR/Cas9 showed reduced woody utilization under various chemical pretreatments.ConclusionsThis study shows that PtoMYB115 plays an important role in specifically regulating lignin biosynthesis and improving lignocellulose features. The enhanced biomass saccharification and bioethanol yield in the PtoMYB115-OE lines suggests that PtoMYB115 is a candidate gene for genetic modification to facilitate the utilization of biomass.

Биоконверсия целлюлозосодержащего сырья. Ферментативный гидролиз целлюлозы (обзор литературы)

Lignocellulose is the most abundant biopolymer on earth. The hydrolysis of lignocellulose to fermentable sugars is a prerequisite for its successful use as a substrate for the large-scale production of value-added industrial products. Enzymatic hydrolysis of lignocellulose is carried out by cellulases, hemicellulases, and ligninases in synergy or individually. The review describes the enzymatic hydrolysis of cellulose, hemicellulose, and lignin. Information on solid-phase and submerged microorganisms fermentation used to obtain cellulases was given. Methods for increasing the level of production and activity of cellulases were characterized. The industrial application of cellulases, including for the production of bioethanol, was described.

Co‑cultivation of anaerobic fungi with Clostridium acetobutylicum bolsters butyrate and butanol production from cellulose and lignocellulose.

A system for co-cultivation of anaerobic fungi with anaerobic bacteria was established based on lactate cross-feeding to produce butyrate and butanol from plant biomass. Several co-culture formulations were assembled that consisted of anaerobic fungi (Anaeromyces robustus, Neocallimastix californiae, or Caecomyces churrovis) with the bacterium Clostridium acetobutylicum. Co-cultures were grown simultaneously (e.g., 'one pot'), and compared to cultures where bacteria were cultured in fungal hydrolysate sequentially. Fungal hydrolysis of lignocellulose resulted in 7-11mM amounts of glucose and xylose, as well as acetate, formate, ethanol, and lactate to support clostridial growth. Under these conditions, one-stage simultaneous co-culture of anaerobic fungi with C. acetobutylicum promoted the production of butyrate up to 30mM. Alternatively, two-stage growth slightly promoted solventogenesis and elevated butanol levels (∼4-9mM). Transcriptional regulation in the two-stage growth condition indicated that this cultivation method may decrease the time required to reach solventogenesis and induce the expression of cellulose-degrading genes in C. acetobutylicum due to relieved carbon-catabolite repression. Overall, this study demonstrates a proof of concept for biobutanol and bio-butyrate production from lignocellulose using an anaerobic fungal-bacterial co-culture system.

Enzymatic formulation strategies unlock highly-efficient saccharification of distinct pretreated corncobs

Optimization of the composition of enzyme cocktail is an effective way to improve the hydrolytic efficiency and reduce the cost during enzymatic hydrolysis of lignocellulose. Herein, the optimal enzymes ratios of enzyme cocktails for efficient hydrolysis of corncobs with distinct pretreatments were quantitatively formulated. As the importance of pretreatment type on xylan extraction, the optimized cellulase/hemicellulase ratios for NaClO2, H2SO4 and steam explosion treated corncob were 1:1.5, 1:0.15 and 1:0.75, respectively. Meanwhile, different requirements for types of cellulases are also determined: endoglucanase and cellobiohydrolase were key enzymes in the hydrolysis of NaClO2 and H2SO4 treated corncobs, respectively, while the synergy of three kinds of cellulases was preferred for steam-exploded corncob. Furthermore, based on the optimized cellulase/hemicellulase ratios, cellulases proportions for hydrolyzing the corncobs pretreated with distinctive methods were determined. Notably, by the enzymatic formulation strategy, 100% yields of glucose and xylose were obtained simultaneously from NaClO2 treated corncob, which was up to 70.1% and 80.1% at high solid loading (20%), respectively. Without detoxification of hydrolysate, high isopropanol yield of 0.45 g/g (of dry matter) was achieved. Hence, this study demonstrated a low cost and high efficient conversion strategy for prospective high-value utilization of lignocellulose.

