Detoxification of corn stover prehydrolysate by different biochars and its effect on lactic acid fermentation.

During the utilization of lignocellulosic biomass such as corn stover, many by-products are produced in the pretreatment process that can severely inhibit the activity of microbes in the fermentation step. To achieve efficient biomass conversion, detoxification is usually required before microbial fermentation. In this study, the prehydrolysate from dilute acid pretreatment of corn stover was used as a lactic acid fermentation substrate. Biochars made from corn stover (CSB), cow manure (CMB), and a mixture of corn stover and cow manure (MB) were applied for the detoxification of the prehydrolysate. All three types of biochar had a porous structure with a specific surface area ranging from 4.08 m2 g-1 (CMB) to 7.03 m2 g-1 (MB). After detoxification, both the numbers of inhibitors and their concentrations in the prehydrolysate decreased, indicating that the biochars prepared in this study were effective in inhibitor removal. The concentration of lactic acid obtained from the prehydrolysate without detoxification was only 12.43 g L-1 after fermentation for 96 h with a productivity of 0.13 g (L h)-1. Although the specific area of CMB was the lowest among the three biochars, the CMB-treated prehydrolysate resulted in the highest lactic acid concentration of 39.25 g L-1 at 96 h with a productivity of 0.41 g (L h)-1. The lactic acid bacteria in the CMB-treated prehydrolysate grew faster than the other two biochars, reaching an OD value of 8.12 at 48 h. The results showed promise for the use of agricultural wastes to make biochar to increase the yield of lactic acid fermentation through the detoxification process.

Enhanced Enzymatic Sugar Recovery of Dilute-Acid-Pretreated Corn Stover by Sodium Carbonate Deacetylation.

The prehydrolysate from dilute acid pretreatment of lignocellulosic feedstocks often contains inhibitory compounds that can seriously inhibit the subsequent enzymatic and fermentation processes. Acetic acid is one of the most representative toxic compounds. In this research, alkaline deacetylation of corn stover was carried out using sodium carbonate under mild conditions to selectively remove the acetyl groups of the biomass and reduce the toxicity of the prehydrolysate. The deacetylation process was optimized by adjusting factors such as temperature, treatment time, and sodium carbonate concentration. Sodium carbonate solutions (2~6 wt%) at 30~50 °C were used for the deacetylation step, followed by dilute acid pretreatment with 1.5% H2SO4 at 121 °C. Results showed that the acetyl content of the treated corn stover could be reduced up to 87%, while the hemicellulose loss remained low. The optimal deacetylation condition was found to be 40 °C, 6 h, and 4 wt% Na2CO3, resulting in a removal of 80.55% of the acetyl group in corn stover and a hemicellulose loss of 4.09%. The acetic acid concentration in the acid prehydrolysate decreased from 1.38 to 0.34 g/L. The enzymatic hydrolysis of solid corn stover and the whole slurry after pretreatment increased by 17% and 16%, respectively.

The transformations of cellulose after concentrated sulfuric acid treatment and its impact on the enzymatic saccharification.

BACKGROUND: The dense structure of cellulose lowers its reactivity and hinders its applications. Concentrated sulfuric acid is an ideal solvent to dissolve cellulose and thus has been used widely to treat cellulose. However, the changes of cellulose after reaction with concentrated sulfuric acid at near-limit S/L ratio and its effect on enzymatic saccharification still need further investigation. RESULTS: In this study, the interactions between cellulose (Avicel) and 72% sulfuric acid at very low acid loading conditions of 1:2 to 1:3 (S/L ratio) were studied for the enhanced production of glucose. The Avicel gradually transformed from cellulose I structure to cellulose II structure during the sulfuric acid treatment. Other physicochemical characteristics of Avicel also changed dramatically, such as the degree of polymerization, particle size, crystallinity index, and surface morphology. After acid treatment, both the yield and productivity of glucose from cellulose increased significantly under a very low enzyme loading of 5 FPU/g-cellulose. The glucose yields for raw cellulose and acid-treated (30 min) were 57% and 85%, respectively. CONCLUSION: Low loadings of concentrated sulfuric acid were proven to be effective to break the recalcitrance of cellulose for enzymatic saccharification. A positive correlation between cellulose CrI and glucose yield was found for concentrated sulfuric acid-treated cellulose, which was opposite to previous reports. Cellulose II content was found to be an important factor that affects the conversion of cellulose to glucose.

A novel in-situ quantitative profiling approach for visualizing changes in lignin and cellulose by stained micrographs.

