Synthetic Hydrolysates Research Articles

Utilization of Noxious Weed Water Hyacinth Biomass as a Potential Feedstock for Biopolymers Production: A Novel Approach

This study aims to utilize a noxious weed water hyacinth biomass (WH) for polyhydroxybutyrate (PHB) production. Alkaline and peracetic acid pretreatment was employed for the hydrolysis of WH and consequently enzymatic saccharification to produce fermentable sugars for PHB production. The pretreatment competence was determined using various operational parameters. By applying ambient conditions, alkaline pretreatment gave higher lignin removal of 65.0%, with 80.8% hydrolysis yield, and on enzyme hydrolysis (40 FPU/g of dry WH), produced total reducing sugar of about 523 mg/g of WH. The resulted WH enzymatic hydolysates were evaluated for the production of PHB by Ralstonia eutropha (ATCC 17699). The WH hydrolysates cultivation was compared to synthetic hydrolysates that contain a similar carbon composition in terms of bacterial growth and PHB synthesis. The effects of various supplements to enhance PHB production were estimated. Supplementation of corn steep liquor (CSL) as a cheap nitrogen source with WH hydrolysates favored a higher amount of PHB synthesis (73%), PHB titer of 7.30 g/L and PHB yield of 0.429 g/g of reducing sugar. Finally, using standard analytical tools, the physical and thermal characteristics of the extracted PHB were evaluated. The findings revealed WH was a promising and technically feasible option for transforming biomass into sustainable biopolymer conversion on a large scale.

Thermophilic Methane Production from Hydrothermally Pretreated Norway Spruce (Picea abies)

Norway spruce (Picea abies) is an industrially important softwood species available in northern Europe and can be used to produce bio-methane after proper pretreatment to overcome its recalcitrant complex structure. Hot water extraction (HWE) pretreatment at two different conditions (170 °C for 90 min (severity 4.02) and 140 °C for 300 min (severity 3.65)) was applied to extract hemicellulosic sugars from Norway spruce for thermophilic anaerobic digestion (AD) of the hydrolysate. The methane yield of hydrolysate prepared at the lower pretreatment severity was found to be 189 NmL/gCOD compared to 162 NmL/gCOD after the higher pretreatment severity suggesting higher pretreatment severity hampers the methane yield due to the presence of inhibitors formed due to sugars and lignin degradation and soluble lignin, extracted partially along with hemicellulosic sugars. Synthetic hydrolysates simulating real hydrolysates (H170syn and H140syn) had improved methane yield of 285 NmL/gCOD and 295 NmL/gCOD, respectively in the absence of both the inhibitors and soluble lignin. An effect of organic loadings (OLs) on the methane yield was observed with a negative correlation between OL and methane yield. The maximum methane yield was 290 NmL/gCOD for hydrolysate pretreated at 140 °C compared to 195 NmL/gCOD for hydrolyate pretreated at 170 °C, both at the lowest OL of 6 gCOD/L. Therefore, both pretreatment conditions and OL need to be considered for efficient methane production from extracted hydrolysate. Such substrates can be utilized in continuous flow industrial AD with well-adapted cultures with stable organic loading rates.

Water-soluble phenolic compounds produced from extractive ammonia pretreatment exerted binary inhibitory effects on yeast fermentation using synthetic hydrolysate.

Biochemical conversion of lignocellulosic biomass to liquid fuels requires pretreatment and enzymatic hydrolysis of the biomass to produce fermentable sugars. Degradation products produced during thermochemical pretreatment, however, inhibit the microbes with regard to both ethanol yield and cell growth. In this work, we used synthetic hydrolysates (SynH) to study the inhibition of yeast fermentation by water-soluble components (WSC) isolated from lignin streams obtained after extractive ammonia pretreatment (EA). We found that SynH with 20g/L WSC mimics real hydrolysate in cell growth, sugar consumption and ethanol production. However, a long lag phase was observed in the first 48 h of fermentation of SynH, which is not observed during fermentation with the crude extraction mixture. Ethyl acetate extraction was conducted to separate phenolic compounds from other water-soluble components. These phenolic compounds play a key inhibitory role during ethanol fermentation. The most abundant compounds were identified by Liquid Chromatography followed by Mass Spectrometry (LC-MS) and Gas Chromatography followed by Mass Spectrometry (GC-MS), including coumaroyl amide, feruloyl amide and coumaroyl glycerol. Chemical genomics profiling was employed to fingerprint the gene deletion response of yeast to different groups of inhibitors in WSC and AFEX-Pretreated Corn Stover Hydrolysate (ACSH). The sensitive/resistant genes cluster patterns for different fermentation media revealed their similarities and differences with regard to degradation compounds.

