Lignocellulosic Hydrolysates Research Articles

Effect of alkali salt-coupled ball milling pretreatment of wheat straw on improving enzymatic hydrolysis and energy efficiency

Pretreatment of lignocellulosic biomass is crucial in producing fermentable sugars in a sustainable biorefinery. In this study, wheat straw was pretreated with a combination of ball milling (BM) and NaAlO2 particles (NaAlO2-BM) for enzymatic hydrolysis within a short hydrolysis period. The effects of energy consumption on modifications of physicochemical proprieties and energy efficiency were elucidated. NaAlO2-BM pretreatment decreased particle size rapidly to tissue scale in 10 min with energy consumption reduced by 0.5 times compared to BM pretreatment. NaAlO2-BM pretreated for 20 min achieved a glucose yield of 95.4% at a cellulase loading of 10 FPU/g dry substrate for 24 h enzymatic hydrolysis and provided the highest energy efficiency of 0.121 kg glucose/kWh, increasing by 1.1 times than BM pretreatment. Energy consumption showed a positive relationship with cellulose content while it negatively correlated with median particle size, cellulose crystallinity and energy efficiency. The combination of BM and NaAlO2 particles was demonstrated to be a promising pretreatment approach for glucose production and improving energy efficiency during hydrolysis of lignocellulose.

Evaluation of Non-Conventional Yeasts Isolated from Rotten Wood for Hydrolytic Activities and Xylose Fermentation

Hydrolysis of lignocellulose to fermentable sugars and their subsequent conversion to ethanol remain great challenges in the biofuel industry. Rotten wood is first colonized by bacteria and molds that possess strong hydrolases. Yeasts are also an important group of microorganisms that may participate in wood hydrolysis. Decaying wood could provide a rich natural reservoir of yeasts possessing promising hydrolytic activities, including xylanases, cellulases, β-glucosidases, or abilities essential for the fermentation of pentose sugars derived from lignocellulose degradation, especially xylose. Therefore, the aim of this work was to screen yeasts isolated from rotten wood samples for the production of hydrolytic enzymes directed at lignocellulose components and the ability to ferment xylose, L-arabinose, and cellobiose. Methods. Yeast strains were isolated from 22 samples of rotten wood and identified by phenotypic characteristics according to Kurtzman et al. Hydrolytic properties and the ability of the isolated strains to ferment xylose, L-arabinose, and cellobiose were determined using conventional methods. Results. 30 strains of yeasts and yeast-like micromycetes were isolated from 22 samples of rotten wood in the Holosiivskyi Forest, Kyiv. Based on phenotypic properties, most of the isolated yeasts belonged to ascomycetous yeasts and were represented by the following genera: Candida (8 strains), Debaryomyces (5 strains), Kluyveromyces (5 strains), Pichia (5 strains), Scheffersomyces (2 strains), Lachancea, Hanseniaspora, Saccharomyces, and Geotrichum/Galactomyces. A strain of yeast-like non-photosynthetic alga Prototheca sp. was also detected. Most of the isolated microfungi (66.6% isolates) exhibited extracellular β-glucosidase activity, two Candida tropicalis strains possessed weak pectinase and xylanase activity. None of the isolates demonstrated extracellular cellulase activity. Two yeast strains preliminarily identified as Scheffersomyces stipitis were able to ferment xylose at a concentration of 20—100 g/L over a wide temperature range up to 37°C. Acetic acid at 0.25—1% (v/v) concentration resulted in the complete inhibition of xylose fermentation. Ethanol production from xylose up to 6 g/L was observed under the microaerobic fermentation conditions for 24 hr at the substrate concentration 40 g/L, but the subsequent fermentation resulted in decreasing ethanol concentration presumably due to ethanol re-assimilation. None of the isolated strains was capable of fermenting cellobiose or L-arabinose under the microaerobic conditions. Conclusions. This work provides the characterization of yeast microbiota of rotten wood that was represented predominantly by ascomycetous yeasts. The dominant extracellular hydrolytic activity of the isolates was β-glucosidase. This is the first report on the isolation of xylose-fermenting yeasts Scheffersomyces stipitis in Ukraine, which comprised 7% of all the microfungi isolated from rotten wood.