Enhancing economic and environmental friendliness of xylonic acid bioproduction from corncob hydrolysate by the combined recycling-technology of detoxifying-resin and catalyzing-cell

The bioproduction of xylonic acid (XA) from acidic lignocellulosic hydrolysate presents a potential booming research area, that XA serves as an important building block of biomass-based chemicals for biomass valorization. However, the existence of inhibiting compounds such as acetic acid in hydrolysate greatly influences bacterial cell metabolism and catalysis, as well as the following electrodialysis separation for XA purification. Therefore, the removal of inhibitors including acetic acid is an essential issue in the industrial bioproduction of XA. In this study, five polymer resins were evaluated on their ability to remove acetic acid and other inhibitors from acidic hydrolysate of corncob (AHC). By comparison, anion exchange resin 335 performed the best on the removal of acetic acid in the model solution and AHC at 100 % and 80 %, respectively, while the sugar loss was also confined within 10 %. Finally, 208.6 g XA was obtained with a productivity of 5.8 g/L/h by 6 rounds of cell-recycling bioconversion. Moreover, recycled resin kept at least 80% toxic removal efficiency after 4 renewed rounds with hot water washing. The combined recycling-technology of detoxifying-resin and catalyzing-cell greatly facilitate economic and environmental bioproduction of XA from lignocellulosic biomass.

Lipid production from lignocellulosic biomass using an engineered Yarrowia lipolytica strain

BackgroundThe utilization of industrial wastes as feedstock in microbial-based processes is a one of the high-potential approach for the development of sustainable, environmentally beneficial and valuable bioproduction, inter alia, lipids. Rye straw hydrolysate, a possible renewable carbon source for bioconversion, contains a large amount of xylose, inaccessible to the wild-type Yarrowia lipolytica strains. Although these oleaginous yeasts possesses all crucial genes for xylose utilization, it is necessary to induce their metabolic pathway for efficient growth on xylose and mixed sugars from agricultural wastes. Either way, biotechnological production of single cell oils (SCO) from lignocellulosic hydrolysate requires yeast genome modification or adaptation to a suboptimal environment.ResultsThe presented Y. lipolytica strain was developed using minimal genome modification—overexpression of endogenous xylitol dehydrogenase (XDH) and xylulose kinase (XK) genes was sufficient to allow yeast to grow on xylose as a sole carbon source. Diacylglycerol acyltransferase (DGA1) expression remained stable and provided lipid overproduction. Obtained an engineered Y.lipolytica strain produced 5.51 g/L biomass and 2.19 g/L lipids from nitrogen-supplemented rye straw hydrolysate, which represents an increase of 64% and an almost 10 times higher level, respectively, compared to the wild type (WT) strain. Glucose and xylose were depleted after 120 h of fermentation. No increase in byproducts such as xylitol was observed.ConclusionsXylose-rich rye straw hydrolysate was exploited efficiently for the benefit of production of lipids. This study indicates that it is possible to fine-tune a newly strain with as minimally genetic changes as possible by adjusting to an unfavorable environment, thus limiting multi-level genome modification. It is documented here the use of Y. lipolytica as a microbial cell factory for lipid synthesis from rye straw hydrolysate as a low-cost feedstock.

Engineering the xylose metabolism in Schizochytrium sp. to improve the utilization of lignocellulose

BackgroundSchizochytrium sp. is a heterotrophic, oil-producing microorganism that can efficiently produce lipids. However, the industrial production of bulk chemicals using Schizochytrium sp. is still not economically viable due to high-cost culture medium. Replacing glucose with cheap and renewable lignocellulose is a highly promising approach to reduce production costs, but Schizochytrium sp. cannot efficiently metabolize xylose, a major pentose in lignocellulosic biomass.ResultsIn order to improve the utilization of lignocellulose by Schizochytrium sp., we cloned and functionally characterized the genes encoding enzymes involved in the xylose metabolism. The results showed that the endogenous xylose reductase and xylulose kinase genes possess corresponding functional activities. Additionally, attempts were made to construct a strain of Schizochytrium sp. that can effectively use xylose by using genetic engineering techniques to introduce exogenous xylitol dehydrogenase/xylose isomerase; however, the introduction of heterologous xylitol dehydrogenase did not produce a xylose-utilizing engineered strain, whereas the introduction of xylose isomerase did. The results showed that the engineered strain 308-XI with an exogenous xylose isomerase could consume 8.2 g/L xylose over 60 h of cultivation. Xylose consumption was further elevated to 11.1 g/L when heterologous xylose isomerase and xylulose kinase were overexpressed simultaneously. Furthermore, cultivation of 308-XI-XK(S) using lignocellulosic hydrolysates, which contained glucose and xylose, yielded a 22.4 g/L of dry cell weight and 5.3 g/L of total lipid titer, respectively, representing 42.7 and 30.4% increases compared to the wild type.ConclusionThis study shows that engineering of Schizochytrium sp. to efficiently utilize xylose is conducive to improve its utilization of lignocellulose, which can reduce the costs of industrial lipid production.

