纖維水解菌株與酒精生產菌株之篩選研究 謝明綸、吳建一

纖維水解菌株與酒精生產菌株之篩選研究 謝明綸、吳建一

E-mail: 322076@mail.dyu.edu.tw

摘 要

隨著世界石油儲量的快速消耗，近年來，生質乙醇已成為最重要的液態替代性燃料資源，並投入了大量的研究在酒精發酵

。可再生纖維素資源能生產乙醇，可以改善能源供應，減少大氣中二氧化碳的累積和空氣污染。因而，本研究主要為利用 微生物轉化纖維廢棄物為酒精，其結果分為以下兩個部分： 第一部分：為了尋找具有高 CMCase 來生產還原糖，我們自 食品工廠與紙廠活性污泥及昆蟲腸內菌中篩選分離出3 株細菌並根據 16S rDNA基因序列鑑定。這 3 株具有高纖維分解能 力之菌株經鑑定後分別命名為 Bacillus subtilis CELL、Bacillus sp. 及 Arthrobacter woluwensis Wu1。另外，利用羧甲基纖維 素(CMC)做為碳源，研究初始pH、溫度及氮源對羧甲基纖維素分解?(CMCase)分解羧甲基纖維素之影響。來自 Arthrobacter woluwensis Wu1、Bacillus subtilis CELL、Bacillus sp. 之羧甲基纖維素分解?(CMCase)最高產量分別於37℃、初始pH 5.0、6.0

、7.0，CMC 濃度皆為15 g/L，以及1、5、5 g/L之yeasy extract作為有機氮源。 第二部分：研究酵母菌株 Candida tropicalis Wu1生產酒精之發酵能力。在批次培養中，以葡萄糖為碳源，探討初始攪拌速率及氮源對酒精生產之影響。自Candida tropicalis Wu1 之酒精最大產量於30℃、靜置培養，葡萄糖濃度為 20 g/L，以及 2.5 g/L 之 (NH4)2SO4 作為氮源。另外，探 討固定化 Candida tropicalis Wu1 細胞顆粒生產酒精。實驗結果顯示，在 30℃ 以及 50 rpm 培養條件下，以及含有 50 g/L葡 萄糖培養基中，固定化 Candida tropicalis Wu1 有最大酒精生產力 0.33 g/L/h。

關鍵詞 : 纖維素、酒精生產、還原糖、固定化

目錄

目錄 封面內頁 簽名頁 授權書iii 中文摘要iv 英文摘要vi 誌謝viii 目錄ix 圖目錄xiv 表目錄xxiv 1.前言1 1.1 緣起1 1.2 研究目的 及架構2 2.文獻回顧4 2.1微生物能源轉換之優缺點4 2.2國內之纖維素廢棄物背景說明5 2.3木質纖維素介紹7 2.3.1纖維素8 2.3.2半纖維素9 2.3.3木質素10 2.4纖維素水解酵素之種類及作用機制12 2.4.1內切型纖維素分解酵素13 2.4.2外切型纖維素分 解酵素14 2.4.3β -葡萄糖?酵素14 2.5纖維分解菌株15 2.5.1纖維分解菌株之類型15 2.5.2纖維分解過程中的抑制影響20 2.5.3 環境因子對纖維分解菌株分解纖維之影響21 2.6生質酒精之重要性24 2.7生質酒精之開發生質酒精之生產25 2.7.1國內生質酒 精之研發概況25 2.7.2國外生質酒精之研發概況27 2.8生質酒精之生產31 2.8.1酒精生產之微生物31 2.8.2固定化酵母菌發酵生 產酒精之相關研究36 2.8.3酒精的抑制影響38 2.9環境因子對酒精生產之影響40 3.材料與方法49 3.1實驗材料49 3.1.1實驗試 藥49 3.1.2實驗設備50 3.2菌株來源與菌種篩選及鑑定51 3.2.1菌株來源51 3.2.2菌株之分離與純化51 3.2.3優勢菌株之16S rDNA鑑定53 3.2.4建立分離菌株之親緣樹狀圖54 3.3固定化微生物顆粒之製備54 3.3.1大量培養菌體並準備固定化54 3.3.2菌 體量之量測55 3.3.3固定化分離菌株之製備及基本性質測定56 3.3.4固定化分離菌株之重複批次試驗57 3.4探討分離菌株之最 適生長條件57 3.4.1不同氮源種類對菌株生長及生產之影響57 3.4.2不同碳、氮源濃度對菌株生長及生產之影響58 3.4.3環境 因子對菌株生長及生產之影響58 3.5分離之纖維分解菌株作用於不同基質下之纖維分解能力59 3.5.1分解不同纖維素來源之 基質59 3.5.2從不同基質測定纖維水解酵素之種類59 3.6分析方法60 3.6.1還原醣定性與定量60 3.6.2Glucose 與 cellobiose 之分 析62 3.6.3剛果紅測試62 3.6.4纖維素分解酵素活性測試63 3.6.4.1內切型纖維素分解酵素(CMCase)活性分析63 3.6.4.2外切型 纖維素分解酵素活性分析64 3.6.4.3β-葡萄糖?酵素(β-glucosidase)活性分析64 3.6.5酒精之檢測64 4.結果與討論65 4.1纖維分 解65 4.1.1纖維分解菌株之篩選及鑑定65 4.1.2纖維分離菌株之最適生長條件及Arrhenius、動力學解析75 4.1.2.1最適的氮源 種類75 4.1.2.2最適的碳源濃度81 4.1.2.3最適的氮源濃度90 4.1.2.4最適的pH值99 4.1.2.5最適的溫度及Arrhenius解析108 4.1.2.6最適的震盪速率121 4.2纖維分離菌株作用於不同基質下之纖維分解能力130 4.2.1菌株分解不同纖維素來源基質之比 較130 4.2.2分析纖維水解酵素之種類133 4.3酒精生產136 4.3.1酒精生產菌株之篩選136 4.3.2固定化及懸浮酒精生產菌株之生 產條件試驗139 4.3.2.1震盪速率對酒精生產菌株生產酒精之影響139 4.3.2.2氮源種類對酒精生產菌株生產酒精之影響144 4.3.2.3氮源濃度對酒精生產菌株生產酒精之影響149 4.3.3不同粒徑之固定化酒精生產菌株Candida tropicalis Wu1 顆粒對生 產酒精之 影響154 4.3.4固定化顆粒之重複批次試驗157 4.3.5碳源濃度對酒精生產菌株生產酒精之影響161 5.結論166 參考文 獻168 圖目錄 Figure 1-1 Schematic of this study procedure3 Figure 2-1 Composition of lignocellulosic materials and their potential hydrolysis products7 Figure 2-2 Partial structure of cellulose9 Figure 2-3 Structure of 5-Carbon sugars in hemicellulose10 Figure 2-4 General structure of hemicellulose10 Figure 2-5 Partial structure of lignin11 Figure 2-6 The synergy effect of cellulose hydrolysis12 Figure 3-1 Schematic diagram of immobilization method of Candida tropicalis Wu1 using PVA materials55 Figure 3-2 The standard calibration curve of glucose61 Figure 4-1 Congo red test (A)-(B): cellulose degradation activites of experimental cellulolytic microbes isolates on CMC agar plate67 Figure 4-2 Congo red test (C)-(E): cellulose degradation activites of experimental cellulolytic

