Efficient Extraction of Fermentation Inhibitors by Means of Green Hydrophobic Deep Eutectic Solvents

The methods for hydrogen yield efficiency improvements, the gaseous stream purification in gaseous biofuels generation, and the biomass pretreatment are considered as the main trends in research devoted to gaseous biofuel production. The environmental aspect related to the liquid stream purification arises. Moreover, the management of post-fermentation broth with the application of various biorefining techniques gains importance. Chemical compounds occurring in the exhausted liquid phase after biomass pretreatment and subsequent dark and photo fermentation processes are considered as value-added by products. The most valuable are furfural (FF), 5-hydroxymethylfurfural (HMF), and levulinic acid (LA). Enriching their solutions can be carried with the application of liquid–liquid extraction with the use of a suitable solvent. In these studies, hydrophobic deep eutectic solvents (DESs) were tested as extractants. The screening of 56 DESs was carried out using the Conductor-like Screening Model for Real Solvents (COSMO-RS). DESs which exposed the highest inhibitory effect on fermentation and negligible water solubility were prepared. The LA, FF, and HMF were analyzed using FT-IR and NMR spectroscopy. In addition, the basic physicochemical properties of DES were carefully studied. In the second part of the paper, deep eutectic solvents were used for the extraction of FF, LA, and HMF from post-fermentation broth (PFB). The main extraction parameters, i.e., temperature, pH, and DES: PFB volume ratio (VDES:VPFB), were optimized by means of a Box–Behnken design model. Two approaches have been proposed for extraction process. In the first approach, DES was used as a solvent. In the second, one of the DES components was added to the sample, and DES was generated in situ. To enhance the post-fermentation broth management, optimization of the parameters promoting HMF, FF, and LA extraction was carried under real conditions. Moreover, the antimicrobial effect of the extraction of FF, HMF, and LA was investigated to define the possibility of simultaneous separation of microbial parts and denatured peptides via precipitation.

Techno-economic analysis of recombinant Endo-β-1,4-Glucanase production from Escherichia coli Eg-RK2 culture using oil palm empty fruit bunch

A techno-economic analysis of recombinant cellulase production from E. coli Eg-RK2 was conducted to support the fulfilling of Indonesia’s energy roadmap for ethanol production. The plant utilizes OPEFB as a primary substrate in cellulase production, with an expected lifetime of 12 years. The plant is assumed to be built in Indonesia and it will fulfill 1% of the total market demand. The effect of different pretreatment processes (alkaline, steam explosion, and sequential acid-alkaline) on the profitability parameter was also studied. A simulation using SuperPro Designer was used to calculate the mass and energy balance based on the kinetic parameters of E. coli EgRK2. A technology evaluation showed that alkaline pretreatment provides the highest yield with no known inhibitors formed. The steam explosion pretreatment offers the lowest rate of lignin and hemicellulose removal, and it is understood to form known fermentation inhibitors. The NPVs of the alkaline, steam explosion and sequential acid-alkaline pretreatments are USD 32,121,000, USD -36,841,000, and USD 384,000, respectively, which means the alkaline pretreatment is economically very feasible for the production of cellulase.

Process Development for the Detoxification of Fermentation Inhibitors from Acid Pretreated Microalgae Hydrolysate

The aim of this study was to remove 5-hydroxymethyl furfural (5-HMF) and furfural, known as fermentation inhibitors, in acid pretreated hydrolysates (APH) obtained from Scenedesmus obliquus using activated carbon. Microwave-assisted pretreatment was used to produce APH containing glucose, xylose, and fermentation inhibitors (5-HMF, furfural). The response surface methodology was applied to optimize key detoxification variables such as temperature (16.5–58.5 °C), time (0.5–5.5 h), and solid–liquid (S-L) ratio of activated carbon (0.6–7.4 w/v%). Three variables showed significant effects on the removal of fermentation inhibitors. The optimum detoxification conditions with the maximum removal of fermentation inhibitors and the minimum loss of sugars (glucose and xylose) were as follows: temperature of 36.6 °C, extraction time of 3.86 h, and S-L ratio of 3.3 w/v%. Under these conditions, removal of 5-HMF, furfural, and sugars were 71.6, 83.1, and 2.44%, respectively, which agreed closely with the predicted values. When the APH and detoxified APH were used for ethanol fermentation by S. cerevisiae, the ethanol produced was 38.5% and 84.5% of the theoretical yields, respectively, which confirmed that detoxification using activated carbon was effective in removing fermentation inhibitors and increasing fermentation yield without significant removal of fermentable sugars.