Lignocellulosic xylitol production from corncob using engineered Kluyveromyces marxianus.

Xylitol production from lignocellulose hydrolysate is a sustainable and environment-friendly process. In this study, a systematic process of converting corncob waste into xylitol is described. First, the corncobs are hydrolyzed with acid to a hydrolysate. Second, Kluyveromyces marxianus YZJQ016 derived from K. marxianus YZJ074, constructed by overexpressing ScGAL2-N376F from Saccharomyces cerevisiae, CtXYL1 from Candida tropicalis, and KmZWF1 from K. marxianus, produces xylitol from the hydrolysate. A total of ten xylose reductase genes were evaluated, and CtXYL1 proved best by showing the highest catalytic activity under the control of the KmGAPDH promoter. A 5L fermenter at 42°C produced 105.22g/L xylitol using K. marxianus YZJQ016-the highest production reported to date from corncob hydrolysate. Finally, for crystallization of the xylitol, the best conditions were 50% (v/v) methanol as an antisolvent, at 25°C, with purity and yield of 99%-100% and 74%, respectively-the highest yield reported to date.

Unraveling the mechanism of furfural tolerance in engineered Pseudomonas putida by genomics.

As a dehydration product of pentoses in hemicellulose sugar streams derived from lignocellulosic biomass, furfural is a prevalent inhibitor in the efficient microbial conversion process. To solve this obstacle, exploiting a biorefinery strain with remarkable furfural tolerance capability is essential. Pseudomonas putida KT2440 (P. putida) has served as a valuable bacterial chassis for biomass biorefinery. Here, a high-concentration furfural-tolerant P. putida strain was developed via adaptive laboratory evolution (ALE). The ALE resulted in a previously engineered P. putida strain with substantially increased furfural tolerance as compared to wild-type. Whole-genome sequencing of the adapted strains and reverse engineering validation of key targets revealed for the first time that several genes and their mutations, especially for PP_RS19785 and PP_RS18130 [encoding ATP-binding cassette (ABC) transporters] as well as PP_RS20740 (encoding a hypothetical protein), play pivotal roles in the furfural tolerance and conversion of this bacterium. Finally, strains overexpressing these three striking mutations grew well in highly toxic lignocellulosic hydrolysate, with cell biomass around 9-, 3.6-, and two-fold improvement over the control strain, respectively. To our knowledge, this study first unravels the furan aldehydes tolerance mechanism of industrial workhorse P. putida, which provides a new foundation for engineering strains to enhance furfural tolerance and further facilitate the valorization of lignocellulosic biomass.

Reconstruction and optimization of a Pseudomonas putida-Escherichia coli microbial consortium for mcl-PHA production from lignocellulosic biomass.

The demand for non-petroleum-based, especially biodegradable plastics has been on the rise in the last decades. Medium-chain-length polyhydroxyalkanoate (mcl-PHA) is a biopolymer composed of 6-14 carbon atoms produced from renewable feedstocks and has become the focus of research. In recent years, researchers aimed to overcome the disadvantages of single strains, and artificial microbial consortia have been developed into efficient platforms. In this work, we reconstructed the previously developed microbial consortium composed of engineered Pseudomonas putida KT∆ABZF (p2-a-J) and Escherichia coli ∆4D (ACP-SCLAC). The maximum titer of mcl-PHA reached 3.98g/L using 10g/L glucose, 5g/L octanoic acid as substrates by the engineered P. putida KT∆ABZF (p2-a-J). On the other hand, the maximum synthesis capacity of the engineered E. coli ∆4D (ACP-SCLAC) was enhanced to 3.38g/L acetic acid and 0.67g/L free fatty acids (FFAs) using 10g/L xylose as substrate. Based on the concept of "nutrient supply-detoxification," the engineered E. coli ∆4D (ACP-SCLAC) provided nutrient for the engineered P. putida KT∆ABZF (p2-a-J) and it acted to detoxify the substrates. Through this functional division and rational design of the metabolic pathways, the engineered P. putida-E. coli microbial consortium could produce 1.30g/L of mcl-PHA from 10g/L glucose and xylose. Finally, the consortium produced 1.02g/L of mcl-PHA using lignocellulosic hydrolysate containing 10.50g/L glucose and 10.21g/L xylose as the substrate. The consortium developed in this study has good potential for mcl-PHA production and provides a valuable reference for the production of high-value biological products using inexpensive carbon sources.