microbes isolates on CMC agar plate68 Figure 4-3 Congo red test (F)-(H): cellulose degradation activites of experimental cellulolytic microbes isolates on CMC agar plate69 Figure 4-4 Congo red test (F)-(H): cellulose degradation activites of experimental cellulolytic microbes isolates on CMC agar plate70 Figure 4-5 Phylogenetic dendrogram showing a comparison of the aligned 16S rDNA sequences of known Bacillus species with that of strain Bacillus sp72 Figure 4-6 Phylogenetic dendrogram showing a comparison of the aligned 16S rDNA sequences of known Bacillus species with that of strain Bacillus subtilis CELL73 Figure 4-7 Phylogenetic dendrogram showing a comparison of the aligned 16S rDNA sequences of known Arthrobacter species with that of strain Arthrobacter woluwensis WU174 Figure 4-8 Effect of nitrogen source on cellulose degradation activities from CMC (10g/L)-agar plate by isolated Arthrobacter woluwensis Wu1, after incubation at 37℃, for 24 h77 Figure 4-9 Effect of nitrogen source on cellulose degradation activities from CMC (10g/L)-agar plate by isolated Bacillus subtilis CELL, after incubation at 37℃, for 24 h78 Figure 4-10 Effect of nitrogen source on cellulose degradation activities from CMC (10g/L)-agar plate by isolated Bacillus sp., after incubation at 37℃, for 24 h79 Figure 4-11 The time course of reducing sugar and biomass by isolated Arthrobacter woluwensis WU1 at different concentration CMC in batch cultures at 150 rpm and 37℃84 Figure 4-12 Effect of initial CMC concentration on specific growth rate and yield of reducing sugar by Arthrobacter woluwensis Wu1 at 37℃85 Figure 4-13 The time course of reducing sugar and biomass by isolated Bacillus subtilis CELL at different concentration CMC in batch cultures at 150 rpm and 37

℃86 Figure 4-14 Effect of initial CMC concentration on specific growth rate and yield of reducing sugar by Bacuillus subtilis CELL at 37℃87 Figure 4-15 The time course of reducing sugar and biomass by isolated Bacillus sp. at different concentration CMC in batch cultures at 150 rpm and 37℃88 Figure 4-16 Effect of initial CMC concentration on specific growth rate and yield of reducing sugar by Bacillus sp. at 37℃89 Figure 4-17 The time course of reducing sugar and biomass by isolated Arthrobacter woluwensis WU1 at different concentration of yeast extract in batch culture at 150 rpm and 37℃93 Figure 4-18 Effect of initial yeast extract concentration on specific growth rate and yield of reducing sugar by Arthrobacter woluwensis WU1 at 37℃94 Figure 4-19 The time course of reducing sugar and biomass by isolated Bacillus subtilis CELL at different concentration of yeast extract in batch culture at 150 rpm and 37℃95 Figure 4-20 Effect of initial yeast extract concentration on specific growth rate and yield of reducing sugar Bacuillus subtilis CELL at 37℃96 Figure 4-21 The time course of reducing sugar and biomass by solated Bacillus sp. at different concentration of yeast extract in batch culture at 150 rpm and 37℃97 Figure 4-22 Effect of initial yeast extract concentration on pecific growth rate and yield of reducing sugar Bacillus sp. at 37℃98 Figure 4-23 The time course of reducing sugar and biomass by solated Arthrobacter woluwensis WU1 at different pH in batch cultures at 37℃ and 150 rpm102 Figure 4-24 Effect of initial pH on specific growth rate and yield of reducing sugar by Arthrobacter woluwensis WU1 at 37℃103 Figure 4-25 The time course of reducing sugar and biomass by isolated Bacillus subtilis CELL at different stirrer speed in batch cultures at 37℃ and 150 rpm104 Figure 4-26 Effect of initial pH on specific growth rate and yield of reducing sugar by Bacuillus subtilis CELL at 37℃105 Figure 4-27 The time course of reducing sugar and biomass by isolated Bacillus sp. at different stirrer speed in batch cultures at 37℃ and 150 rpm106 Figure 4-28 Effect of initial pH on specific growth rate and yield of reducing sugar by Bacillus sp. at 37℃107 Figure 4-29 The time course of reducing sugar and biomass by isolated Arthrobacter woluwensis WU1 at varying temperature in batch culture at 150 rpm112 Figure 4-30 Effect of initial temperature on specific growth rate and yield of reducing sugar by Arthrobacter woluwensis WU1 at 150 rpm113 Figure 4-31 Arrhenius plots for specific growth rate and yield of reducing sugar by Arthrobacter woluwensis WU1114 Figure 4-32 The time course of reducing sugar and biomass by isolated Bacillus subtilis CELL at varying temperature in batch culture at 150 rpm115 Figure 4-33 Effect of initial temperature on specific growth rate and yield of reducing sugar by Bacuillus subtilis CELL at 150 rpm116 Figure 4-34 Arrhenius plots for specific growth rate and yield of reducing sugar by Bacuillus subtilis CELL117 Figure 4-35 The time course of reducing sugar and biomass by isolated Bacillus sp. at varying

temperature in batch culture at 150 rpm118 Figure 4-36 Effect of initial temperature on specific growth rate and yield of reducing sugar by Bacillus sp. at 150 rpm119 Figure 4-37 Arrhenius plots for specific growth rate and yield of reducing sugar by Bacillus sp120 Figure 4-38 The time course of reducing sugar and biomass by isolated Arthrobacter woluwensis WU1 at different stirred speed in batch cultures at 37℃124 Figure 4-39 Effect of stirred speed on specific growth rate and yield of reducing sugar by Arthrobacter woluwensis WU1 at 37℃125 Figure 4-40 The time course of reducing sugar and biomass by isolated Bacillus subtilis CELL at different stirred speed in batch cultures at 37℃126 Figure 4-41 Effect of stirred speed on specific growth rate and yield of reducing sugar by Bacillus subtilis CELL at 37℃127 Figure 4-42 The time course of reducing sugar and biomass by isolated Bacillus sp. at different stirred speed in batch cultures at 37℃128 Figure 4-43 Effect of stirred speed on specific growth rate and yield of reducing sugar by Bacillus sp. at 37℃129 Figure 4-44 Effect of carbon sources on cellulose degradation activities by isolated