Physiological and Molecular Characterization of Yeast Cultures Pre-Adapted for Fermentation of Lignocellulosic Hydrolysate

Economically feasible bioethanol process from lignocellulose requires efficient fermentation by yeast of all sugars present in the hydrolysate. However, when exposed to lignocellulosic hydrolysate, Saccharomyces cerevisiae is challenged with a variety of inhibitors that reduce yeast viability, growth, and fermentation rate, and in addition damage cellular structures. In order to evaluate the capability of S. cerevisiae to adapt and respond to lignocellulosic hydrolysates, the physiological effect of cultivating yeast in the spruce hydrolysate was comprehensively studied by assessment of yeast performance in simultaneous saccharification and fermentation (SSF), measurement of furaldehyde reduction activity, assessment of conversion of phenolic compounds and genome-wide transcription analysis. The yeast cultivated in spruce hydrolysate developed a rapid adaptive response to lignocellulosic hydrolysate, which significantly improved its fermentation performance in subsequent SSF experiments. The adaptation was shown to involve the induction of NADPH-dependent aldehyde reductases and conversion of phenolic compounds during the fed-batch cultivation. These properties were correlated to the expression of several genes encoding oxidoreductases, notably AAD4, ADH6, OYE2/3, and YML131w. The other most significant transcriptional changes involved genes involved in transport mechanisms, such as YHK8, FLR1, or ATR1. A large set of genes were found to be associated with transcription factors (TFs) involved in stress response (Msn2p, Msn4p, Yap1p) but also cell growth and division (Gcr4p, Ste12p, Sok2p), and these TFs were most likely controlling the response at the post-transcriptional level.

Zirconium Phosphate Assisted Phosphoric Acid Co-Catalyzed Hydrolysis of Lignocellulose for Enhanced Extraction of Nanocellulose.

The high mechanical strength, large specific surface area, favorable biocompatibility, and degradability of nanocellulose (CNC) enable it to be a potential alternative to petroleum-based materials. However, the traditional preparation of CNCs requires a large amount of strong acid, which poses a serious challenge to equipment maintenance, waste liquid recycling, and economics. In this study, a solid and easily recoverable zirconium phosphate (ZrP) was used to assist in the phosphoric acid co-catalyzed hydrolysis of lignocellulose for extracting CNCs. Due to the presence of acidic phosphate groups, ZrP has a strong active center with a high catalytic activity. With the assistance of ZrP, the amount of phosphoric acid used in the reaction is significantly reduced, improving the equipment's durability and economic efficiency. The effects of the process conditions investigated were the phosphate acid concentration, reaction temperature, and reaction time on the yield of CNCs. The Box-Behnken design (BBD) method from the response surface methodology (RSM) was applied to investigate and optimize the preparation conditions. The optimized pre-treatment conditions were 49.27% phosphoric acid concentration, 65.38 °C reaction temperature, and 5 h reaction time with a maximal cellulose yield (48.33%). The obtained CNCs show a granular shape with a length of 40~50 nm and a diameter of 20~30 nm, while its high zeta potential (-24.5 mV) make CNCs present a stable dispersion in aqueous media. Moreover, CNCs have a high crystallinity of 78.70% within the crystal type of cellulose Ⅰ. As such, this study may pioneer the horizon for developing a green method for the efficient preparation of CNC, and it is of great significance for CNCs practical production process.