Mathematical modeling of the biotechnological production of xylitol from hydrolysates of agro-industrial waste

Xylitol is widely used in the pharmaceutical and food industries since it has cariogenic properties and low caloric value compared to sucrose. In recent decades, its demand has increased, which has motivated the generation of alternatives for its production. The biotechnological route is an attractive alternative since in addition to reducing production costs, it uses lignocellulosic hydrolysates from agro-industrial residues as raw material. However, due to the inherent variability in the composition of the waste, the implementation of the process is not a simple task, i. e., the determination of the operating conditions that maximize production yield requires a broad experimental design. In this sense, mathematical modeling can help reduce experimental work, and allows for predicting and evaluating the dynamic behavior of key variables, making it a useful tool for the implementation of process optimization and control schemes. Therefore, the objective of this work is to propose a generalized mathematical model that can be adapted to different study cases (i.e., different substrates and microorganism strains), operating conditions, and fermenter configuration. The results show high determination coefficients R2 > 0.90 for each case study, indicating that the proposed model is easily adaptable to different substrates and operating conditions. In addition, the model allows the incorporation of different effects such as pH, temperature, and agitation.

Identification of acetic acid sensitive strains through biosensor-based screening of a Saccharomyces cerevisiae CRISPRi library

BackgroundAcetic acid tolerance is crucial for the development of robust cell factories for conversion of lignocellulosic hydrolysates that typically contain high levels of acetic acid. Screening mutants for growth in medium with acetic acid is an attractive way to identify sensitive variants and can provide novel insights into the complex mechanisms regulating the acetic acid stress response.ResultsAn acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1, was used to screen a CRISPRi yeast strain library where dCas9-Mxi was set to individually repress each essential or respiratory growth essential gene. Fluorescence-activated cell sorting led to the enrichment of a population of cells with higher acetic acid retention. These cells with higher biosensor signal were demonstrated to be more sensitive to acetic acid. Biosensor-based screening of the CRISPRi library strains enabled identification of strains with increased acetic acid sensitivity: strains with gRNAs targeting TIF34, MSN5, PAP1, COX10 or TRA1.ConclusionsThis study demonstrated that biosensors are valuable tools for screening and monitoring acetic acid tolerance in yeast. Fine-tuning the expression of essential genes can lead to altered acetic acid tolerance.

Effect of process mode, nitrogen source and temperature on L-malic acid production with Aspergillus oryzae DSM 1863 using acetate as carbon source.

Malic acid, mainly used as acidulant and taste enhancer in the food industry, is currently produced from fossil resources. In this study, microbial L-malate production with the filamentous fungus A. oryzae using the carbon source acetate was evaluated. Acetate is for example contained in biomass-derived substrates such as lignocellulosic hydrolysates and condensates of fast pyrolysis, thus avoiding competition with food production. Since research on malic acid synthesis from acetate is limited and reported productivities and yields are low, this work aimed to improve the process. First, different cultivation temperatures were tested. This parameter was found to affect the ratio between malic and succinic acid, which is the major by-product of organic acid production with A. oryzae. At 32°C, the malate share was highest (53.7 ± 1.6%), while it was lowest at 38°C (43.3 ± 1.1%) whereas succinate represented the main product (51.5 ± 1.0%). Besides the temperature, the type of nitrogen source was also found to affect malate synthesis as well as biomass production. In the pre-culture, the biomass concentration was increased by a factor of 3.4–3.9, and germination started earlier with the complex nitrogen sources yeast extract, casein hydrolysate and peptone compared to the defined nitrogen source (NH4)2SO4. Especially with yeast extract, malate synthesis in the main culture was accelerated and the titer obtained after 48 h was about 2.6 times higher than that quantified with (NH4)2SO4. To reduce substrate inhibition in acetate medium, fed-batch and repeated-batch processes were evaluated using (NH4)2SO4 or yeast extract as nitrogen source. In the fed-batch process, the period of malate production was extended, and the maximum product concentration was increased to 11.49 ± 1.84 g/L with (NH4)2SO4 and 12.08 ± 1.25 g/L with yeast extract. In the repeated-batch process, the total acid production was highest within the first 240 h of fermentation, but optimization is required to maintain high production rates in later cycles. The lessons learned in this study will help in the development of further process strategies to maximize malate production using acetate as alternative substrate to the commonly used glucose.