Arthrobacter woluwensis WU1 and Bacillus sp. and Bacillus subtilis CELL, after incubation at 37℃132 Figure 4-45 Effects of added different substrates on reducing sugar product by Bacillus subtilis CELL at 40℃ for 24 h134 Figure 4-46 Effects of added different substrates on cellubiose degradation by Bacillus subtilis CELL at 37℃ for 6 day135 Figure 4-47 The time course of ethanol product by suspended yeast at batch and static cultures using glucose as carbon source137 Figure 4-48 Phylogenetic dendrogram showing a comparison of the aligned 16S rDNA sequences of known Candida species with that of strain Candida tropicalis Wu1138 Figure 4-49 The time course of biomass and ethanol product by suspended and immobilized Candida tropicalis Wu1 cell beads (25

g-bead/250 ml) at different stirred speed in batch cultures141 Figure 4-50 The results of average specific growth rate, cell mass yield, ethanol yield on glucose, and ethanol yield on cell mass at different stirred speed in suspended cell system142 Figure 4-51 The results of ethanol yield on glucose, and ethanol yield on immobilized-cell beads at different stirred speed in immobilized-cell beads

system143 Figure 4-52 The time course of biomass and ethanol product by suspended and immobilized Candida tropicalis Wu1 cell beads (25 g-bead/250 ml) at different nitrogen source in batch cultures146 Figure 4-53 The results of average specific growth rate, cell mass yield, ethanol yield on glucose, and ethanol yield on cell mass at different nitrogen source in suspended cell system147 Figure 4-54 The results of ethanol yield on glucose, and ethanol yield on immobilized-cell beads at different nitrogen source in immobilized-cell beads system148 Figure 4-55 The time course of biomass and ethanol product by suspended and immobilized Candida tropicalis Wu1 cell beads (25 g-bead/250 ml) at different concentration of (NH4)2SO4 in batch cultures151 Figure 4-56 The results of average specific growth rate, cell mass yield, ethanol yield on glucose, and ethanol yield on cell mass at different (NH4)2SO4 concentration in suspended cell system152 Figure 4-57 The results of ethanol yield on glucose, and ethanol yield on immobilized-cell beads at different (NH4)2SO4 concentration in immobilized-cell beads system153 Figure 4-58 The time course of biomass and ethanol product by immobilized Candida tropicalis Wu1 cell beads (25 g-bead/250 ml) at different beads size in batch cultures155 Figure 4-59 The results of ethanol yield on glucose, and ethanol yield on immobilized-cell beads at different beads size in immobilized-cell beads system156 Figure 4-60 The results of the production of ethanol by immobilized Candida tropicalis Wu1 cell beads during repeat-batch fermentation158 Figure 4-61 Microbial population development and distribution of PVA gel beads during continuous operation. (A) beads prior to start-up; (B) beads after 5 times of incubation. Dash 1 and 2 respectively indicate the whole beads and surface area159 Figure 4-62 Microbial population development and distribution of PVA gel beads during

continuous operation. (A) beads prior to start-up; (B) beads after 5 times of incubation. Dash 1 and 2 interior of beads160 Figure 4-63 The time course of biomass and ethanol product by suspended and immobilized Candida tropicalis Wu1 cell beads (25 g-bead/250 ml) at different concentration of glucose in batch cultures163 Figure 4-64 (a) Effect of the ethanol product of different glucose concentration in suspended cell system (b) Lineweaver-Burk plot of the different concentration of glucose164 Figure 4-65 (a) Effect of the ethanol product of different glucose concentration in immobilized-cell beads system.(b) Substrate inhibition of the different oncentration of glucose165 表目錄 Table 2-1 The contents of cellulose, hemicellulose, and lignin in common a gricultural residues and wastes5 Table 2-2纖維分解菌株之種類18 Table 2-3國內纖維分解及纖維分解菌株的相關研究19 Table 2-4國內生 質酒精之推廣政策27 Table 2-5國外生質酒精之推動情形30 Table 2-6 Yeast species which produce ethanol as the main

fementation product33 Table 2-7不同固定化方法之優缺點比較38 Table 3-1 Modified Mandels-Reese medium52 Table 3-2 Yeast Extract Peptone Dextrose (YPD) medium53 Table 3-3 PCR program53 Table 3-4 PCR primers used in this study54 Table 3-5 The composition of DNS reagent61 Table 4-1 Comparison with cellulose degrading ability of different cellulose degrading bacteria66 Table 4-2 Comparison with cellulose degrading ability of different cellulose degrading bacteria under various nitrogen source80 參考文獻

王三郎。1991。生物工學入門。第57-72頁。藝軒圖書出版社。台北市。 2 王俊豪。2005。德國再生能源與生物能源之發展。農政與農 情(175):78-83。 3 王鳳英。1991。利用固態發酵以玉米穗軸生產纖維素分解酵素。國立台灣大學農業化學研究所博士論文。台北。 4 台 灣區造紙工業同業公會。 http://60.244.127.66/big5/tpia/htm/index2.html。 5 台灣綜合研究院。2007。國際生質柴油推展之初步探討。

石油策略油研究中心。 6 左峻德，蘇美惠和朱浩。2006。我國發展生質酒精之推動策略探討。在中華民國經濟學會主辦，中華民國經濟 學會 95 年年會暨學術研討會，台北市。 7 左峻德。2007。我國發展生質能源產業之可行性，農業生技產業季刊，第九期:56-61。 8 吳滕 文。2003。生物產業技術概論。清大出版社。新竹。 9 宋賢良，溫其標和朱江。2001。纖維素?水解的研究進展 22:第67-84頁。鄭州工程 學院學報。 10 周建，羅學剛和蘇林。2006。纖維素?法水解的研究現狀及展望。化工科技 14:51-56。 11 孟祥傑。2006。1 公斤向日葵煉 半公斤油 ，Sina NEWS， http://news.sina.com/udn/101-102-101-101/2005-09-18/1509148440.html。 12 秦全貴。1997。纖維素?造紙工 業 3:第12-15頁。廣西:廣西輕工業出版社。 13 陳威廷。2004。纖維素水解菌之培養策略與纖維素水解酵素之鑑定。國立成功大學化學工 程學系碩士論文。台南。 14 氣候變化綱要公約資訊網。2006。溫室效應與氣候變化， http://sd.erl.itri.org.tw/fccc/ch/intro/intro_2.htm