There is a strong need for low-cost lignocellulosic composition simultaneous localization methodologies to benefit deeper understandings of crop stalk morphology. This study developed a robust quantitative safranin O-fast green staining-based optical microscopy imaging methodology for in-situ simultaneously generating digital profiles of lignin and cellulose in stalk tissues. Foreground extraction and dye residue removal of stained images were adapted. The ratios of normalized red (R), green (G), and blue (B) channel signal intensity, R/B and G/B, were defined as quantitative indicators of lignin and cellulose, respectively. The method was validated on model rice with known bioinformatics, and the results were consistent with those of fluorescence microscopy and immunogold labeling methods. The high-definition spatial in-situ simultaneous profiles of lignin and cellulose in alkali-treated maize stalk tissues and their variations were visualized. This low-cost, cell-scale method is expected to contribute to new discoveries in many areas of biomass refining and plant science.

New rapid detection method of total chlorogenic acids in plants using SERS based on reusable Cu2O-Ag substrate.

A new method for rapidly detecting of total chlorogenic acids (CGAs) in plants by surface-enhanced Raman spectroscopy (SERS) based on reusable Cu2O-Ag substrate was developed in this study. The Cu2O-Ag substrate prepared by the in-situ growth method had high uniformity with peak intensity relative standard deviation (RSD) of 5.27%, repeatability with peak intensity RSD of 3.58%, and sensitivity with an analytical enhancement factor of 1.27 × 105 for detecting CGAs. Furthermore, the substrate had excellent reusability, after it was reused for seven cycles, the signal strength of CGAs was still above 80% of initial. Compared with the standard method of high-performance liquid chromatography (HPLC), the SERS method can successfully analyze the contents of total CGAs in plants, such as Stevia rebaudiana leaves, coffee beans, Lonicera japonica leaves, and Eucommia ulmoides flowers, with recovery rate from 93.26% to 112.65%, and the limit of detection was 0.13 µg/mL. The total CGAs content of Stevia rebaudiana leaves samples detected by HPLC and SERS have good consistency with R = 0.9760 and RMSE = 3286 mg/kg. Furthermore, the SERS method only needed less than 1 min, one standard and reusable substrate in this study to analyze, which can further reduce the cost of method analysis. Therefore, the SERS method with the appropriate substrate can provide a rapid, accurate, and economic way to detect the total CGAs in plants.

Effect of combined wet alkaline mechanical pretreatment on enzymatic hydrolysis of corn stover and its mechanism.

BACKGROUND: To further optimize the mechanochemical pretreatment process, a combined wet alkaline mechanical pretreatment of corn stover was proposed with a short time and less chemical consumption at room temperature. RESULTS: The combined alkaline mechanical pretreatment significantly enhanced enzymatic hydrolysis resulting a highest glucose yield (YG) of 91.9% with 3% NaOH and ball milling (BM) for 10 min. At this optimal condition, 44.4% lignin was removed and major portion of cellulose was retained (86.6%). The prehydrolysate contained by-products such as monosaccharides, oligosaccharides, acetic acid, and lignin but no furfural and 5-HMF. The alkaline concentration showed a significant impact on glucose yield, while the BM time was less important. Quantitative correlation analysis showed that YG (%) = 0.68 × BM time (min) + 19.27 × NaOH concentration (%) + 13.71 (R2 = 0.85), YG = 6.35 × glucan content - 231.84 (R2 = 0.84), and YG = - 14.22 × lignin content + 282.70 (R2 = 0.87). CONCLUSION: The combined wet alkaline mechanical pretreatment at room temperature had a boosting effect on the yield of enzymatic hydrolysis with short treatment time and less chemical consumption. The impact of the physical and chemical properties of corn stover pretreated with different BM times and/or different NaOH concentrations on the subsequent enzymatic hydrolysis was investigated, which would be beneficial to illustrate the effective mechanism of the mechanochemical pretreatment method.

A particle scale micro-CT approach for 3D in-situ visualizing the Pb (II) adsorption in different crop residue-derived chars.

It is crucial to develop a new characterization method to provide insight into the complex adsorption mechanism of crop residue-derived char. This study established a novel 3D in-situ visualization method for qualitative and semi-quantitative characterizing Pb (II) adsorption profiles in crop residue-derived char particles. First, coconut shell activated carbon, rice husk biochar, and wheat biochar after Pb (II) adsorption was used for X-ray micro-CT imaging. Then, the K-means clustering algorithm was developed for segmenting the volume image of samples, and the optimized segmentation thresholds for the 3 samples were 6000HU, 7000HU, and 1300HU, respectively. The rendered images for qualitative illustrating the adsorption profile of Pb (II) were presented. Finally, based on the derived quantitative formula, the Pb (II) distribution in the biochar particle was presented for the first time. This method provided a new perspective and methodology for analysis and simulations of the adsorption behavior of heavy metals onto chars.

