Ultra-high Pressure Explosion Pretreatment And Ethanol Fermentation Research Of Lignocellulose

Lignocellulosic biomass, which is majorly composed of cellulose (accounted for 35～ 50%), hemicellulose (accounted for 25～35%) and lignin (accounted for 15～40%), is the largest renewable organic carbon resource around the world. In theory, the cellulose and hemicellulose components in llignocellulose can be hydrolyzed into glucose, C5 or C6 sugar respectively, and then further converted to ethanol product by microbial fermentation. It is very significance to produce the ethanol from lignocellulosic resource since the enormous resource of lignicellulose will guarantee raw material supply for sustainable development of ethanol fermentation industry, including the traditional alcohol fermentation and the novel rising bio-fuel ethanol production. However, realizing commerce industrial production of lignocellulosic ethanol still faced three technological bottlenecks need to be campaigned:1, the component separation of lignocellulose and the hydrolysis of cellulose in effective cost; 2, the high gravity ethanol fermentation of cellulose hydrant; 3, the high efficiently conversion of C5 sugar to ethanol. To campaign the technique challenges for realizing industrial production of lignocellulose ethanol, the Ultra-high pressure explosion (UHPE) pretreatment of lignocellulose and the ethanol fermentation of cellulose hydrant were explored in this paper. The investigation included four respects:1, the UHPE pretreatment and enzymatic hydrolysis of lignocellulose; 2, the breeding of Saccharomyces cerevisiae strain for very high gravity ethanol fermentation of cellulose hydrant; 3, demonstrating ethanol fermentation of screened high-yield strain; and 4, the genome comparing analysis of serial S. cerevisiae strains with different physiology features relevant to ethanol fermentation.1, The UHPE pretreatment and enzymatic hydrolysis of lignocelluloseA novel technology for pretreatment of lignocellulose materials was innovated by exploding the lignocellulose with a homogenizer. Sugarcane bagasse was ground to 40 meshes, suspended in 0.2% NaOH solution (containing 0.2% xanthan gum) in 3% content, refluxed at 100℃ for 10min, and then realized UHPE by conducting through a homogenizer. The maximum explosion pressure was reached to 100MPa. Since homogenization is achieved by passing a material suspending solution under a very high pressure through an adjustable, restricted orifice discharge valve, the material in suspension solution is first forced by a very high pressure and then exploded by releasing the pressure transiently, the process is very similar to the steam explosion, ammonia explosion and carbon dioxide explosion of lignocellulose pretreatment except that its functioning pressure is much higher and being released much faster than that of steam explosion or ammonia explosion, and the explosion in homogenization is achieved by liquid pressure, the innovated method is defined as a Ultra-high pressure explosion (UHPE) method.After UHPE treatment the light yellow lignocellulose suspension was changed into a dark-brown fluid, i.e., the lignocellulose supension was fluidized, changed from a suspension into a viscous fluid. The scanning electron microscope (SEM) observation showed that the micro-structure of bagasses, rice straw and switch grass was tremendously disrupted by UHPE. The non-UHPE-treated samples (including control and sample only heated at 100℃ for 10min in 0.2% NaOH) exhibited a rigid and highly ordered fibrous structure, and all UHPE-treated samples exhibited a microstructure of distorted, poly-porous and "empty-inside" appearances. The particle of bagasses sample before and after UHPE treatment was determined. The particles of un-treated control sample and the sample only heated at 100℃ for 10min in 0.2% NaOH exhibited a Gauss distribution with the average diameter (Dav) were 583.69um and 605.49um respectively and did not have statistics significant difference between each other (P>0.05). After UHPE-treatment, the particle diameter of bagasse decreased with the pressure increased, exhibited a Poisson distribution at 30～70MPa pressure and recovered Gauss distribution at 80～100MPa pressure. After UHPE-treated at 100MPa the Dav was decreased to 65.08um,88.85～89.25% was decreased comparing with control sample. The explosion pressure (P) and the reciprocal of particle diameter exhibited a linear