。 15 偕慶璋，劉嘉哲和顏政瑋。2006。巴西、美國生質能源(biomass, bioenergy)與汽車酒精產業之發展考察報告。OPEN 政府出版資料 回應網， http://open.nat.gov.tw/OpenFront/report/show_file.jsp?sysId=C09502510&fileNo= 001，(2007/04/23)。 16 郭家倫，門立中，陳 威希和湯俊彥。2005。我國發展生質酒精之推動策略報告。 核能研究所。 17 陳國誠。1989。微生物酵素工程學。藝軒圖書出版社。台 北。 18 曾豐祥。2007。纖維素分解產氫之微生物社會結構與動態變化。國立成功大學生命科學研究所碩士論文。台南。 19 黃恆信和黃 文松。2005。生質酒精之製造與運用。核能研究所。 20 楊紹榮。農業廢棄物處理與再利用。台南區農業改良場，

http://www.tndais.gov.tw/Soil/b1.htm。 21 經濟部。2006。發展綠色能源－生質燃料執行計畫。在行政院第 3010 次院會。台北市。 22 農委會農糧署。2006b。96 年度建立能源作物產銷體系計畫，農糧署全球資訊網，

http://www.afa.gov.tw/Public/DownLoadTree/200612211425457055。 23 劉朱松。2006。農委會深耕生質能源作物。HiNet 新聞網，

http://times.hinet.net/news/20060227/finance/f9ec3acef544.htm。 24 鄭作林和蕭耀基。2006。台糖再生質酒精的發展策略。能源報 導:11-13。 25 鄭作林，張謙裕和蕭耀基。2006。台灣甘蔗酒精醱酵產業之回顧與展望。生質能源開發與利用，台北:台大生技系。 26 鄭

煥斌。2007。世界各國生物燃料開發你追我趕，科技日報， http://www.stdaily.com/big5/kebaishidian/2007-04/12/content 657058.htm。

27 簡宣裕，張明暉和劉禎祺。2007。木質纖維素產生能源方法之探討。行政院農業委員會農業試驗所。木質纖維素產生能源方法之探 討:103-114。 28 蘇宗振。2007。我國生質能源發展趨勢與農業政策-生物產業機電工程之機會與挑戰，台灣大學生物產業機電工程專題 演講。 29 蘇遠志，莊育梩和鄞雪麗。1989。利用醋酸菌之固定化細胞進行山梨糖生產之研究. 中國農業化學會誌 27:1-11。 30 Aiello, C., Ferrer, A. and Ledesma, A. 1996. Effect of alkaline treatments at various temperatures on cellulase and biomass production using submerged sugarcane bagasse fermentation with Trichoderma reesei QM 9414. Bioresource Technology. 57: 13-18. 31 Alexander, M. A. and Jeffries, T. W.

1990. Respiratory efficiency and metabolite partitioning as regulatory phenomena in yeasts. Enzyme and Microbial Technology. 12: 2-19. 32 Almeida, J., Modig, T., Petersson, A., Ha"hn-Ha"gerdal, B., Lide'n, G. and Gorwa-Grauslund, M. 2007. Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. Journal of Chemical Technology and Biotechnology. 82: 340-9. 33 An, D. S., Im, W. T., Yang, H. C., Kang, M. S., Kim, K. K., Jin, L., Kim, M. K. and Lee, S. T. 2005. Cellulomonas terrae sp. nov., a cellulolytic and xylanolytic bacterium isolated from soil. International Journal of Systematic and Evolutionary Microbiology. 55: 1705-1709. 34 Anon. 2006. Malta

’s annual report for 2005 submitted to fulfill requirements of Article 4 of Directive 2003/30/EC. Ministry for Resources and Infrastructure, Valletta, Malta. 35 Anuradha, R., Suresh, A. K. and Venkatesh, K. V. 1999. Simultaneous saccharification and fermentation of starch to lactic acid. Proc Biochemical. 35: 367-375. 36 Bakalidou, A., Kampfer, P., Berchtold, M., Kuhnigk, T., Wenzel, M. and Konig, H. 2002.

Cellulosimicrobium variabile sp. nov., a cellulolytic bacterium from the hindgut of the termite Mastotermes darwiniensis. International Journal of Systematic and Evolutionary Microbiology. 52: 1185-1192. 37 Banerjee, N., Bhatnagar, R. and Viswanathan, L. 1981. Inhibition of glycolysis by furfural in Saccharomyces cerevisiae. European Journal of Applied Microbiology and Biotechnology. 11: 224-228. 38 Barker, B. T. P. and Hillier, V. F. 1912. Cider sickness. The Journal of Agricultural Science. 5: 67-85. 39 Bartkowiak, A. and Hunkeler, D. 1999. Alginate-oligo-chitosan microcapsules: a mechanistic study relating membrane and capsule properties to reaction conditions. Chemistry of Materials. 11: 2486-2492. 40 Bartkowiak, A. and Hunkeler, D. 2001. Carrageenan–oligochitosan microcapsules: optimization of the formation process. Colloids and Surfaces.

B: Biointerfaces. 21: 285-298. 41 Beguin P. and Lemaire, M. 1996. The cellulosome: an exocellular, multiprotein complex specialized in cellulose degradation. CRC critical reviews in biochemistry and molecular biology. 31: 201-236. 42 Bhat, M. K., and Bhat, S. 1997. Cellulose degrading enzymes and their potential industrial applications. Biotechnology Advances. 15: 583-620. 43 Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3 : 286-290. 44 Bisaria, V. S., and Ghose, T. K., 1981. Biodegradation of cellulostic materials: substrates, microorganisms, enzymes and products. Enzyme and Microbial Technology. 3: 90-104. 45 Bisaria, V. S., and Mishra, S. 1989. Regulatory aspects of cellulose biosynthesis and secretion. Critical Reviews in Biotechnology. 9: 61-103. 46 Bobleter, O., 1994. Hydrothermal degradation of polymers derived from plants.

Progress in Polymer Science. 19: 797-841. 47 Bok, J. D., Yernool, D. A., and Eveleigh, D. E. 1998. Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana. Applied and Environmental Microbiology. 64: 4774-4781. 48 Boles, E. and Hollenberg, C. P. 1997. The molecular genetics of hexose transport in yeasts. FEMS Microbiology reviews. 21: 85-111. 49 Boopathy, R. 1998. Biological treatment of swine waste using anaerobic baffled reactors. Bioresource Technology. 64: 1-6. 50 Brown, S. W., Oliver, S. G., Harrison, D. E. F. and Righelato, R. C. 1981. Ethanol inhibition of yeast growth and fermentation: Differences in the magnitude and complexity of the effect. European Journal of Applied Microbiology and Biotechnology. 11: 151-155. 51 Camacho-Ruiz, L., Perez-Guerra, N. and Roses, RP.