Genome shuffling to generate recombinant yeasts for tolerance to inhibitors present in lignocellulosic hydrolysates

To investigate the use of genome shuffling to generate recombinants from previously generated hydrolysates-tolerant strains to improve tolerance of Saccharomyces cerevisiae to one or more inhibitory by-products present in lignocellulosic hydrolysates. Recombinants of previously evolved strains of S. cerevisiae were generated and analyzed for their relative performance in the individual inhibitors furfural, acetic acid, 5-(hydroxymethyl)-furfural (HMF) and in synthetic hydrolysates. One recombinant exhibited a 100% fitness increase in the presence of HMF as compared to the wild-type diploid, while another stain exhibited a 13% fitness increase in the presence of furfural. Furthermore, for one of these recombinants, these increases in fitness were specific to the inhibitor HMF and to synthetic hydrolysates rather than being due to a general increase in fitness. Mutations present in the evolved hydrolysates-tolerant mutants were identified via whole-genome resequencing. Recombinants of S. cerevisiae were produced with increased tolerance to inhibitory by-products present in hydrolysates of lignocellulosic biomass and identified potential genetic determinants associated with this phenotype.

Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification.

Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts.

Xeno-Free Plant-Derived Hydrolysate-Based Freezing of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are one of the most interesting cell types for tissue engineering and cell therapy. The large-scale banking of hESCs for research and future clinical application requires economic, defined, and xeno-free cryopreservation protocols. In this study, the possibility to substitute knockout serum replacement (KO-SR) in the cryopreservation process with vegetal and synthetic hydrolysates was investigated. To our knowledge, the use of hydrolysates in hESC cryopreservation has not been yet explored. Initially, 3 different hydrolysates (Ultrapep Soy, Hypep 4601 and EX-CELL(®) CD Hydrolysate Fusion) were tested in the cryopreservation solution. A concentration of 8 mg/mL EX-CELL CD Hydrolysate Fusion in the cryopreservation solution leads to the highest recovery ratio; thus, this solution was selected for additional cryopreservation experiments. To ensure reproducibility of the results, 3 hESC lines (HS181, H9, and BG01) were used. The hESCs were collected prefreezing by application of collagenase IV and cell dissociation solution. Experiments showed that it was feasible to substitute the KO-SR in both the cryopreservation solution as the thawing solution. The obtained recovery ratios were comparable to those obtained with KO-SR (no statistical significant difference; Student's t-test, P<0.05). Further optimization protocols showed a doubling of the obtained recovery ratio after addition of Rock-inhibitor Y-27632 post-thawing. The expansion profile and pluripotency analysis revealed no changes in normal hESC behavior. We conclude that the application of vegetal or synthetic hydrolysates is suitable for xeno-free hESC cryopreservation.

Biological detoxification of waste house wood hydrolysate using Ureibacillus thermosphaericus for bioethanol production

Hydrolysates of lignocelluloses hydrolyzed by diluted sulfuric acid contain toxic compounds that inhibit ethanol production by Saccharomyces cerevisiae and the ethanologenic recombinant Escherichia coli KO11. We investigated the biological detoxification of a hydrolysate of waste house wood (WHW) by a thermophilic bacterium, Ureibacillus thermosphaericus. When the hydrolysate was treated with this bacterium at 50 degrees C for 24 h, the ethanol production rate by S. cerevisiae increased markedly and was comparable to that for the hydrolysate treated with an excess amount of calcium hydroxide (overliming). Chromatographic analysis of synthetic hydrolysates containing furfural or 5-hydroxymethyl furfural that are considered to be major toxic compounds in hydrolysates revealed that U. thermosphaericus degrades these compounds. In the WHW hydrolysates, however, the concentrations of these compounds were not decreased markedly by the bacterium. These results suggest that the bacterium degrades minor but more toxic compounds or phenolic compounds in the WHW hydrolysates. The combination of bacterial and overliming treatments of hydrolysates minimized significantly the decrease in ethanol production rate by E. coli KO11 as fermentation proceeded. Because the bacterium grows rapidly and does not consume sugars, our biological detoxification should be useful for bioethanol production from acid hydrolysates of lignocelluloses.