correlation with the correlation coefficient had significance (P< 0.01). After liquid-solid separation, the crystallinity indexe (CrI) of bagasse solid sample, determined by X-ray Diffraction (XRD), was decreased 17.34%, from 54.83% to 45.34% with the UHPE-treatment pressure was enhanced from 0 to 1 OOMPa.With the microstructure was disrupted and the material feature was significantly changed by UHPE-treatment, the enzymatic digestibility (ED) of bagasse was significantly enhanced. The ED of bagasse had linear relationship with the pressure of UHPE treatment with the correlation coefficient had statistical significance (P<0.01).With the cellulase complex from Trichoderma viride, which containing 5.8FPU of cellulase,6.6IU of xylanase and 0.5CBU of glucosidase, was loaded for 1g solid sample in dry weight basis, the ED of bagasse at 72h hydrolysis was reached to 59.43% after 100MPa UHPE treatment,101.18% more than the sample only heated at 100℃ for 10min in 0.2% NaOH and 389.14% more than the control sample without any pretreatment.The determination of components showed that, with the explosion pressure enhanced the reduced sugar content in both liquid and solid bagasses sample was increased, and the Klason lignin and acid soluble lignin in solid sample were decreased. After 100MPa UHPE treatment, the reduced sugar in solid sample was increased from 73.67% to 78.93%,7.14% was increased, the Klason lignin and acid soluble lignin in solid sample were decreased respectively from 14.64% and 7.74% to 12.30% and 6.87%,15.98% and 11.24% were decreased, and the reduced sugar in liquid was increased from 0 to 0.16%. The recovery of reduced sugar of process was 98.38%～97.20%, indicating UHPE-treatment has negligible affect on the components of lignicellulose.By increasing the NaOH treatment intensity, a complex pretreatment (CP) method by combining UHPE and dilute alkaline treatment was investigated. The lignocellulose was first heated at 125℃ for 120min in 0.5～1.0% NaOH and then UHPE-treated at 100MPa. After UHPE treatment the NaOH consuming of lignocellulose was significantly increased with the hemicellulose and lignin solved out by NaOH was also significantly enhanced. The NaOH consumed by bagasse, rice straw and switchgrass was enhanced 30.66%,163.41% and 53.94% respectively. Since the solving out function of NaOH, the reduced sugar content in solid samples of bagasse, rice straw and switchgrass was increased from 73.67% to 90.60%, from 51.00% to 91.16% and from 59.01% to 89.49%, with 22.98%,78.75% and 51.65% was enhanced respectively, the Klason lignin content in solid sample of bagasse, rice straw and switchgrass was decreased from 14.64%、26.10% and 18.70% to 5.78%,4.69% and 4.15%, with 60.52%、82.03% and 77.81% was decreased respectively, and the acid soluble lignin in solid sample was also decreased from 7.74%、10.23% and 13.03% to 6.70%,6.02% and 5.99%, with 13.44%(bagasse)、41.15% (rice straw) and 54.03% (switchgrass) was decreased respectively.0.34% of reduced sugar in liquid sample of bagasse was determined. The reduced sugar recovery of bagasse during CP pretreatment was reaching to 96.97%, indicating the combining pretreatment of UHPE and dilute alkaline also has negligible affect on the components of lignocellulose.With same amount of cellulase complex was loaded, i.e,5.8FPU of cellulsas,6.6IU of xylanase and 0.5CBU of β-glucosidase for per gram of dry solid sample, after CP the ED of bagasse at 36h,48h,60h and 72h hydrolysis was reaching to 90.03%,90.48%,94.50% and 95.53% respectively, the ED at 72h hydrolysis was 6.86 times (686.26%) enhanced comparing with the un-pretreated control sample (with ED of 12.15%), the ED of rice straw at 48h、60h and 72h hydrolysis was reaching to 90.53%、95.98% and 100.09% respectively, the ED at 72h hydrolysis was enhanced 2.02 times (202.66%) comparing with the un-pretreated control sample, and the ED of switchgrass at 48h hydrolysis was reaching to maximum of 97.60%, which was enhanced 3.15 times (314.97%) comparing with un-pretreated control sample.The Accessible surface area (ASA) of bagasse sample was determined with a cellulase absorption experiment according to the Langmuir isotherm equation. With the pressure of UHPE was enhanced the absorption of cellulase by bagasse samples was enhanced. The maximum absorption and ED had a linear correlation, and the pressure of UHPE and maximum absorption of cellulase also had a linear correlation as well. Both