Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
  • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention relates to simultaneous saccharification and co-fermentation of cellulosic biomass substrates hydrolysable to produce glucose and xylose using a genetically 0 engineered Saccharomyces cerevisiae
• Bioethanol is a fuel that not only reduces negative environmental impacts from the transportation sector including reductions in greenhouse gas emissions but it can also be 5 manufactured from a wide range of available renewable feedstocks using established industrial processes.
• the aim of the present invention is thus to improve the yield of expression of ethanol when fermenting a biomass comprising glucose and xylose. According to the present invention this will be possible by using a simultaneous saccharification fermentation process.
• the present invention thus relates to a method for the manufacture of ethanol by fermenting a xylose and glucose containing biomass using a strain of Saccharomyces cerevisiae, wherein the method encompasses
• corn stover is used as an example of raw material.
• Corn stover is an abundant agricultural by-product with a low commercial value, thus it is a good raw material for bio-ethanot production.
• corn stover consists of mainly two types of sugars: glucose and xylose.
• Both these sugars can be hydrolysed at high yield using steam pretreatment and subsequent enzymatic hydrolysis and the glucose can then, using Saccharomyces cerevisiae (a yeast strain used widely within the industry), be fermented to ethanol at high yield, preferably in a combined hydrolysis and fermentation (SSF- simultaneous saccharification and fermentation),
• Saccharomyces cerevisiae a yeast strain used widely within the industry
• SSF- simultaneous saccharification and fermentation a combined hydrolysis and fermentation
• the pretreatment indicated above is not necessary per se, but a common enzymatic hydrolysis may be used. It is however, time-consuming if the biomass raw material is a fresh wooden material. Even a chemical hydrolysis is possible as such.
• Saccharomyces cerevisiae has been used for a long time in ethanol producing industry. It has also been shown to be a very good and tolerant yeast for fermentation of lignocellulosic hydrolysate to ethanol. However, S. cerevisiae does not, in its natural occurring state, ferment xylose. Thus, a lot of work has been put into genetically modifying it and other yeast strains to ferment xylose.
• S. cerevisiae strain generated by random mutagenesis at the Department of Applied Microbiology, Lund University in Lund, Sweden on industrial process applicable condition.
• TMB3400 was co-fermenting glucose and xylose during simultaneous saccharification and fermentation (SSF) of steam pretreated corn stover with lower by-product formation than during fermentation of xylose in well-defined solutions. Furthermore, this work showed that TM33400 is very tolerant to harsh process conditions and that the process configuration 3SF is to be preferred when it comes to co-fermentation of glucose and xylose due to the increased fermentation rate of xylose by TMB3400 in the presence of the low amount of glucose constantly liberated in the hydrolysis.
• the upper limit of glucose in the hydrolysate should not exceed 3 g/l, preferably not 2 g/l, more preferably not 1 g/l, still more preferably 0,6 g/l.
• TMB3400 was used in fed-batch SSF at final concentration of water insoluble substances (WIS) of 12 % resulting in an ethanol concentration in the fermented slurry of 17.5 g/l. This is the highest concentration in the slurry after an SSF ever published.
• WIS water insoluble substances
• the material Prior to pretreatment the material was milled and sieved and the fraction between 2 and 10 mm was recovered and used. Since the raw material used was very dry (95 % DM, owing to the dry storage) it had to be remoistened. This was done by presteaming with saturated steam at 10000 for 30 mm, after which the material was immersed in cold water and then allowed to drain off. The dried and re-wetted material may differ slightly from fresh, moist material since some collapsed pores may not regain their original shape, and the pore size has an impact on the subsequent enzymatic hydrolysis.
• the material was pressed and the water pressed off was measured to assure that the drj matter content landed in the right region whereupon the material was thoroughly mixed to assure an even moisture distribution. To double-check the dry matter content, it was also 'measured by drying in 10500 till constant weight.
• the rewetted and pressed com stover was impregnated with SO 2 (used as a catalyst to enhance reaction speed) in plastic bags and the uptake was measured by weighing the material before and after impregnation. Adding 3% SO 2 (w/w, based on the water content of the corn stover) resulted in an actual uptake of around 2% SO 2 .
• the impregnated raw material was steam pretreated in a 10-1 reactor and was then collected for subsequent analysis and SSF-tests.
• composition of the pretreated solid fraction was determined in the same way as was the composition of the raw material., Analyses was made on the liquid fraction after pretreatment with respect to monomeric sugars, oligomeric sugars and inhibitors such as acetic acid, furfural and 5 ⁇ hydroxymethyl-2-furfural (HMF). Simultaneous saccharification and fermentation (SSF)
• the SSF-tests were performed in 2 I fermentors (Labfors® Infers AG, Switzerland) with a working weight of 1.5 kg using the whole slurry from the pretreatment.
• the enzyme mixture used was a commercial cellulase mixture, Cefluclast I 51(65 FPU/g and 17 ⁇ -glucosidase IU/g ⁇ supplemented with the ⁇ -glucosidase preparation Novozyme 188 (376 ⁇ -glucosidase IU/g), both kindly donated by Novozymes A/S (Bagsvaerd, Denmark).
• the enzyme activity used in all experiments was 15 FPU and 25 IU/g water insoluble solids.
• the yeasts used was compressed baker's yeast, Saccharomyses cerevisiae from Jastbolaget AB, Rotebro (hexose fermenting) Sweden, USM21 (glucose fermenting) and TMB3400, a glucose and xylose fermenting yeast generated by random mutagenesis. All yeast was grown on the substrate used in the SSF.