Synthesis of Recyclable UCST-Type Copolymer PPSP and Its Application in Enhancing Lignocellulosic Enzymatic Hydrolysis and Recycling Cellulase

The cost of lignocellulosic enzymatic hydrolysis can be reduced by adding additives with upper critical solution temperature (UCST) response. A recyclable UCST-type copolymer PPSP was synthesized, for enhancing lignocellulosic enzymatic hydrolysis and the cellulase recovery effect of substrate reabsorption. For the hydrolysis system of corncob residue without adding PPSP, only 50% of cellulase amount was saved at a room temperature of 25 °C by adding the fresh corncob residue. The substrate enzymatic digestion efficiency for 72 h was increased by 1.3 times with only adding 0.5 g/L PPSP10-3. After the hydrolysis, 70% of cellulase amount was saved at 25 °C by using the above method. Both lignocellulosic hydrolysis and recovery of cellulase performances were improved by adding PPSP. This may be attributed to the fact that PPSP preferentially adsorbed on lignin to form a hydration membrane to reduce the no-productive adsorption of cellulase and enhanced lignocellulosic enzymatic hydrolysis, while increasing the concentration of free cellulase in the system and strengthening recovery of cellulase. This work will provide one way to reduce the cost of lignocellulosic enzymatic hydrolysis.

Исследование виноградных субпродуктов для получения биоэтанола на основе новых устойчивых штаммов

Technological development of environmentally friendly biofuels is highly desirable due to the depletion of fossil fuel reserves and increased environmental awareness. Currently, bioethanol appears to be the key alternative fuel, most of which is made from sugar cane, corn, and wheat. However, the use of these sugar- and starch-basedmaterials is still controversial due to their alternative use as animal feed and as raw materials for human staple foods. Various detoxification strategies have been developed to remove these inhibitors from lignocellulose hydrolysates and reduce their levels, such as alkali treatment, sulfite treatment, evaporation, anion exchange and laccase addition.However, these methods create 2 main problems in terms of their technical and economic feasibility: the addition of expensive processing stages and the loss of easily digestible sugars.

PRODUCTION OF BIOETHANOL FROM SUGARCANE BAGASSE THROUGH HYDROLYSIS AND FERMENTATION: THE EFFECT OF FE2+ AND CA2+ IONS ADDITION ON BIOETHANOL CONCENTRATION

Fossil energy sources are one of the driving factors in the economic development and prosperity of the nation. The availability of fossil energy sources is currently shrinking and is expected to run out quickly, so an alternative is needed to replace it. Sugarcane bagasse is a lignocellulosic biomass which can be utilized for bioethanol production as an alternative energy source. Lignocellulosic hydrolysis with the addition of H2SO4 catalyst was carried out to obtain glucose. The addition of Fe2+ and Ca2+ ions was performed to increase the glucose concentration. Commonly, Saccharomyces cerevisiae is used to convert glucose into bioethanol using fermentation process. The objective of this study was to investigate the effect of Fe2+ and Ca2+ ions on reducing sugar concentration, and the concentration of distillate bioethanol. The effect of yeast type on the concentration of bioethanol was also discussed in this paper. The result shows that the addition of Fe2+ and Ca2+ ions in the hydrolysis process can increase the concentration of glucose produced. This is because the higher concentration of cellulose, the more substrate is converted into reducing sugars. The use of Fe2+ and Ca2+ ions in the fermentation process will increase ethanol concentration compares with fermentation without metal ions. This is because Ca2+ ions have an important role in the flocculation process of bioethanol fermentation and Fe2+ ions are important nutrients for eukaryotes, which play a role in cell oxidationreduction reactions. The type of yeast that can produce higher concentrations of ethanol is tapai yeast. This is because tapai’s yeast has three supporting ingredients for the fermentation process, whereas baker’s yeast only contains two microorganisms.

Subcritical Water Process for Reducing Sugar Production from Biomass: Optimization and Kinetics

The competitive reactions of lignocellulose hydrolysis and monosaccharide degradation in the subcritical water (SCW) hydrolysis of coconut husk were investigated to optimize the reducing sugar yield. Optimization analysis was performed by response surface methodology (RSM) and kinetics studies. Parameters of process optimization were varied at 130-170 °C for 15-45 min. The reducing sugars were measured using the Dinitro salicylic acid method. The sugar yield increased when the temperature increased from 130 °C to 170 °C. The highest reduction sugar yield of 4.946 g/L was obtained at 183.6 °C for 4.8 min and 23.4 liquid/solid ratio (LSR). Kinetics studies were carried out at temperature variations of 150, 170, and 190 °C and pressures of 60, 80, and 100 bar for 5 to 60 min. The yield of reducing sugar decreased with increasing temperature. The kinetic model 2B is the best method to explain the competitive reaction kinetics of coconut husk hydrolysis. This research is an innovation to increase the reducing sugar to make the process more commercially viable. Copyright © 2022 by Authors, Published by BCREC Group. This is an open access article under the CC BY-SA License (https://creativecommons.org/licenses/by-sa/4.0).