correlation coefficient were statistical significance (P<0.01).Summarize the above results, we argue that UHPE is the only pretreatment method or technology that can significantly disrupt the microstructure and change the material feature of lignicellulose materials in current investigation around world. With the advantage of low process cost, mild process condition and above 95% enzymatic digestibility after pretreatment, the combining pretreatment of UHPE and dilute alkaline is much potential for industrial production of lignocellulose ethanol.2. Breeding of S. cerevisiae strain for high-gravity ethanol fermentation from cellulose hydrantAccording to the requirement of high gravity ethanol fermentation with cellulose hydrant as feedstock to the strain, three yeast strains, nominated as MF1001, MF1002 and MF1003, were screened from years old molasses and identified as belong to different strains of Saccharomyces cerevisiae by aligning ITS sequences using the program Blast with GenBank databases. According to the comparison of the three strains in their features of carbon metabolism, sugar using ability, growth in molasses, and ethanol fermentation, the MF1001, which was with the most superior phenotype in ethanol fermentation, was assigned as the strain for ethanol fermentation of cellulose hydrant, and its phenotype relevant to ethanol fermentation of cellulose hydrant was investigated.The result of optimizing ethanol fermentation condition showed that, the cell growth and ethanol fermentation of MF1001 was not defected in 20°Bx molasses, but was delayed 8h in 30°Bx molasses and seriously repressed in 40°Bx molasses. The optimum fermentation temperature was 30℃, and the result of ethanol fermentation at pH4.0 was better than that at pH3.8. Passage cultures (16 times) and different inoculation amount had a minor influence on the strain’s ethanol fermentation. By using the industrial process of molasses ethanol fermentation, i.e., the strain was grown in medium of 20°Bx molasses first for preparing the seed culture, followed by mixing the seed culture with medium of 55°Bx molasses at 1:1 ratio to proceed fermentation, the ethanol concentration occurred at 50h,60h and 72h of fermentation was reached to 13.2%(V/V),13.8%(V/V) and 14.3%(V/V) respectively. The residual fermentable sugar was low to 0.44% at the end of fermentation (72h). The conversion efficiency was 88.6%(50h),94.3%(60h) and 98.6%(72h) of the theoretical ethanol yield.Based on lignocellulose was pretreated by UHEP combining dilute NaOH, the harmful inhibiting chemicals in hydrant was major lignin-like components, the tolerance of MF1001 to residual lignin in hydrant was investigated. The cell growth, ethanol fermentation and sugar utilization of MF1001 was not affected by 3600PPM lignin, was delayed 8h by 7200PPM lignin, and was completely inhibited by 14400PPM lignin. The ethanol concentration in broth was reached to 7.20%(V/V), and the fermentation efficiency was reached to 91.84% of theoretical conversion when MF1001 was used to ferment the cellulose hydrant for 40h. With the mix of the cellulose hydrant and molasses as substrate, the ethanol concentration at 48h fermentation was reached to 14.77%(V/V),13.44% higher than that with pure molasses as substrate. The results indicate that MF1001 is a superior strain suitable for high gravity ethanol fermentation of cellulose hydrant. Since it has the advantage of high tolerance, high yield and high efficiency for ethanol fermentation, we argue MF1001 is much potential for high gravity ethanol fermentation of cellulose hydrant.3. Demonstrating production of ethanol fermentation using high-yield S. cerevisiae strainBased on the above results, the performance of MF1001 in ethanol industrial fermentation was investigated. The process was double feeding continue fermentation. The fermentation scale was 50000.00 ton ethanol (95%, V/V) per year. The results of 15days continue production showed that, the maximum average ethanol concentration in broth was 14.1%(V/V) for one shift production and 13.8%(V/V) for one day (3 shift) production, the fermentable sugar in broth was 0-0.7%(W/V) with the average of 0.2%(W/V), the fermentation efficiency calculated basing on the fermentable sugar in medium was average 97.72% and maximum 98.89% of theoretic conversion rate, the waste water effluent for 1 ton ethanol (95%,W/V) production was average 8.17 tons with the minimum was 7.57 tons. The fermentation