2003. Factors affecting the growth of Saccharomyces cerevisiae in batch culture and in solid sate fermentation. Electron Journal of Agricultural and Food Chemistry. 2: 531-542. 52 Campbell, C. J. and Laherrere, J. H. 1998. The end of cheap oil. Scientific American. 3: 78-83. 53 Carpita, N. C.

and Gibeaut, D. M. 1993. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant journal. 3: 1-30. 54 Cartwright, C. P., Juroszek, J. R., Beavan, M. J., Ruby, F. M. S., de Morais, S. M. F. and Rose, A. H. 1986. Ethanol dissipates the proton-motive force across the plasma membrane of Saccharomyces cerevisiae. The Journal of General Microbiology. 132: 369-377. 55 Cartwright, C. P., Veazy, F. J. and Rose, A. H. 1987. Effect of ethanol on activity of the plasma membrane ATPase in, and accumulation of glycine by Saccharomyces cerevisiae. The Journal of General Microbiology. 133: 857-865. 56 Casey, G. P., Ingledew, W. M. 1986. Ethanol tolerance in yeasts. Critical Reviews in Microbiology. 13: 219-280. 57 Caylak, B. and Vardar, S. F. 1996.

Comparison of different production processes for bioethanol. Turk Journal of Chemistry. 22: 351-359. 58 CEC. 2002. Commission staff working paper: Inventory of public aid granted to different energy sources. Commission of the European Communities (CEC), Brussels, Belgium. 59 Chang, H. N., Seong, G. H., Yoo, I. K. and Park, J. K. 1996. Microencapsulation of recombinant Saccharomyces cerevisiae cells with invertase activity in liquid-core alginate capsules. Biotechnology and Bioengineering. 51: 157-162. 60 Cheung, S. W. and Anderson, B. C. 1997. Laboratory

investigation of ethanol production from municipal primary wastewater. Bioresource Technology. 59: 81-96. 61 Chung, I. S. and Lee, Y. Y. 1985.

Ethanol fermentation of crude acid hydrolyzate of cellulose using high-level yeast inocula. Biotechnology and Bioengineering. 27: 308-315. 62 Chyi, Y. T. andd Dague, R. R. 1994. Effects of particulate size in anaerobic acidogenesis using cellulose as a sole carbon source. Water Environmental Research. 66: 670-678. 63 Converti, A., Perego, P. and Dominguez, J. M. 1999. Microaerophilic metabolism of Pachysolen tannophilus at different pH values. Biotechnology Letters. 21: 719-723. 64 Cosgrove, D. J. 1998. Cell Walls: Structures, Biogenesis, and Expansion.

In: Plant Physiology. p. 409-443., Taiz L., Zeiger E., Eds., Sinauer Associates, Inc, Sunderland. 65 da Cruz, S. H., Batistote, M. and Ernandes, J.

R. 2003. Effect of sugar catabolite repression in correlation with the structural complexity of the nitrogen source on yeast growth and fermentation.

Journal of the Institute of Brewing. 109: 349-355 66 Dawes, I. W. and Sutherland, I. W. 1992. Microbial Physiology, Blackwell Scientific Publications, Oxford. 67 Demain, A. L., Newcomb, M. and Wu, J. H. 2005. Cellulase, clostridia, and ethanol. Microbiology and Molecular

Biology Reviews. 69: 124-154. 68 Desvaux, M. 2005. Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia. FEMS Microbiology Reviews. 29: 741-764. 69 Dickensheets, P. A., Chen, L. F. and Tsao, G. T. 1977. Characteristics of yeast invertase immobilized on porous cellulose beads. Biotechnology and Bioengineering. 19: 365-375. 70 Distel, D. L., Morrill, W., MacLaren-Toussaint, N., Franks, D. and Waterbury, J. 2002. Teredinibacter turnerae gen. nov., sp. nov., a dinitrogen-fixing, cellulolytic, endosymbiotic gamma-proteobacterium isolated from the gills of wood-boring molluscs (Bivalvia: Teredinidae). International Journal of Systematic and Evolutionary Microbiology. 52: 2261-2269.

71 DSIRE. 2006. Database of state incentives for renewables & efficiency. NC State University, Raleigh NC, USA. 72 du Preez, J. C. 1994. Process parameters and environmental factors affecting u-xylose fermentation by yeasts. Enzyme and Microbial Technology. 16: 944-956. 73 du Preez, J.

C., Bosch, M. and Prior, B. A. 1986a. Xylose fermentation by Candida shehatae and Pichia stipitis: Effects of pH, temperature and substrate concentration. Enzyme and Microbial Technology. 8: 360-364. 74 du Preez, J. C., Bosch, M. and Prior, B. A. 1986b. The fermentation of hexose and pentose sugars by Candida shehatae and Pichia stipitis. Applied Microbiology and Biotechnology. 23: 228-233. 75 du Preez, J. C., Bosch, M.

and Prior, B. A. 1987. Temperature profiles of growth and ethanol tolerance of the xylose-fermenting yeasts Candida shehatae and Pichia stipitis.

Applied Microbiology and Biotechnology. 25: 521-525. 76 du Preez, J. C., van Driessel, B. and Prior B. A. 1989. Ethanol tolerance of Pichia stipitis and Candida shehatae strains in fed-batch cultures at controlled low dissolved oxygen levels. Applied Microbiology and Biotechnology. 30: 53-58.

77 Dunlop, A. P. 1948. Furfural formation and behaviour. Industrial and Engineering Chemistry. 40: 204±209. 78 El Sheikh Idris, E. and Berry, D. 1980. Selection of thermotolerant yeast strains for biomass production from Sudanese molasses. Biotechnology Letters. 2: 61-6. 79 Enayati, N.

and Parulekar, S. J., 1995. Enzymatic saccharification of soybean hull based materials. Biotechnology Progress. 11: 708-711. 80 Ergun, M. and Mutlu, S. F. 2000. Application of a statistical technique to the production of ethanol from sugar beet molasses by Saccharomyces cerevisiae.

Bioresource Technology. 73: 251-255 81 Eriksson, T., Borjesson, J. and Tjerneld, F. 2002. Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme and Microbiology Technology. 31: 353-364. 82 Esteban, R., Willanueva, J. R. and Villa, T. G. 1982. β-D-Xylanases of Bacillus circulans WL-12. Can. Microbiology. 28: 733-739. 83 Fengel, D. and Wegener, G., 1984. Wood: Chemistry, Ultrastructure, Reactions. De Gruyter, Berlin. 84 Galazzo, J. and Bailey, J. 1990. Growing Saccharomyces cerevisiae in calciumalginate beads induces cell alterations which accelerate glucose conversion to ethanol. Biotechnology and Bioengineering. 36: 417-426. 85 Garrote, G., Dominguez, H. and Parajo, J. C. 1999.

Hydrothermal processing of lignocellulosic materials. Holz Roh Werkst. 57: 191-202. 86 Ghasem, N., Habibollah, Y., Ku, S. and Ku, I. 2004.