Carotenoids production and genome analysis of a novel carotenoid producing Rhodococcus aetherivorans N1

Carotenoids are a series of natural pigments with unique structure and physiological functions. In this study, a novel Rhodococcus aetherivorans strain N1 was discovered, which can produce 6.4 mg/g carotenoids including β-carotene, zeaxanthin and isorenieratene from glucose. Moreover, strain N1 can directly produce 3.0 mg/g carotenoids from the undetoxified straw hydrolysate, representing the highest carotenoids production from the undetoxified lignocellulosic hydrolysate. The crude carotenoid extracts of strain N1 showed efficient free radical scavenging activity and stability. Strain N1 has complete methylerythritol 4-phosphate (MEP) pathway and related genes for carotenoid synthesis, especially the rare aromatic carotenoid of isorenieratene. Genomic comparison between strain N1 and other carotenoid producing Rhodococcus sp. strains showed the conservatism and universality of carotenoids synthesis gene. These results proved that R. aetherivorans strain N1 can serve as a promising producer for the industrialization of carotenoid production.

Overexpressing GRE3 in Saccharomyces cerevisiae enables high ethanol production from different lignocellulose hydrolysates.

The efficiently renewable bioethanol can help to alleviate energy crisis and environmental pollution. Genetically modified strains for efficient use of xylose and developing lignocellulosic hydrolysates play an essential role in facilitating cellulosic ethanol production. Here we present a promising strain GRE3OE via GRE3 overexpressed in a previously reported Saccharomyces cerevisiae strain WXY70. A comprehensive evaluation of the fermentation level of GRE3OE in alkaline-distilled sweet sorghum bagasse, sorghum straw and xylose mother liquor hydrolysate. Under simulated corn stover hydrolysate, GRE3OE produced 53.39 g/L ethanol within 48 h. GRE3OE produced about 0.498 g/g total sugar in sorghum straw hydrolysate solution. Moreover, GRE3OE consumed more xylose than WXY70 in the high-concentration xylose mother liquor. Taken together, GRE3OE could be a candidate strain for industrial ethanol development, which is due to its remarkable fermentation efficiency during different lignocellulosic hydrolysates.

Tailoring the expression of Xyr1 leads to efficient production of lignocellulolytic enzymes in Trichoderma reesei for improved saccharification of corncob residues

BackgroundThe filamentous fungus Trichoderma reesei is extensively used for the industrial-scale cellulase production. It has been well known that the transcription factor Xyr1 plays an important role in the regulatory network controlling cellulase gene expression. However, the role of Xyr1 in the regulation of cellulase expression has not been comprehensively elucidated, which hinders further improvement of lignocellulolytic enzyme production.ResultsHere, the expression dosage of xyr1 was tailored in T. reesei by differentially overexpressing the xyr1 gene under the control of three strong promoters (Pegl2, Pcbh1, and Pcdna1), and the transcript abundance of xyr1 was elevated 5.8-, 12.6-, and 47.2-fold, respectively. We found expression of cellulase genes was significantly increased in the Pegl2-driven xyr1 overexpression strain QE2X, whereas relatively low in the Pcbh1- and Pcdna1-driven overexpression strains. We also found that the Pegl2-driven overexpression of xyr1 caused a more significant opening of chromatin in the core promoter region of the prominent cellulase genes. Furthermore, the cellulase activity showed a 3.2-fold increase in the strain QE2X, while insignificant improvement in the Pcbh1- and Pcdna1-driven strains. Finally, the saccharification efficiency toward acid-pretreated corncob residues containing high-content lignin by the crude enzyme from QE2X was increased by 57.2% compared to that from the parental strain. Moreover, LC–MS/MS and RT-qPCR analysis revealed that expression of accessory proteins (Cip1, Cip2, Swo1, and LPMOs) was greatly improved in QE2X, which partly explained the promoting effect of the Pegl2-driven overexpression on enzymatic hydrolysis of lignocellulose biomass.ConclusionsOur results underpin that the precise tailoring expression of xyr1 is essential for highly efficient cellulase synthesis, which provide new insights into the role of Xyr1 in regulating cellulase expression in T. reesei. Moreover, these results also provides a prospective strategy for strain improvement to enhance the lignocellulolytic enzyme production for use in biorefinery applications.