level reflected from parameter of production was significant better than that of current ethanol production. The statistical analysis exhibited that the cell number, ethanol concentration and fermentable sugar concentration in the broth of No.1 tank (for strain culture) respectively has a "bell curve", a negative linear correlation and a linear correlation with the final ethanol fermentation concentration, the fermentable sugar concentration in the broth of No.2 tank(for mixing the strain culture and high gravity medium to proceed the fermentation) has a linear correlation with the final ethanol fermentation concentration. All correlation had statistical significance (P<0.001). With the cell number in No.1 tank maintained in 2.10～ 2.15×108/ml, the ethanol was reached to maximum. The result indicates MF1001 is suitable completely for industrial ethanol fermentation.4. The genome comparing analysis of serial S. cerevisiae strains with different ethanol fermentation featuresThe genome DNA sequence of three S. cerevisiae strains, MF1001, ME1313 and MC1415, which was screened from same high osmotic environment and had different ethanol fermentation feature, was sequenced and comparatively analyzed.Their ethanol fermentation features were investigated first. Comparing with the low-yield strains ME1313 and MC1415, the high-yield strain MF1001 exhibited a serial characters relevant to ethanol fermentation, including superior features of higher ethanol fermentation concentration, stronger respiration, higher tolerance to the high-osmosis of molasses, and longer time of cell alive in medium, and inferior features of lower capacity of utilizing residual sugar, metabolizing lesser carbon resource, having not ability to metabolize maltose, and lower cell growth rate et al.The genomic structure of MF1001, ME1313 and MC1415 was analyzed and compared. The number of "Single Nucleotide Polymorphisms" (SNP) and "Genomic Structure Variation" (SV) and their heterologous mutation proportion of MF1001 were much higher than that of ME1313 and MC1415.59467 SNP and 811SV was identified in the genome sequence of MF1001, accounting for 0.49% and 0.0067% of total genomic DNA sequence respectively. Of which, the 45.05% of SNP and 71.89% of SV was belong to heterologous or insertion, the homologous SNP and deleted SV were 54.96% and 28.11% respectively. However, only 39674 SNP and 556 SV in the genome sequence of ME1313, and 42338 SNP and 507 SV in the genome sequence of MC1313 were identified. Comparing with MF1001, the SNP and SV of ME1313 were less 32.7% and 31.44%, and the SNP and SV of MC1415 were less 28.6% and 37.48% respectively. Unlike the high yield strain MF1001, the SNP of both low yield strains ME1313 and MC1415 was only 1.57% and 1.50% respectively belong to heterologous. Their SV were also only 63.49%(ME1313) and 50.3%(MC1415) belong to insertion SV, much less than that of MF1001(71.89%).The number of "Inserted Deletion" (InDel) in the genome sequence of three strain was 2225 (MF1001),2429 (ME1313 and 2299 (MC1415) respectively. The difference was not so significance. However, the heterologous InDel in genome sequence of high yield strain was significant more than that in the genome sequence of both high yield strains. The heterologous InDel was reaching to 23.06% for high yield strain MF1001 and only 5.31% and 4.39% for both low yield strains ME1313 and MC1415 respectively.The mutated homogenous genes of MF1001, ME1313 and MC1415 were further assayed. The number of mutated homogenous genes in the genome of MF1001 was much more than that in the genome of ME1313 and MC1414. Of the mutated homogenous genes, the ratio of sense mutated gene in high yield strain MF1001 was also a little more than that in both low yield strains ME1313 and MCI415. When comparing the genome sequences of MF1000l and ME1313,4234 mutated homogenous genes was identified, involving 3155 sense mutated genes and 1079 non-sense mutated genes, the ratio of sense mutation to non-sense mutation was 74.52%:25.48%. And when comparing the genome sequences of MF1001 with MC1415, 4223 mutated homogenous genes were identified, of which 3253 gens were sense mutation and 970 genes were non-sense mutation, the ratio of sense mutation to non-sense mutation was 77.03% :22.97%. However, when comparing the genome sequence of ME1313 and MC1415, only 3661 mutated homogenous genes were indentified, including 2617 sense mutated genes and 1044 non-sense