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae. Bioresource Technology. 92: 251-260 87 Gijzen, H. J., Lubberding, H. J., Verhagen, F. J. M., Zwart, K. B. and Vogels, G. D. 1987. Application of rumen microorganisms for an enhanced anaerobic degradation of solid organic waste materials. Biological Wastes. 22: 81-95. 88 Gikas, P. and Livingston, A. G. 1997. Specific ATP and specific oxygen uptake rate in immobilized cell aggregates: experimental results and theoretical analysis using a structured model of immobilized cell growth. Biotechnology and Bioengineering. 55: 660-673. 89 Gilkes, N., Henrissat, B., Kilburn, D., Miller, R. and Warren, R. 1991. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiological Reviews. 55: 303-315. 90 Goldemberg, J., Coelho, S. T., Nastari, P. M. and Lucon, O. 2004. “Ethanol learning curve—the Brazilian experience”, Biomass and Bioenergy. 26: 301-304. 91 Goldman, J. C., Carpenter, E. J. 1974. A kinetic approach to effect of temperature on algal growth. Oceanol Limnol. 19: 756-766. 92 Goldstein, I.

S. 1983. Wood and Agricultural Residues. p. 315-328. New York: Academic Press. 93 Gomes, D. J., Gomes, J. and Steiner, W. 1994. Production of highly thermostable xylanase by a wild strain of thermophilic fungus Thermoascus aurantiacus and partial characterization of the enzyme. Journal of Biotechnology. 37: 11-22. 94 Gong, C. S., McCracken, L. D. and Tsao, G. T. 1981. Direct fermentation of D-xylose to ethanol by a

xylose-fermenting yeast mutant, Candida sp. XF 217. Biotechnology Letters. 3: 245-250. 95 Grabber, J. H. 2005. How do lignin composition, structure, and cross-linking affect degradability? A review of cell wall model studies. Crop Science. 45: 820-831. 96 Green, K. D., Gill, I. S., Khan, J. A. and Vulfson, E. N. 1996. Microencapsulation of yeast cells and their use as a biocatalyst in organic solvents. Biotechnology and

Bioengineering. 49: 535-543. 97 Gruninger, H. and Fiechter, A. 1986. A novel, highly thermostable D-xylanase. Enzyme and Microbial Technology. 8: 309-314. 98 Hahn-Ha"gerdal, B., Jeppsson, H., Skoog, K. and Prior, B. A. 1994. Biochemistry and physiology of xylose fermentation by yeasts. Enzyme and Microbial Technology. 16: 933-943. 99 Hahn-Ha"gerdal, B., Linden, T., Senac, T. and Skoog, K. 1991.

Ethanolic fermentation of pentoses in lignocellulose hydrolysates. Applied Biochemistry and Biotechnology. 28/29: 131-144. 100 Hall, D.O. 1997.

Biomass Energy in Industrialized Countries: A View of the Further. Forest Ecology and Management. 91: 17-45. 101 Hilge-Rotmann, B. and Rehm, H. J. 1990. Relationship between fermentation capability and fatty acid composition of free and immobilized Saccharomyces cerevisiae.

Applied Microbiology and Biotechnology. 34: 502-508. 102 Hiraishi, H., Mochizuki, M. and Takagi, H. 2006. Enhancement of stress tolerance in Saccharomyces cerevisiae by overexpression of ubiquitin ligase Rsp5 and ubiquitin-conjugating enzymes. Bioscience Biotechnology and

Biochemistry. 70: 2762-5. 103 Hoppe, G. K. and Hansford, G. S. 1982. Ethanol inhibition of continuous anaerobic yeast growth. Biotechnology Letters. 4: 39-44. 104 Hossack, J. A. and Rose, A. H. 1976. Fragility of plasma membranes in Saccharomyces cerevisiae enriched with different sterols. Journal of Bacteriology. 127: 67-75. 105 Hossack, J. A., Belk, D. M. and Rose, A. H. 1977. Environmentally induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis. Archives of Microbiology. 114: 137-142. 106 Ingram, L.

O. 1986. Microbial tolerance to alcohols: Rple of the cell membrane. Tibtech. 40-44. 107 Ingram, L. O. 1990. Ethanol tolerance in bacteria.

Critical Reviews in Biotechnology. 9: 305–319. 108 International Energy Agency (IEA). 2003. Renewable information. IEA, Paris. Jackson, R. S.

2000. Wine Science. Academic Press, USA. 109 Jain, W. K., Diaz, I. T. and Barattif, J. 1985. Continuous production of ethanol from fructose immobilized growing cells of Zymomonas mobilis. Biotechnology and Bioengineering. 27: 613-620. 110 Jeffries, T. W. 1981. Conversion of xylose

to ethanol under aerobic conditions by Candida tropicalis. Biotechnology Letters. 3: 213-218. 111 Jeffries, T. W. 1983. Utilization of xylose by bacteria, yeasts, and fungi. Advance Biochemical Engineering Biotechnology. 27: 1-32. 112 Jeffries, T. W. and Alexander, M. A. 1990. Production of ethanol from xylose by Candida shehatae grown under continuous or fed-batch conditions. In “Biotechnology in Pulp and Paper Manufacture

” (T. K. Kirk and H. M. Chang, eds.). p. 311-321. Butterworth-Heinemann, Boston. 113 Jeffries, T. W., Fady, J. H. and Lightfoot, E. N. 1985.

Effect of glucose supplements on the fermentation of xylose by Pachysolen tannophilus. Biotechnology and Bioengineering. 27: 171-176. 114 Jimenez, J. and van Uden, N. 1985. Use of extracellular acidification for the rapid testing of ethanol tolerance in yeasts. Biotechnology and Bioengineering. 27: 1596-1598. 115 Jirku, V., Masak, J. and Cejkova, A. 2003. The potential of functional changes in attached biomass. Advance Environ Research. 7: 635-639. 116 Jo"bses, I. M. L. and Roels, J. A. 1986. The inhibition of the maximum specific growth and fermentation rate of Zymomonas mobilis by ethanol. Biotechnology and Bioengineering. 28: 554-563. 117 Jones, R. P. and Greenfield, P. F. 1987. Ethanol and fluidity of the yeast plasms membrane. Yeast. 3: 223-232. 118 Jones, R. P. 1989. Biological principles for the effects of ethanol. Enzyme and Microbial Technology. 11:130-153. 119 Juhasz, T., Szengyel, Z., Szijarto, N. and Reczey, K. 2004. The effect of pH on the cellulase production of Trichoderma reesei RUT C30. Applied Biochemistry and Biotechnology. 113-116: 201-11. 120 Kastner, J. R., Jones, W. J. and Roberts, R. S.