• the SSF-tests were performed under semi-sterile conditions. After the addition of the material to the fermentor, water was added to adjust the dry matter concentration, the pH was adjusted to 5.0 with 25% NH 3 and the fermentor was sterilised. The equipment was left to cool down over night. Nutrients were added so that the concentrations in the fermentor were 0.5 g/l (NH 4 ) 2 HP0 4 0.025 g/l MgSO 4 x7H 2 O and 1.0 g/l yeast extract. The experiments were run at 35°C for 96 hours with the pH maintained at 5.0 by 5% NH 3 . Samples were withdrawn after 0, 2, 4, 6, 8, 24, 28, 32, 48, 72 and 96 hours, and analyzed for ethanol, sugars, glycerol, acetic acid, lactic acid and sugar degradation products.
• a small amount of pure Baker'.s yeast culture (Jastbolaget, Rotebro, Sweden) from an agar plate was added to a 300 ml Erlenmeyer flask, which contained 100 ml of sterile medium with a pH of 55.
• the medium composition was as follows; Glucose: 16.6 g/l, (NhU) 2 SO 4 ; 7.5 g/l, KH 2 PO 4 : 3.5 g/l, MgSO 4 : 075 g/l, Trace Metal Solution: 10 ml/I and Vitamin Solution: 1 ml/I.
• the culture was incubated at 30°C for 24 h.
• the Erlenmeyer flask was sealed with a cotton plug.
• the stirrer speed was kept at 750 rpm and the aeration was maintained at 1 .4 1/mm. Adding 45 ml of inoculum culture started the cultivation.
• the dissolved oxygen concentration (DOT) was measured with a DOT-electrode, The DOT never decreased below 20 % during the batch and fed-batch phases.
• the liquid was enriched with 64 g glucose/1 and pH-adjusted to 4.7 with NaOH.
• 1.0 I of glucose enriched pretreatment liquid was added during 16 h.
• the feed rate was initially set to 0.040 l/h and was increased linearly to 0.10 l/h after 16 h.
• the pH was maintained at 5.0 by automatic addition of 3 M NaOH.
• the stirrer speed was kept at 700 rpm and the aeration was maintained at 1.5 I/mm.
• the cultivation liquid containing the yeast was transferred from the fermentor into a sterile glass flask.
• the cultivation liquid was centrifuged (1,000 g) in 700 ml containers using a HERMLE Z 513 K (HERMLE Labortechnik, Wehingen, Germany). The supernatant was discarded and the pellets were transferred to a sterile glass flask.
• Sterile 0.9% NaCI-solution was added in order to obtain a cell suspension with a cell mass concentration of about 75 dw /I.
• the time lapse between the end of the fed-batch phase and the addition of the harvested cells to the 3SF-fermentations was less than 2 h.
• Glucose, arabinose, galactose and xylose were separated using an Aminex HPX-87-Pb column (Bio- Red, Hercules, USA) at 85°C and a flow rate of 0.5 ml/mm with water as eluent.
• Glucose, arabinose, lactic acid, glycerol, acetic acid, ethanol, HMF and furfural were separated on an Aminex HFX-87-H column at 65 0 C using 5 mmol/l H 2 SO 4 as eluent at a flow rate of 0.5 ml/mm.
• the samples from the liquid fraction after pretreatment were neutralised using CaCO 3 and Ba(OH) 2 and diluted 3 times.
• Ba(OH) 2 was used to precipitate sulphur CaCO 3 was used for the final pH-adjustment. All samples were filtered through a 0.20-pm filter before analysis.
• the amount of acid-soluble lignin was determined using an absorption spectrophotometer at a wavelength of 205 nm with a 4 % H 2 SO 4 solution as a reference.
• the sugar content in the raw mate al used is presented in Table I
• the raw material used in the two studies was delivered from the same place in Italy but at different times thus the raw material content in the two batches differ slightly, as can be expected, but the contents are still in a normal range for corn stover.
• Figure 1 denoted SSF 5 % with cultivated BY shows the result of fermenting 5 % SSF with bakers yeast with regard to outcome of ethanol, glucose, xylose, galactose, arabinose, xylitol, lactic acid, glycerol, acetic acid, HMF and furfural, respectively.
• Figure 2 denoted SSF 5 % with cultivated USM21 shows the result of fermenting 5 % SSF with USM21 with regard to outcome of ethanol, glucose, xylose, galactose, arabinose, xylitol, lactic acid, glycerol, acetic acid, HMF and furfural, respectively.
• Figure 3 denoted SSF 5 % with cultivated TMB3400 shows the result of fermenting 5 % SSF with TMB3400 with regard to outcome of ethanol, glucose, xylose, galactose, arabinose, xylitol, lactic acid, glycerol, acetic acid, HMF and furfural, respectively.
• Figure 4 denoted SSF 5 % with cultivated BY (2) shows the result of fermenting 5 % SSF with bakers yeast with regard to outcome of ethanol, glucose, xylose, galactose, arabinose, xylitol, lactic acid, glycerol, acetic acid, HMF and furfural, respectively.
• Figure 5 denoted SSF 5 % with cultivated USM21 (2) shows the result of fermenting 5 % SSF with USM21 with regard to outcome of ethanol, glucose, xylose, galactose, arabinose, xylitol, lactic acid, glycerol, acetic acid, HMF and furfural, respectively.
• Figure 6 denoted SSF 5 % with cultivated TMB3400 shows the result of fermenting 5 % SSF with TMB3400 with regard to outcome of ethanol, glucose, xylose, galactose, arabinose, xylitol, lactic acid, glycerol, acetic acid, HMF and furfural, respectively

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• 2006
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