mutated genes, the ratio of sense mutation to non-sense mutation was 71.48%:39.89%.By using BWA on line software, the genes in genome were divided into three gene groups according to their function, i.e., "Cellular Component", "Molecular Function" and "Biological Process". The enrichment of mutated homogenous genes in genome of three strains was assayed. On gene group level, the mutated homogenous genes were majorly enriched in both "Molecular Function" group and "Biological process" group. In both groups the gene mutation ratio in all mutation rich area was significant higher than the average mutation ratio of total genome. In "Cellular Component" the enrichment of mutated homogenous genes was relative lower. During evolution the enrichment of mutated homogenous genes in the genome of three strains exhibited some extent discipline. The gene groups relevant to "Cell", "Cell part", "Envelope", "Transcription regulator activity", "Molecular transducer activity", "Multicellular process", "Growth", "Pigmentation", and "Response to stimulus", et al., were the major rich areas of mutated homogenous genes. In the gene groups relevant to "Enzyme regulator activity", "Transporter activity", "Localization", "Establishment of localization", "Cellular component organization", Developmental process", "Cellular process" and "Metabolic process", the enrichment of mutated homogenous genes was also very high. However, in the gene groups relevant to "Structure molecule activity", "Electron carrier activity", "Antioxidant activity", "Translation regulator activity", "Metallochaperone activity", "Reproduction", "Anatomic structure formation", "Death),and "Cellular component biogenesis", the mutation ratio of homogenous genes was very low.Since MF1001, ME1313 and MC1415 exhibited the significant difference in the ethanol fermentation, the genes in their genome were blasted (Nucleitide—Nucleitide BLAST) using BLAST 2.0 server of NCBI, with the genome sequence of S288c strain published in GeneBank as reference. The 56 genes relevant to regulation of ethanol fermentation were identified. Of which 43 were the genes relevant to cell cycle, other was relevant to sugar metabolism. The replacement of genes between MF1001 and MC1415 has preliminarily verified the identification.Basing on the above results we argue that, the genes mutation of high-yield strain MF1001 was majorly caused by the insertion of heterogenous genes (or DNA fragments), the influence of InDel on the genome function was less significance than that of SNP and SV. The SNP caused by heterogenous genes insertion and the SV caused by insertion might be the major causes for the change of ethanol fermentation feature of MF1001. During the evolution the genes in the gene groups relevant to the function of basic metabolism, cell structure, were also happening significant mutation, implying that the ethanol fermentation of S. cerevisiae is not only regulated by the genes in the metabolism route, but also regulated by the genes relevant to cell basic metabolism and cell structure.Summary, a novel ultra-high pressure explosion (UHPE) technology for lignocellulose treatment was innovated around world. By combining the UHPE and dilute NaOH heating treatment a novel lignocellulose pretreatment technology with significant potential of to be used in industry was established. An ethanol fermentation high-yield strain MF 1001 that has the advantage of high-yield, high-efficiency and high tolerance to stresses and suitable to high-gravity ethanol fermentation of cellusoe hydrant was screened. The ethanol fermentation process of MF 1001 was optimized and the feasibility of MF 1001 used in industrial ethanol fermentation was verified. Finally, the genome of three S. cerevisiae strains, MF1001, ME1313 and MC1415, which were screened from same osmotic environment and were with different features in ethanol fermentation, was compared and analyzed. The 56 genes relevant to regulation of ethanol fermentation of S. cerevisae were indetified. The research in this paper is very significance since the result will provide a novel and feasible technology for lignocellulose treatment and provide a high-yield S. cerevisiae strain MF1001 for realizing the industrial high gravity ethanol fermentation from cellulose hydrant, and also provide the support for the gene modification and reconstruction of ethanol fermentation high-yield S. cerevisiae strain in theory and technology.