1999. Oxygen starvation induces cell death in Candida shehatae fermentations of D-xylose, but not D-glucose. Applied Microbiology and Biotechnology. 51: 780-785. 121 Kavinaugh, K. and Whittaker, P. A. 1994. Application of the Melle-Boinot process to the fermentation of xylose by Pachysolen tannophilus. Applied Microbiology and Biotechnology. 42: 28-31. 122 Kiran, S., Sikander, A. and Lkram-ul-Haq. 2003. Time course study for yeast invertase production by submerged fermentation. Journal of Biological. 3: 984-988 123 Kiss, T. 2006. Update on promotion of the use of biofuels. Memorandum 286/V/Adm./ 2006. Budapest, Hungary. 124 Ko"tter, P., Amore, R., Hollenberg, C. P. and Ciriacy, M.

1990. Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, Xyl2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant. Current Genetics. 18: 463-500. 125 Kruse, B. and Schugerl, K. 1996. Investigation of ethanol formation by Pachysolen tannophilus from xylose and glucose-xylose cosubstrates. Proceedings Biochemistry. 31: 389-408. 126 Kubicek, C. P. and Penttila", M. E. 1998.

Regulation of production of plant polysaccharide degrading enzymes by Trichoderma. In: Harman, GE, Kubicek, CP, editors. Trichoderma and Gliocladium, Enzymes, biological control and commercial applications, vol. 2: 49-67. Bristol: Taylor & Francis Ltd. 127 Laplace, J. M., Delgenes, J.

P., Moletta, R. and Navarro, J. M. 1991. Simultaneous alcoholic fermentation of D-xylose and D-glucose by four selected microbial strains: process considerations in relation with ethanol tolerance. Biotechnology Letters. 13: 445-450. 128 Larsson, S., Palmqvist, E., Hahn-Ha"gerdal, B., Tengborg, C., Stenberg, K., Zacchi, G. and Nilvebrant, N. O. 1998. The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme and Microbial Technology. 24: 151±159. 129 Lea~o, C. and van Uden, N. 1982. Effects of ethanol and other alkanols on the glucose transport system of Saccharomyces cerevisiae. Biotechnology and Bioengineering. 24. 2601-2604. 130 Lea~o, C. and van Uden, N. 1983.

Effects of ethanol and other alkanols on the ammonium transport system of Saccharomyces cerevisiae. Biotechnology and Bioengineering. 25:

2085-2090. 131 Lea~o, C. and van Uden, N. 1984a. Effects of ethanol and other alkanols on the general amino acid permease of Saccharomyces cerevisiae. Biotechnology and Bioengineering. 26: 403-405. 132 Lea~o, C. and van Uden, N. 1984b. Effects of ethanol and other alkanols on passive proton influx in the yeast Saccharomyces cerevisiae. Biochimica et Biophysica Acta. 774: 43-48. 133 Lea~o, C. and van Uden, N. 1985.

Effects of ethanol and other alkanols on the temperature relations of glucose transport and fermentation in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 22: 359-363. 134 Lee, R. L., Charles, E. W., and Tillman U. G. 1999. Biocommodity engineering. Biotechnology Progress. 15: 777-793. 135 Lee, S. 1996. Alternative Fuels, (Taylor & Francis, Washington, DC). 136 Leticia, P., Miguel, C., Humberto, G. and Jaime, AJ. 1997. Fermentation parameters influencing higher alcohol production in the tequila process. Biotechnology Letters. 19: 45-47. 137 Levan, S. L., Ross, R. J. and Winandy, J. E. 1990. Effects of fire retardant chemicals on bending properties of wood at elevated temperatures.

Research Paper FPL-RP-498. p. 24. Madison, WI: U.S. Department of agriculture, Forest service, Forest Products Laboratory. 138 Lichts, F. O.

2006. World Ethanol Biofuels Report. 5: 40. 139 Lichts, F. O. 2006. World Ethanol Biofuels Report. 5: 48. 140 Lim, F. and Sun, AM. 1980.

Microencapsulated islets as bioartificial endocrine pancreas. Science. 210: 908-910. 141 Liu, Z. 2006. Genomic adaptation of ethanologenic yeast to biomass conversion inhibitors. Applied Microbiology and Biotechnology. 73: 27-36. 142 Loureiro-Dias, M. C. and Peinado, J. M. 1982. Effects of ethanol and other alkanols on the maltose transport ystem of Saccharomyces cerevisiae. Biotechnology Letters. 4: 721-724. 143 Loureiro-Dias, M. C. and Santos, H. 1990. Effects of Ethanol on Saccharomyces cerevisiae as monitored by in vivo P-31 and C-13 nuclear magnetic resonance.

Archives of Microbiology. 153: 384-391. 144 Lucas, C. and van Uden, N. 1985. The temperature profiles of growth, thermal death and ethanol tolerance of the xylose-fermenting yeast Candida shehatae. Journal of Basic Microbiology. 8: 547-850. 145 Lynd, L. R., Weimer, P. J., Zyl, W. H.

V. and Pretorius, I. S. 2002. Microbial cellulose utilization: Fundamentals and biotechnology. Microbiology and Molecular Biology Review. 66:

506-577. 146 Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S. 2002. Microbial cellulose utilization: fundamentals and biotechnology.

Microbiology and Molecular Biology Reviews. 66: 506-577. 147 Maleszka, R. and Schneider, H. 1984. Involvement of oxygen and mitochondrial function in the metabolism of D-xylulose by Saccharomyces cerevisiae. Archives of Biochemistry and Biophysics. 228: 22-30. 148 Margaritis, A., Bajpai, P. K. and Wallace, J. K. 1981. High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis.

Biotechnology Letters. 3: 613-615. 149 McMillan, J. D. 1994. Conversion of hemicellulose hydrolysates to ethanol. American Chemical Society Symposium Series. 566: 411-437. 150 Meinander, N. Q. and Hahn-Ha"gerdal, B. 1997. Influence of cosubstrate concentration on xylose

conversion by recombinant, XYL1-expressing Saccharomyces cerevisiae: A comparison of different sugars and ethanol as cosubstrates. Applied and Environmental Microbiology. 63: 1959-1964. 151 Mergaert, J., Lednicka, D., Goris, J., Cnockaert, M. C., De Vos, P. and Swings, J. 2003.