Production of biological pest control agents on hydrolysates of switchgrass

Recently, biological products have outpaced chemicals for pest control because they are more compatible with the environment, and they offer multiple modes of action not easily overcome by resistance mechanisms. Pseudomonas spp. have been developed as biocontrol agents (BCAs) to replace chemicals no longer effective against dry rot disease and other maladies of postharvest potatoes, including sprouting, late-blight, pink rot, and Pythium leak. It is proposed that Pseudomonas spp., which can use the diverse carbon sources in lignocellulosic hydrolysates, have aptitude as valuable coproducts compatible with lignocellulosic biorefining. Our results showed that three Pseudomonas fluorescens strains could rapidly consume all switchgrass hydrolysate sugars, including the pentoses, and also acetic acid, furfural, and hydroxymethylfurfural. If pH was controlled near neutral and sugar concentrations were kept near 10 g/L to prevent osmotic inhibition, high yields of efficacious BCAs could be obtained on xylose- or glucose-rich fractions and unfractionated switchgrass hydrolysates.

Co-Fermentation of Glucose-Xylose Mixtures from Agroindustrial Residues by Ethanologenic Escherichia coli: A Study on the Lack of Carbon Catabolite Repression in Strain MS04.

The production of biofuels, such as bioethanol from lignocellulosic biomass, is an important task within the sustainable energy concept. Understanding the metabolism of ethanologenic microorganisms for the consumption of sugar mixtures contained in lignocellulosic hydrolysates could allow the improvement of the fermentation process. In this study, the ethanologenic strain Escherichia coli MS04 was used to ferment hydrolysates from five different lignocellulosic agroindustrial wastes, which contained different glucose and xylose concentrations. The volumetric rates of glucose and xylose consumption and ethanol production depend on the initial concentration of glucose and xylose, concentrations of inhibitors, and the positive effect of acetate in the fermentation to ethanol. Ethanol yields above 80% and productivities up to 1.85 gEtOH/Lh were obtained. Furthermore, in all evaluations, a simultaneous co-consumption of glucose and xylose was observed. The effect of deleting the xyIR regulator was studied, concluding that it plays an important role in the metabolism of monosaccharides and in xylose consumption. Moreover, the importance of acetate was confirmed for the ethanologenic strain, showing the positive effect of acetate on the co-consumption rates of glucose and xylose in cultivation media and hydrolysates containing sugar mixtures.

Molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression in Zymomonas mobilis.

Zymomonas mobilis is a promising microorganism for industrial bioethanol production. However, ethanol produced during fermentation is toxic to Z. mobilis and affects its growth and bioethanol production. Although several reports demonstrated that the RNA-binding protein Hfq in Z. mobilis contributes to the tolerance against multiple lignocellulosic hydrolysate inhibitors, the role of Hfq on ethanol tolerance has not been investigated. In this study, hfq in Z. mobilis was either deleted or overexpressed and their effects on cell growth and ethanol tolerance were examined. Our results demonstrated that hfq overexpression improved ethanol tolerance of Z. mobilis, which is probably due to energy saving by downregulating flagellar biosynthesis and heat stress response proteins, as well as reducing the reactive oxygen species induced by ethanol stress via upregulating the sulfate assimilation and cysteine biosynthesis. To explore proteins potentially interacted with Hfq, the TEV protease mediated Yeast Endoplasmic Reticulum Sequestration Screening system (YESS) was established in Z. mobilis. YESS results suggested that Hfq may modulate the cytoplasmic heat shock response by interacting with the heat shock proteins DnaK and DnaJ to deal with the ethanol inhibition. This study thus not only revealed the underlying mechanism of enhanced ethanol tolerance by hfq overexpression, but also provided an alternative approach to investigate protein-protein interactions in Z. mobilis.