Integrated production of optically pure l-lactic acid from paper mill sludge by simultaneous saccharification and co-fermentation (SSCF).

Paper mill sludge (PMS) raises critical environmental issues due to its disposal problem, but its high sugar content and well-dispersed structure make it a great feedstock for biochemical production. The technical feasibility of integrating cellulase enzyme production into lactic acid (LA) fermentation from PMS was investigated in this study. The low ash content of PMS suggests a great potential for cellulase production. The enzyme produced using PMS without any treatment gave an activity of 7.8 FPU/ml, a performance comparable to the commercial enzyme, Cellic CTec 2. The LA yield from PMS with in-house enzyme was 64.7% and 73.7% at the enzyme loading of 10 and 15 FPU/g-glucan, respectively. The LA obtained was optically pure L- isomer with over 99% purity. The optimal condition of LA production by Bacillus coagulans was found to be 50 °C and pH 5.3 (with 50 g/L CaCO3). The nutrient effect of yeast extract (YE) and corn steep liquor (CSL) was substrate dependent, and CSL could substitute YE as an inexpensive nutrient when using PMS as a substrate.

Effect of residual extractable lignin on acetone-butanol-ethanol production in SHF and SSF processes.

BACKGROUND: Lignin plays an important role in biochemical conversion of biomass to biofuels. A significant amount of lignin is precipitated on the surface of pretreated substrates after organosolv pretreatment. The effect of this residual lignin on enzymatic hydrolysis has been well understood, however, their effect on subsequent ABE fermentation is still unknown. RESULTS: To determine the effect of residual extractable lignin on acetone-butanol-ethanol (ABE) fermentation in separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes, we compared ABE production from ethanol-washed and unwashed substrates. The ethanol organosolv pretreated loblolly pine (OPLP) was used as the substrate. It was observed that butanol production from OPLP-UW (unwashed) and OPLP-W (washed) reached 8.16 and 1.69 g/L, respectively, in SHF. The results showed that ABE production in SHF from OPLP-UW prevents an "acid crash" as compared the OPLP-W. In SSF process, the "acid crash" occurred for both OPLP-W and OPLP-UW. The inhibitory extractable lignin intensified the "acid crash" for OPLP-UW and resulted in less ABE production than OPLP-W. The addition of detoxified prehydrolysates in SSF processes shortened the fermentation time and could potentially prevent the "acid crash". CONCLUSIONS: The results suggested that the residual extractable lignin in high sugar concentration could help ABE production by lowering the metabolic rate and preventing "acid crash" in SHF processes. However, it became unfavorable in SSF due to its inhibition of both enzymatic hydrolysis and ABE fermentation with low initial sugar concentration. It is essential to remove extractable lignin of substrates for ABE production in SSF processes. Also, a higher initial sugar concentration is needed to prevent the "acid crash" in SSF processes.

Detoxification of Organosolv-Pretreated Pine Prehydrolysates with Anion Resin and Cysteine for Butanol Fermentation.

Bioconversion of lignocellulose to biofuels suffers from the degradation compounds formed during pretreatment and acid hydrolysis. In order to achieve an efficient biomass to biofuel conversion, detoxification is often required before enzymatic hydrolysis and microbial fermentation. Prehydrolysates from ethanol organosolv-pretreated pine wood were used as substrates in butanol fermentation in this study. Six detoxification approaches were studied and compared, including overliming, anion exchange resin, nonionic resin, laccase, activated carbon, and cysteine. It was observed that detoxification by anion exchange resin was the most effective method. The final butanol yield after anion exchange resin treatment was comparable to the control group, but the fermentation was delayed for 72 h. The addition of Ca(OH)2 was found to alleviate this delay and improve the fermentation efficiency. The combination of Ca(OH)2 and anion exchange resin resulted in completion of fermentation within 72 h and acetone-butanol-ethanol (ABE) production of 11.11 g/L, corresponding to a yield of 0.21 g/g sugar. The cysteine detoxification also resulted in good detoxification performance, but promoted fermentation towards acid production (8.90 g/L). The effect of salt on ABE fermentation was assessed and the possible role of Ca(OH)2 was to remove the salts in the prehydrolysates by precipitation.

Utilization of solid catfish manure waste as carbon and nutrient source for lactic acid production.