Identification of the major fermentation inhibitors of recombinant 2G yeasts in diverse lignocellulose hydrolysates

Background Presence of inhibitory chemicals in lignocellulose hydrolysates is a major hurdle for production of second-generation bioethanol. Especially cheaper pre-treatment methods that ensure an economical viable production process generate high levels of these inhibitory chemicals. The effect of several of these inhibitors has been extensively studied with non-xylose-fermenting laboratory strains, in synthetic media, and usually as single inhibitors, or with inhibitor concentrations much higher than those found in lignocellulose hydrolysates. However, the relevance of individual inhibitors in inhibitor-rich lignocellulose hydrolysates has remained unclear. Results The relative importance for inhibition of ethanol fermentation by two industrial second-generation yeast strains in five lignocellulose hydrolysates, from bagasse, corn cobs and spruce, has now been investigated by spiking higher concentrations of each compound in a concentration range relevant for industrial hydrolysates. The strongest inhibition was observed with industrially relevant concentrations of furfural causing partial inhibition of both D-glucose and D-xylose consumption. Addition of 3 or 6 g/L furfural strongly reduced the ethanol titer obtained with strain MD4 in all hydrolysates evaluated, in a range of 34 to 51% and of 77 to 86%, respectively. This was followed by 5-hydroxymethylfurfural, acetic acid and formic acid, for which in general, industrially relevant concentrations caused partial inhibition of D-xylose fermentation. On the other hand, spiking with levulinic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid or vanillin caused little inhibition compared to unspiked hydrolysate. The further evolved MD4 strain generally showed superior performance compared to the previously developed strain GSE16-T18. Conclusion The results highlight the importance of individual inhibitor evaluation in a medium containing a genuine mix of inhibitors as well as the ethanol that is produced by the fermentation. They also highlight the potential of increasing yeast inhibitor tolerance for improving industrial process economics.

Microwave-assisted hydrotropic pretreatment as a new and highly efficient way to cellulosic ethanol production from maize distillery stillage

Aim of the study was to assess the suitability of the combined use of microwave radiation and sodium cumene sulfonate under optimized process conditions for the preparation of maize stillage biomass as a raw material for the production of cellulosic ethanol. The key parameter guaranteeing a high level of lignin removal from biomass (ca. 44%) was concentration of hydrotrope. Even at high biomass concentration (16% w/v) and a cellulase enzyme dose of about 4 filter-paper units/g, maize stillage biomass subjected to microwave-assisted hydrotropic pretreatment was highly susceptible to enzymatic degradation, which resulted in 80% hydrolysis yield. It is possible to obtain a fermentation medium with a very high glucose concentration (up to 80 g/L), without fermentation inhibitors and, as a consequence, to reach a very high level of sugar conversion to ethanol (concentration above 40 g/L), even as much as 95% of theoretical yield. Microwave hydrotropic treatment with sodium cumene sulfonate is a very effective way to prepare waste maize stillage biomass for the production of cellulosic ethanol. The degradation of the lignocellulose structure by the simultaneous use of microwaves and hydrotropes ensured a high degree of conversion of structural polysaccharides to bioethanol. The method provides a high level of enzymatic degradation of cellulose, leading to a medium with high content of released sugars suitable for bioconversion, which is in line with assumptions of the second-generation ethanol production technology. Key points • Microwave-assisted hydrotropic pretreatment is a new way to cellulosic ethanol production. • Microwave-assisted hydrotropic delignification removes 44% of lignin from biomass. • No fermentation inhibitors are obtained after microwave-assisted hydrotropic pretreatment. • High ethanol concentration (above 40 g/L) and fermentation yield (95% of theoretical yield) from biomass after microwave-assisted hydrotropic pretreatment.

Study of Carbohydrate Hydrolysis From Arracacha Roots (Arracacia Xanthorriza Bancroft) to Produce Fermentable Sugars

In Colombia, approximately 855 840 tons of arracacha are produced each year. The unsalable postharvest arracacha root (Arracacia xanthorriza Bancroft) is not commercialized, mainly due to mechanical damage or small and misshapen roots. In this work, dry samples were characterized and subjected to two treatments: one using thermal hydrolysis, applying saturated steam at pressures of 0,1034 MPa, 0,2068 MPa, and 0,4137 MPa; and another one using hydrolysis with sulfuric acid in concentrations between 0,252,00 M. Then, the cake resulting from the hydrolysis and filtration process was enzymatically hydrolyzed (Liquozyme SC DS, Novozymes) at 1,5, 5 and 10 KNU/g (pH 6, 80 _C, 2 h). Fermentation inhibitors (acetic acid and furfural) were evaluated in the best pretreatment. The results showed that the treatment with sulfuric acid at 1,00 M (2 h) has high yields in reducing sugars added to enzymatic hydrolysis. The maximum level of fermentable carbohydrates per gram of dry sample (1,04 g/g) was also reached. Regarding the fermentation inhibitors of the reducing sugar, a higher concentration of acetic acid was found with a lower furfural content. Therefore, arracacha discards are a promising raw material to increase the supply of bioethanol.