Synergistic Enhancement Effect of Compound Additive of Organic Alcohols and Biosurfactant on Enzymatic Hydrolysis of Lignocellulose

The insufficient of lignocellulose degradation enzymes, such as cellulase and hemicellulase, is the major obstacle that hinders the bioconversion of lignocellulosic biomass to monosaccharides, especially during the woody biomass hydrolysis process. The addition of additives has received significant attention due to their enhancement of the enzymatic degradation efficiency of lignocellulose. In the present study, a combination of organic alcohols and a biosurfactant could synergistically enhance the saccharification of the cellulose substrate of Avicel, as well as that of pretreated poplar. Results showed that compound additives can greatly improve the conversion rate of enzymatic hydrolysis. The combination of 0.1% (v/v) n-decanol and 1% (v/v) sophorolipid dramatically increased the poplar enzymatic conversion rate from 17.9% to 85%, improving it by 67.1%. Enzyme-rich Hypocrea sp. W63 was fermented to obtain beta-glucosidase (BGL) and xylanase (XYL), which were used as auxiliary enzymes during enzymatic hydrolysis. It was found that the effects of such a combination of additives improved the filter paper activity, stability, and longevity, helping in the recovery of the cellulase cocktail. The compound additives associated with the commercial cellulase and Hypocrea sp. W63 enzyme solution formed an excellent formula for improving the stability of BGL and XYL. The results provide insight into compound additives and the use of a cellulase and auxiliary enzyme cocktail to improve enzymatic hydrolysis for lignocellulose conversion into biofuels.

Anaerobic microbial cocktail of lignocellulolytic fungi and bacteria with methanogens for boosting methane production from unpretreated rice straw

Rice straw (RS) has been recognized as a sustainable renewable energy resource for converting into sugars and volatile fatty acids (acetate, propionate, and butyrate) and subsequently to produce biogas. Enhanced production of these intermediates from RS by the different combinations of two consortia was investigated. Anaerobic microbial cocktails of fungi, bacteria, and methanogens were evaluated for performance and stability in the anaerobic digestion of untreated RS. The best-defined anaerobic microbial cocktail for high RS degradation and methane production, consisting of anaerobic bacteria (mainly Proteiniphilum acetatigenes, Pyramidobacter piscolens, and Mesotoga prima) and anaerobic lignocellulolytic/fermentative fungi (uncultured Neocallimastigales, Orpinomyces, Anaeromyces, and Feramyces sp.) at a copy number ratio of 103-105 copies/mL, including hydrogenotrophic and acetoclastic methanogens (Methanosarcina mazei, Methanoculleus marisnigri, Methanofollis liminatans, Methanoculleus bourgensis, and Methanosaeta harundinacea) concentration of 106 copies/mL, was successfully constructed. The system performance was 80% VS (volatile solids) RS degradation, 34mL/day methane production rate, 318mL/g VSadded methane yield, and a pH range of 6.90-7.70 within a short time of 14days. A defined microbial cocktail has been proven as a potential alternative process for lignocellulose hydrolysis and methane production.

Advanced one-pot deconstruction and valorization of lignocellulosic biomass into triacetic acid lactone using Rhodosporidium toruloides.