The aim of this work was to study the solid waste (manure) produced by catfish as a potential feedstock for the production of lactic acid (LA) via fermentation. The solid waste contains high levels of both carbohydrates and nutrients that are sufficient for LA bacteria. Simultaneous saccharification and co-fermentation (SSCF) was applied using enzyme and Lactobacillus pentosus, and different loadings of enzyme and solid waste were tested. Results showed LA concentrations of 35.7 g/L were obtained at 15% solids content of catfish waste. Because of the high nutrient content in the fish waste, it could also be used as supplementary substrate for nitrogen and carbon sources with other lignocellulosic materials. A combined feedstock of catfish waste and paper mill sludge was tested, increasing the final LA concentration to 43.1 g/L at 12% solids loading. The catfish waste was shown to be a potential feedstock to provide both carbon and nutrients for LA production, suggesting its use as a sole substrate or in combination with other lignocellulosic materials.

Nutrient value of fish manure waste on lactic acid fermentation by Lactobacillus pentosus.

The aim of this work was to study the feasibility of using fish manure waste as a nutrient source for lactic acid fermentation. Fish waste contains nitrogen and minerals that could support the growth of lactic acid bacteria (LAB), making it a good candidate as the nutrient source for lactic acid fermentation. Two different fish manure wastes, from Nile tilapia and channel catfish aquaculture, were investigated for their performance on different sugar substrates. Both fish waste types showed low efficiency in the direct fermentation of glucose, but satisfactory efficiencies in simultaneous saccharification and fermentation (SSF) of cellulosic materials, such as pure cellulose and paper sludge. The highest lactic acid yield obtained was 87% and 91%, with a corresponding volumetric productivity of 1.006 and 0.580 g L-1 h-1, and corresponding lactic acid concentration of 96 and 56 g L-1 for cellulose and paper sludge, respectively. Fish waste concentrations did not show much impact on lactic acid production for the SSF process, where increasing fish waste from 10 to 30 g L-1 resulted in less than a 10% yield increase. In the present study, fish manure waste was shown to be an effective and economic nutrient source for lactic acid production by SSF.

Acetone-butanol-ethanol production from Kraft paper mill sludge by simultaneous saccharification and fermentation.

Paper mill sludge (PS), a solid waste from pulp and paper industry, was investigated as a feedstock for acetone-butanol-ethanol (ABE) production by simultaneous saccharification and fermentation (SSF). ABE fermentation of paper sludge by Clostridium acetobutylicum required partial removal of ash in PS to enhance its enzymatic digestibility. Enzymatic hydrolysis was found to be a rate-limiting step in the SSF. A total of 16.4-18.0g/L of ABE solvents were produced in the SSF of de-ashed PS with solid loading of 6.3-7.4% and enzyme loading of 10-15FPU/g-glucan, and the final solvent yield reached 0.27g/g sugars. No pretreatment and pH control were needed in ABE fermentation of paper sludge, which makes it an attractive feedstock for butanol production. The results suggested utilization of paper sludge should not only consider the benefits of buffering effect of CaCO3 in fermentation, but also take into account its inhibitory effect on enzymatic hydrolysis.

Production of lactic acid from the mixture of softwood pre-hydrolysate and paper mill sludge by simultaneous saccharification and fermentation.

Paper mill sludge is a solid waste material composed of pulp residues and ash generated from pulping and paper making process. The carbohydrate portion of the sludges from Kraft/Recycle paper mill has chemical and physical characteristics similar to those of commercial wood pulp. Because of its high carbohydrate content and well-dispersed structure, the sludge can be biologically converted to value-added products without pretreatment. In bioconversion of solid feedstock such as paper mill sludge, a certain amount of water must be present to attain fluidity. In this study, hemicellulose pre-hydrolysate, in place of water, was added to the sludge to increase the concentration of the final product. Pre-hydrolysate was obtained by hot-water treatment of pine wood in which the total sugar concentration reached 4 wt.%. The mixture was processed by simultaneous saccharification and fermentation (SSF) using enzymes (cellulase and pectinase) and Lactobacillus rhamnosus (ATCC-10863). Pectinase was added to hydrolyze mannose oligomers in the pre-hydrolysate to monomers. During the SSF of the mixture, calcium carbonate in the paper sludge acted as a buffer, yielding calcium lactate as the final product. External pH control was unnecessary due to the buffer action of calcium carbonate that maintained the pH near optimum for the SSF. The lactic acid yield in the range of 80-90 % of the theoretical maximum was obtained. Use of the mixed feed of pre-hydrolysate and pulp mill sludges in the SSF raised the product concentration to 60 g of lactate/L.