Taxonomic study of Cellvibrio strains and description of Cellvibrio ostraviensis sp. nov., Cellvibrio fibrivorans sp. nov. and Cellvibrio gandavensis sp. nov. International Journal of Systematic and Evolutionary Microbiology. 53: 465-471. 152 Mes-Hartree, M. and Saddler, J. N. 1983. The nature of inhibitory materials present in pretreated lignocellulosic substrates which inhibit the enzymatic hydrolysis of cellulose. Biotechnology Letters. 5: 531-536. 153 Meyrial, V., Delegenes, J. P., Romieu, C., Moletta, R. and Gounot, A. M. 1995a. Ethanol tolerance and activity of plasma-membrane ATPase in Pichia stipitis grown on D-xylose or on D-glucose. Enzyme and Microbial Technology. 17: 535-540. 154 Meyrial, V., Delegenes, J. P., Romieu, C., Moletta, R. and Gounot, A. M. 1995b. In vivo ethanol activation of the plasma membrane ATPase of Pichia stipitis:

Effect of the carbon source. Journal of Biotechnology. 42: 109-116. 155 Moreira, J. R. and Goldemberg, J. 1999. Energ Policy. 27: 229. 156 Morrison, M. and Miron, J. 2000. Adhesion to cellulose by Ruminococcus albus: a combination of cellulosomes and Pil-proteins? FEMS

Microbiology Letters. 185: 109-115. 157 Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y. Y., Holtzapple, M. and Ladisch, M. 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technology. 96: 673-686. 158 Mukataka, S., Tada, M. and Takahashi, J. 1983. Effects of agitation on enzymatic hydrolysis of cellulose in a stirredtank reactor. Journal of Fermentation Technology. 61: 615 –621. 159 Myers, H. M. and Montgomery, D. C. 1991. Response surface methodology: process and product optimisation using designed experiments. Wiley±Interscience, New York. 160 Najafpour, G. D. 1990. Immobilization of microbial cells for the production of organic acids.

Journal of Science Islam Repub Iranica. 1: 172-176. 161 Navarro, A. R. 1994. Effects of furfural on ethanol fermentation by Saccharomyces cerevisiae: mathematical models. Current Microbiology. 29: 87±90. 162 Navarro, A. R., Sepulveda, M. C. and Rubio, M. C. 2000.

Bio-concentration of vinasse from the alcoholic fermentation of sugar cane molasses. Waste Management. 20: 581-585 163 Neirinck, L. G., Maleszka, R. and Schneider, H. 1984. The requirement of oxygen for incorporation of carbon from D-xylose and D-glucose by Pachysolen tannophilus. Archives of Biochemistry and Biophysics. 228: 13-21. 164 Nguyen, Q. 1998. Tower Reactors for Bioconversion of Lignocellulosic Material. US Patent 5,733,758, USA. 165 Nigam, S. C., Tsao, I. F., Sakoda, A. and Wang, H. Y. 1988. Techniques for preparing hydrogel membrane capsules. Biotechnology Techniques. 2: 271-276. 166 Nishise, H., Ogawa, A., Tani, Y. and Yamada, H. 1985. Studies on microbial glycerol dehydrogenase 4: glycerol dehydrogenase and glycerol dissimilation in Cellulomonas sp. NT3060. Agricultural and Biological Chemistry.

49: 629-636. 167 Nitisinprasert, S. and Temmes, A. 1991. The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CMC126 isolated from municipal sewage sludge. Journal Applied Bacteriolgy. 71: 154-161. 168 Nogi, Y., Takami, H. and Horikoshi, K. 2005.

Characterization of alkaliphilic Bacillus strains used in ndustry: Proposal of five novel species. International Journal of Systematic and Evolutionary icrobiology. 55(6): art. no. 63649, 2309-2315. 169 Norton, S., Watson, K. and D’Amore, T. 1995. Ethanol tolerance of immobilized brewer’s yeast cells. Applied Microbiology and Biotechnology. 43: 18-24. 170 Novak, M., Strehaiano, P., Moreno, M. and Goma, G. 1981. Alcoholic fermentation: on the inhibitory effect of ethanol. Biotechnology and Bioengineering. 23: 201-211. 171 Nunez, M. J. and Lema, J. M. 1987. Cell immobilization: application to alcohol production. Enzyme and Microbial Technology. 9: 642-651. 172 Olsson, L. and Hahn-Hagerdal, B. 1996.

Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme and Microbial Technology. 18: 312-331. 173 Palmqvist, E. and Hahn-Ha"gerdal, B. 1999. Fermentation of lignocellulosic hydrolysates: Inhibition and detoxification. Bioresource Technol. submitted. 174 Palmqvist, E., Almeida, J. and Hahn-Ha"gerdal, B. 1999a. Influence of furfural on anaerobic glycolytic kinetics of Saccharomyces cerevisiae in batch culture. Biotechnology and Bioengineering. 62: 447±454. 175 Palmqvist, E., et al. 1996. The effect of water-soluble inhibitors from steam-pretreated willow on enzymatic hydrolysis and ethanol fermentation. Enzyme and Microbial Technology. 19: 470-476. 176 Palmqvist, E., Grage, H., Meinander, N. and Hahn-Ha"gerdal, B. 1999. Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts. Biotechnology and Bioengineering. 63: 46-55. 177 Park, J. K. and Chang, H. N. 2000.

Microencapsulation of microbial cells. Biotechnology Advances. 18: 303-319. 178 Pascual, C., Alonso, A., Garcia, I., Roman, C. and Kotyk, A.

1988. Effect of ethanol on glucose transport, key glycolytic enzymes and proton extrusion in Saccharomyces cerevisiae. Biotechnology and Bioengineering. 32: 374-378. 179 Pason, P., Kyu, K. L. and Ratanakhanokchai, K. 2006. Paenibacillus curdlanolyticus strain B-6

xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides. Applied and Environmental Microbiology. 72: 2483-2490.. 180 Pettersen, R. R. 1984. The chemical composition of wood. In “The Chemistry of Solid Wood” (R. Rowell, ed.). p. 58-126. American Chemical Society, Washington, DC. 181 Prior, B. A., Kilian, S. G. and du Preez, J. C. 1989. Fermentation of D-xylose by the yeast Candida shehatae and Pichia stipitis: prospects and problems. Process Biochemistry. 24: 21-32. 182 Raabo, E. and Terkildsen, T. C. 1960. On the enzymatic

determination of blood glucose. Scandinavian Journal Clinical Laboratory Investigation. 12: 402-407. 183 Reese, E. T. and Ryu, D. Y., 1980.

Shear inactivation of cellulose of Trichoderma reesei. Enzyme and Microbial Technology. 2: 239-240. 184 Reese, E., Siu, T. and Levinson, H. S.

1950. The biological degradation of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolysis. Journal of Bacteriology. 59: 485-497. 185 Reifenberger, E., Boles, E. and Ciriacy, M. 1997. Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. European Journal of Biochemistry. 245: 324-333.

186 Reshamwala, S., Shawky, B. T. and Dale, B. E. 1995. Ethanol production from enzymatic hydrolysates of AFEX-treated coastal Bermuda grass and switchgrass. Applied Biochemistry and Biotechnology. 51/52: 43-55. 187 Rikhvanov, E., Varakina, N., Rusaleva, T., Rachenko, E., Kiseleva, V. and Voinikov, V. 2001. Heat shock-induced changes in the respiration of the yeast Saccharomyces cerevisiae. Microbiology. 70: 462-5.

188 Riley, M. R., Muzzio, F. J., Buettner, H. M. and Reyes, S. C. 1996. A simple