Ethanol Production from Wheat Straw Hydrolysate by Issatchenkia Orientalis Isolated from Waste Cooking Oil

The interest in using non-conventional yeasts to produce value-added compounds from low cost substrates, such as lignocellulosic materials, has increased in recent years. Setting out to discover novel microbial strains that can be used in biorefineries, an Issatchenkia orientalis strain was isolated from waste cooking oil (WCO) and its capability to produce ethanol from wheat straw hydrolysate (WSHL) was analyzed. As with previously isolated I. orientalis strains, WCO-isolated I. orientalis KJ27-7 is thermotolerant. It grows well at elevated temperatures up to 42 °C. Furthermore, spot drop tests showed that it is tolerant to various chemical fermentation inhibitors that are derived from the pre-treatment of lignocellulosic materials. I. orientalis KJ27-7 is particularly tolerant to acetic acid (up to 75 mM) and tolerates 10 mM formic acid, 5 mM furfural and 10 mM hydroxymethylfurfural. Important for biotechnological cellulosic ethanol production, I. orientalis KJ27-7 grows well on plates containing up to 10% ethanol and media containing up to 90% WSHL. As observed in shake flask fermentations, the specific ethanol productivity correlates with WSHL concentrations. In 90% WSHL media, I. orientalis KJ27-7 produced 10.3 g L−1 ethanol within 24 h. This corresponds to a product yield of 0.50 g g−1 glucose (97% of the theoretical maximum) and a volumetric productivity of 0.43 g L−1 h−1. Therefore, I. orientalis KJ27-7 is an efficient producer of lignocellulosic ethanol from WSHL.

Impact of Lignocellulose Pretreatment By-Products on S. cerevisiae Strain Ethanol Red Metabolism during Aerobic and An-aerobic Growth

Understanding the specific response of yeast cells to environmental stress factors is the starting point for selecting the conditions of adaptive culture in order to obtain a yeast line with increased resistance to a given stress factor. The aim of the study was to evaluate the specific cellular response of Saccharomyces cerevisiae strain Ethanol Red to stress caused by toxic by-products generated during the pretreatment of lignocellulose, such as levulinic acid, 5-hydroxymethylfurfural, furfural, ferulic acid, syringaldehyde and vanillin. The presence of 5-hydroxymethylfurfural at the highest analyzed concentration (5704.8 ± 249.3 mg/L) under aerobic conditions induced the overproduction of ergosterol and trehalose. On the other hand, under anaerobic conditions (during the alcoholic fermentation), a decrease in the biosynthesis of these environmental stress indicators was observed. The tested yeast strain was able to completely metabolize 5-hydroxymethylfurfural, furfural, syringaldehyde and vanillin, both under aerobic and anaerobic conditions. Yeast cells reacted to the presence of furan aldehydes by overproducing Hsp60 involved in the control of intracellular protein folding. The results may be helpful in optimizing the process parameters of second-generation ethanol production, in order to reduce the formation and toxic effects of fermentation inhibitors.

Removal of fermentation inhibitors from pre-hydrolysis liquor using polystyrene divinylbenzene resin

Background The presence of soluble lignin, furfural and hydroxymethylfurfural (HMF) in industrial pre-hydrolysis liquor (PHL) from the pulping process can inhibit its bioconversion into bioethanol and other biochemicals. Although various technologies have been developed to remove these inhibitors, certain amounts of sugars are also inevitably removed during the treatment process. Hence, polystyrene divinylbenzene (PS-DVB) resin was used as an adsorptive material to simultaneously remove fermentation inhibitors while retaining sugars with high yields to improve the fermentability of PHL after acid hydrolysis by enriching its xylose concentration. The fermentability of acid-hydrolyzed PHL (A-PHL) was evaluated by the bioconversion into ethanol and xylosic acid (XA) after treatment with PS-DVB resin. Results The results showed that the highest xylose concentration (101.1 g/L) in PHL could be obtained by acid hydrolysis at 100 °C for 80 min with 4% acid, while the concentration of fermentation inhibitors (furfural, HMF and lignin) in PHL could also be significantly improved during the acid-hydrolysis process. After treatment with PS-DVB resin, not only were 97% of lignin, 92% of furfural, and 97% of HMF removed from A-PHL, but also 96% of xylose was retained for subsequent fermentation. With resin treatment, the fermentability of A-PHL could be improved by 162–282% for ethanol production from A-PHL containing 30–50 g/L xylose and by 18–828% for XA production from A-PHL containing 90–150 g/L xylose. Conclusions These results confirmed that PS-DVB resin can remove inhibitors from PHL before producing value-added products by bioconversion. In addition, this work will ideally provide a concept for producing value-added chemicals from pre-hydrolysis liquor, which is regarded as the waste stream in the pulping process.