β-Farnesene Production from Low-Cost Glucose in Lignocellulosic Hydrolysate by Engineered Yarrowia lipolytica

β-Farnesene is value-added acyclic volatile sesquiterpene with wide applications in energy, industry, and agriculture. Producing high-value-added compounds from low-cost renewable feedstocks in engineered microbial cell factories is an environmentally friendly and economical process for β-farnesene biosynthesis. In this study, the potential for using engineered Yarrowia lipolytica to produce β-farnesene from lignocellulosic hydrolysate as the carbon source was investigated. An efficient biosynthetic pathway for β-farnesene production was established via iterative enhancement of multiple genes based on the high endogenous acetyl-CoA flux in Yarrowia lipolytica. Overexpression of mevalonate pathway genes and screening of β-farnesene synthase resulted in a β-farnesene titer of 245 mg L−1 in glucose media. Additional copies of mevalonate pathway genes and enhanced expression of HMG-CoA reductase and β-farnesene synthase further increased the titer of β-farnesene to 470 mg L−1. In addition, by combining metabolic engineering strategies using the lignocellulosic hydrolysate utilization strategy, the addition of Mg2+ promoted the production of β-farnesene, and the best-performing strain produced 7.38 ± 0.24 g L−1 β-farnesene from lignocellulosic hydrolysate media in a 2 L fermenter after 144 h. This study shows great potential for the sustainable production of β-farnesene from lignocellulosic biomass via engineered Yarrowia lipolytica.

Metabolic Engineering for the Comprehensive Utilization of N,N-Dimethylformamide-Containing Wastewater.

N, N-dimethylformamide is frequently present in industrial wastewater and is environmentally detrimental. The current study aims to assess the utilization and biodegradation of N, N-dimethylformamide-containing wastewater to lessen the associated environmental load. Results show that addition of wastewater containing N, N-dimethylformamide to Trichoderma reesei fermentation media enhances cellulase production and facilitates cellulose hydrolysis. However, N, N-dimethylformamide is a cellulase enhancer that is not degraded during cellulase production in T. reesei fermentation and is retained in the N, N-dimethylformamide-enhanced cellulase solution. Indeed, the cellulosic sugar solution generated via lignocellulose hydrolysis with N, N-dimethylformamide-enhanced cellulase retains N, N-dimethylformamide. We further identified three core enzyme modules─N, N-dimethylformamidase, dimethylamine dehydrogenase, and methylamine dehydrogenase enzyme─which were inserted into Escherichia coli to develop metabolically engineered strains. These strains degraded N, N-dimethylformamide and produced succinate using N, N-dimethylformamide-enhanced cellulosic sugar as the substrate. The platform described here can be applied to effectively convert waste into valuable bioproducts.

