Production of bioethanol, part 1

Implemented, the technology would constitute a great environmental impact in both industrial and developing countries. The Danish Bioethanol Concept is a recently developed lignocellulosic conversion concept that produces ethanol and biogas from biomass wastes (wheat straw) and manure. The concept has been developed in collaboration between the Technical University of Denmark and RISOS National Laboratories, Denmark, and combines ethanol and biogas production in an integrated process, obtaining almost complete biomass utilization. Wheat straw, together with the fiber fraction from manure, are opened using wet oxidation, and the liberated sugars are utilized for ethanol production. The non-fibrous fraction of the manure and the residual compounds from the ethanol fermentation are subsequently converted to methane The pre-treatment and characterisation of products were carried out at Risø National Laboratory. Previous investigations have shown that the pre-treatment of wheat straw is effectively carried out by wet oxidation at alkaline conditions e.g. by addition of sodium carbonate. In order to reduce the cost price of pre-treatment by wet oxidation, the replacement of oxygen with compressed air was examined. Additionally, pre-soaking prior to wet oxidation to reduce reaction time during wet oxidation, the use of H_20_2 was examined during presoaking for the replacement of oxygen by air. It was shown that air could not replace oxygen at the same pressure. However, hydrogen peroxide pre-soaking improved the enzymatic convertibility of cellulose in pretreatments of wheat straw with air at 24 bar and addition of 2 g/L Na_2C0_3. The addition of carbonate during pre-treatment of wheat straw was possible to reduce from 6.5 g/l to 2-4 g/L. The best pre-treatment condition for wheat straw was 12 minutes reaction time at 195 deg. C, with addition of 4 g/L Na_2C0_3, and 12 bar oxygen or pre-soaking in 1% H_20_2 for 15 minutes at 50 deg. C combined with 24 bar air with addition of 2 g/L Na_2C0_3. The pretreatment of lignocellulose generates inhibitors that limit the enzymatic conversion of the polymeric sugars. Experiments were done to examine the impact on the enzymatic hydrolysis working with wet oxidized wheat straw. It was shown that at the low enzyme loadings required for feasible ethanol from lignocellulosic biomasses enzymatic feedback inhibition occurred. The results represent an important point for the future research activities and upcoming experiments can be planned to take into account the observed nonirreversible enzymatic inhibition. A screening test of thermophilic (70 deg. C) anaerobic microorganism from DTU strain collection was conducted to identify potential candidates to produce thermostable enzymes, able of hydrolyzing wet oxidized wheat straw. The screening program of 48 pure cultures, reviled thermophilic anaerobic strains wich were able to hydrolysate the lignocellulosic hydrolysate. However, when these strains were tested for their performances to undiluted WO hydrolysate, three strains A5, A10 and X7B were found to have the best performance. Three strains, A5, Al0 and X7B were able to grow in WO fibers media without any available monosaccharides and to have ethanol, acetate and lactate production, whereas xylose and glucose accumulation was not observed. Furthermore it was demonstrated that those three organisms were able to produce xylanolytic enzymes at simultaneous ethanol production. Two basal tools are necessary to do genetic improvements on a given strain: Tools for chromosomal integration or deletion, and tools for over expression of genes. The aim of screening for plasmids is to broaden the variety of tools for extremely thermophilic microorganisms - a necessity for doing genetic improvements. It was found that Anaerocellum thermophilum contains to plasmids of 3653 and 8294 base pairs respectively. These two plasmids were fully sequenced and analyzed. The two plasmids, pBAS2 and pBAL, contain 10 and 17 potential genes respectively, encoding peptides of more than 70 amino acids. Since both plasmids are relatively small, they have the potential to be used as part of shuttle vectors. The smaller of the two plasmids was successfully inserted into an E. coli low copy number vector as the first step to make a true E. coli - thermophilic bacterium shuttle vector. To investigate the possibility of ethanol production at extreme loading rates and temperature (70 deg. C), it was attempted to immobilize the thermophilic strain Thermoanaerobacter HY10 onto sterile mesophilic granules in UASB reactors. It was shown that in less than 10 hydraulic retention times Thermoanaerobacter HY10 was immobilized in lab-scale UASB reactors using the organisms' own natural microbial attachment processes. Product concentration and the xylose conversion were found to be unaffected by gradually decreased hydraulic retention time (HRT) down to 4 hr. The highest substrate conversion was 99.4% corresponding to a. product/substrate yield of 0.33 g-ethanol/g-xylose and it was achieved at 4 hr HRT and 63 g-COD/(L*d) OLR. The maximum OLR applied to the reactors was 243 g-COD/(L*d) at a HRT of only 1 hr. After an initial drop in performance the reactors recovered and stabilized to a xylose conversion of 93.8% and a yield of 0.27 g-ethanol/g-xylose. In order to substitute the addition of nutrients in the different fermentation steps different approaches were pursued. The aim was to substitute costly nutrients by utilizing the nutrients that are enclosed in manure, and to use the liquid fraction of manure as process water in the final concept set-up. From the experiments it was shown that ethanol production from the yeast - Saccharomyces cerevisiae - was independent of nutrient supplements such as yeast extract and corn steep liquor. It became also evident that the Saccharomyces cerevisiae was able to ferment glucose to ethanol in a manure media in which the redistilled water normally used in synthetic media was replaced by the liquid fraction of manure. In addition, it was shown that the yeast additions could be substituted by compounds in the manure media. In experiments using Thermoanaerobacter mathranii it was showed that the bacteria fermented xylose optimally at a nutrients supplement of 2 g-YE/L and l g-CSL/L in wheat straw hydrolysate. Ethanol production of bacteria was inhibited significantly at concentrations above 50% manure. It is expected that the bacteria can be adapted to the higher concentrations of manure