Rhodosporidium toruloides is capable of co-utilization of complex carbon sources and robust growth from lignocellulosic hydrolysates. This oleaginous yeast is therefore an attractive host for heterologous production of valuable bioproducts at high titers from low-cost, deconstructed biomass in an economically and environmentally sustainable manner. Here we demonstrate this by engineering R. toruloides to produce the polyketide triacetic acid lactone (TAL) directly from unfiltered hydrolysate deconstructed from biomass with minimal unit process operations. Introduction of the 2-pyrone synthase gene into R. toruloides enabled the organism to produce 2.4g/L TAL from simple media or 2.0g/L from hydrolysate produced from sorghum biomass. Both of these titers are on par with titers from other better-studied microbial hosts after they had been heavily engineered. We next demonstrate that filtered hydrolysates produced from ensiled sorghum are superior to those derived from dried sorghum for TAL production, likely due to the substantial organic acids produced during ensiling. We also demonstrate that the organic acids found in ensiled biomass can be used for direct synthesis of ionic liquids within the biomass pretreatment process, enabling consolidation of unit operations of in-situ ionic liquid synthesis, pretreatment, saccharification, and fermentation into a one-pot, separations-free process. Finally, we demonstrate this consolidation in a 2L bioreactor using unfiltered hydrolysate, producing 3.9g/L TAL. Many steps involved in deconstructing biomass into fermentable substrate can be combined into a distinct operation, and directly fed to cultures of engineered R. toruloides cultures for subsequent valorization into gram per liter titers of TAL in a cost-effective manner.

Selective adsorption of ferulic acid and furfural from acid lignocellulosic hydrolysate by novel magnetic lignin-based adsorbent

In this study, two novel adsorbents (M-0 and M-1) were obtained by one-step synthesis of magnetic nanoparticles with alkaline lignin and oxidized alkaline lignin, respectively. The core-shell structure of the two adsorbents was demonstrated by various characterization results. The adsorption performance of M-0 and M-1 for ferulic acid and furfural at different pH, contact time, initial concentration, and temperature were investigated. The results showed that the adsorbents were pH-sensitive for ferulic acid but not for furfural, and the adsorption process was rapid and thermodynamically unfavorable. Meanwhile, the simulated acid hydrolysate was detoxified by different concentrations of M-1 and its reusability was investigated under mild regeneration conditions (pH = 10, 328 K). M-1 exhibited 93.41 % removal of ferulic acid and 53.12 % removal of furfural with almost no sugar loss during the adsorption process, as well as excellent reusability. The adsorbent M-1 (before and after adsorption) was determined by suitable characterization techniques and the results illustrated π-π interactions and hydrogen bonding as the major forces. Above all, adsorbents have promising applications for the detoxification of acidic lignocellulosic hydrolysates.

Optimization of l-malic acid production from acetate with Aspergillus oryzae DSM 1863 using a pH-coupled feeding strategy

BackgroundMalic acid, a dicarboxylic acid mainly used in the food industry, is currently produced from fossil resources. The utilization of low-cost substrates derived from biomass could render microbial processes economic. Such feedstocks, like lignocellulosic hydrolysates or condensates of fast pyrolysis, can contain high concentrations of acetic acid. Acetate is a suitable substrate for l-malic acid production with the filamentous fungus Aspergillus oryzae DSM 1863, but concentrations obtained so far are low. An advantage of this carbon source is that it can be used for pH control and simultaneous substrate supply in the form of acetic acid. In this study, we therefore aimed to enhance l-malate production from acetate with A. oryzae by applying a pH-coupled feeding strategy.ResultsIn 2.5-L bioreactor fermentations, several feeding strategies were evaluated. Using a pH-coupled feed consisting of 10 M acetic acid, the malic acid concentration was increased about 5.3-fold compared to the batch process without pH control, resulting in a maximum titer of 29.53 ± 1.82 g/L after 264 h. However, it was not possible to keep both the pH and the substrate concentration constant during this fermentation. By using 10 M acetic acid set to a pH of 4.5, or with the repeated addition of NaOH, the substrate concentration could be maintained within a constant range, but these strategies did not prove beneficial as lower maximum titers and yields were obtained. Since cessation of malic acid production was observed in later fermentation stages despite carbon availability, a possible product inhibition was evaluated in shake flask cultivations. In these experiments, malate and succinate, which is a major by-product during malic acid production, were added at concentrations of up to 50 g/L, and it was found that A. oryzae is capable of organic acid production even at high product concentrations.ConclusionsThis study demonstrates that a suitable feeding strategy is necessary for efficient malic acid production from acetate. It illustrates the potential of acetate as carbon source for microbial production of the organic acid and provides useful insights which can serve as basis for further optimization.

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.