2-Naphthol modification alleviated the inhibition of ethanol organosolv lignin on enzymatic hydrolysis

It has been reported that the lignins deposited on surfaces of pretreated biomass directly affected the enzyme adsorption, thus impeding enzymatic hydrolysis of lignocellulose. Alleviating the surface lignin inhibition is urgently needed for establishing an efficient lignocellulose biorefinery process. In this study, ethanol organosolv lignins (EOLs) from masson pine and poplar were employed as representatives of surface lignins. Aiming to mitigate the EOLs inhibition on enzymatic hydrolysis, 2-naphthol as a carbocation scavenger was added in the organosolv pretreatment. The function of 2-naphthol for suppressing lignin condensation was verified by investigating the lignin structural changes with NMR analysis. Moreover, it was verified that 2-naphthol modification remarkably eliminated the negative effects of EOLs from both two wood species, leading to a 6.8–34.4% increase in hydrolysis yields. The effects of 2-napthol modified lignins were even reversed to slightly promote the enzymatic hydrolysis of pure cellulose. The mechanism of 2-naphthol modification for relieving lignin inhibition was revealed by evaluating the relationship between lignin structural features and hydrolysis performance with Pearson's correlation heatmap. Results enrich our understanding of surface lignin inhibition, thereby providing ideas for promoting enzymatic hydrolysis by alleviating the surface lignin inhibition.

Investigation of stress tolerance of Pichia kudriavzevii for high gravity bioethanol production from steam–exploded wheat straw hydrolysate

This study investigated a newly isolated thermotolerant strain of Pichia kudriavzevii with respect to its stress tolerance and fermentation performance. Response surface methodology was applied to evaluate the combined effects of furfural, osmotic and thermal stress on ethanol yield. The proposed model shows that P. kudriavzevii has a natural resistance against multiple stress factors. Further evolutionary adaptation of the isolated strain in lignocellulosic hydrolysates improved the ethanol yield by ≥ 24 %. The adapted strain HYPK213_ELA was able to produce ethanol from wheat straw hydrolysates at a high solid loading of 37 %ww–1 at 40 °C and anaerobic conditions. The highest ethanol concentration of 56.8 ± 1.0 gL–1 was reached at 40°C with an inoculum size of 2.5 × 106cellsmL−1. The results show that Pichia kudriavzevii has the potential to enable high gravity bioethanol production under conditions where most yeast strains are unable to grow.

A smart self-balancing biosystem with reversible competitive adsorption of in-situ anion exchange resin for whole-cell catalysis preparation of lignocellulosic xylonic acid

Xylonic acid (XA) bioproduction via whole-cell catalysis of Gluconobacter oxydans is a promising strategy for xylose bioconversion, which is hindered by inhibitor formation during lignocellulosic hydrolysates. Therefore, it is important to develop a catalytic system that can directly utilize hydrolysate and efficiently produce XA. Determination of the dynamic adsorption characteristics of 335 anion exchange resin resulted in a unique and interesting reversible competitive adsorption between acetic acid-like bioinhibitor, fermentable sugar and XA. Xylose in crude lignocellulosic hydrolysates was completely oxidized to 52.52 g/L XA in unprecedented self-balancing biological system through reversible competition. The obtained results showed that in-situ resin adsorption significantly affected the direct utilization of crude lignocellulosic hydrolysate for XA bioproduction (p ≤ 0.05). In addition, the resin adsorbed ca. 90 % of XA during bioconversion. The study achieved a multiple functions and integrated system, “detoxification, neutralization and product separation” for one-pot bioreaction of lignocellulosic hydrolysate.

Enhanced tolerance of Cupriavidus necator NCIMB 11599 to lignocellulosic derived inhibitors by inserting NAD salvage pathway genes.

Polyhydroxybutyrate (PHB) is a bio-based, biodegradable and biocompatible plastic that has the potential to replace petroleum-based plastics. Lignocellulosic biomass is a promising feedstock for industrial fermentation to produce bioproducts such as polyhydroxybutyrate (PHB). However, the pretreatment processes of lignocellulosic biomass lead to the generation of toxic byproducts, such as furfural, 5-HMF, vanillin, and acetate, which affect microbial growth and productivity. In this study, to reduce furfural toxicity during PHB production from lignocellulosic hydrolysates, we genetically engineered Cupriavidus necator NCIMB 11599, by inserting the nicotine amide salvage pathway genes pncB and nadE to increase the NAD(P)H pool. We found that the expression of pncB was the most effective in improving tolerance to inhibitors, cell growth, PHB production and sugar consumption rate. In addition, the engineered strain harboring pncB showed higher PHB production using lignocellulosic hydrolysates than the wild-type strain. Therefore, the application of NAD salvage pathway genes improves the tolerance of Cupriavidus necator to lignocellulosic-derived inhibitors and should be used to optimize PHB production.