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Production of bioethanol using lignocellulosic hydrolysate by the white rot fungus Hohenbuehelia sp. ZW-16

Production of bioethanol using lignocellulosic hydrolysate by the white rot fungus Hohenbuehelia... Ann Microbiol (2013) 63:719–723 DOI 10.1007/s13213-012-0524-6 ORIGINAL ARTICLE Production of bioethanol using lignocellulosic hydrolysate by the white rot fungus Hohenbuehelia sp. ZW-16 Xiaohui Liang & Dongliang Hua & Zhixin Wang & Jie Zhang & Yuxiao Zhao & Haipeng Xu & Yan Li & Mintian Gao & Xiaodong Zhang Received: 13 May 2012 /Accepted: 30 July 2012 /Published online: 17 August 2012 Springer-Verlag and the University of Milan 2012 Abstract A novel white rot fungus strain Hohenbue- Introduction helia sp. ZW-16 was identified and first used for bio- ethanol production in this study. It was found that the Bioethanol is a type of promising alternative new energy, strain could produce bioethanol with glucose, xylose which has recently attracted more and more attention, and is and arabinose under limited oxygen condition. Then, mainly produced from starch of corn which competes with corn straw hydrolysate and corncob hydrolysate (mainly food resources. As a kind of sustainable and renewable composed of glucose, xylose, and arabinose) were used resources, lignocellulose was recently used for bioethanol for bioethanol production; the former substrate could production (Lin et al. 2010). After pretreatment, these produce more bioethanol in the experiment. The opti- resources could be used for ethanol production by yeasts mal sugar concentration and nitrogen sources were se- (Tang et al. 2006). The hydrolysate of lignocellulose was lected (50 g/L corn straw hydrolysate and 10 g/L soybean composed not only of glucose but also of pentoses such as meals, respectively) and the maximum yield of bioethanol xylose, and arabinose (Olsson and Hahn-agerdal 1996), reached 4.6 g/L after 8 days of fermentation. which were not fermented by Saccharomyces cerevisiae. So it is very necessary to find new microorganism strains for ethanol production with the pentoses mentioned above. . . . Except for some species of yeasts (for example, Pachysolen Keywords Bioethanol Production Hydrolysate Hohenbuehelia sp. tannophilus, Pichia stipitis), filamentous fungi (including the genus Aspergillus, Rhizopus, Monilia, Neurospora, Fusarium, Trichoderma and Mucor) have also been used for ethanol production with biomass (Skory et al. 1997; Millati et al. 2005; Zhang et al. 2003; Christakopoulos et al. 1989; Stevenson and Weimer 2002; Sues et al. 2005; : : : : : : : X. Liang D. Hua J. Zhang Y. Zhao H. Xu Y. Li M. Gao Okamoto et al. 2010). White rot fungi, such as Peniophora X. Zhang Energy Research Institute of Shandong Academy of Sciences, cinerea, Trametes suaveolens, and Phanerochaete chryso- Jinan 250014, China sporium, can produce ethanol directly from biomass (Okamoto et al. 2010; Kenealy and Dietrich 2004), and the : : : : : : X. Liang (*) D. Hua J. Zhang Y. Zhao H. Xu Y. Li production can reach over 5 g/L in 2 weeks or more. M. Gao X. Zhang (*) Key Laboratory for Biomass Gasification Technology Okamotoetal(2011) also reported that the white rot of Shandong Province, fungus Trametes hirsuta can produce ethanol directly Jinan 250014, China from biomass such as wheat bran and rice straw, and e-mail: liang_xiaohui2002@yahoo.com.cn that the maximum yield can reach 3.4 g ethanol/20 g e-mail: xd_zhang77@yahoo.com.cn ball-milled rice straw. However, there has been no re- Z. Wang port on bioethanol production from lignocellulosic hy- College of Bioscience and Bioengineering, drolysate, which has been mainly used for biofuels Hebei University of Science and Technology, production. In this study, a novel white rot fungus Shijiazhuang 050018, China 720 Ann Microbiol (2013) 63:719–723 Table 1 Sugars in the lignocellulosic hydrolysate describedbyOkamoto et al (2011)The strain was incubated in 30 °C for 8 days on a reciprocal shaker at Material Glucose (g/L) Xylose (g/L) Arabinose (g/L) Total 100 rpm. In order to produce bioethanol, the the flasks (g/L) were sealed tightly with plastic film to obtain oxygen- Corn straw 4.9 (73.1 %) 1.6 (23.9 %) 0.2 (3.0 %) 6.7 limited condition after 24 h of incubation. Corncob 0.6 (12.5 %) 3.9 (81.3 %) 0.3 (6.3 %) 4.8 Genomic DNA extraction and 18S rDNA analysis strain, Hohenbuehelia sp. ZW-16, was identified and Samples used for DNA extraction were collected by liquid first used for bioethanol production from glucose, xy- state fermentation in the shake flask, and the medium com- lose and arabinose. Then, lignocellulosic hydrolysate position was: glucose 20 g/L, potato extract 20 % (w/v), pH (from corn straw and corncob) was used for bioethanol 6.0. Strain ZW-16 was incubated in a 250-mL Erlenmeyer production and the highest yield reached 4.6 g/L in flask containing of 50 mL medium at 28 °C on a reciprocal 8 days of fermentation when the corn straw hydrolysate shaker at 200 rpm for 72 h. The fresh mycelia were precip- wasusedascarbonsource. itated by centrifugation at 10,000 g,4°C for15min. Genomic DNA was isolated as described by Paavanen- Huhtala et al. (1999). The 18S rDNA was amplified using Materials and methods the universal primer pairs of NS1 (5′-GTAGT CATAT GCTTG TCTC-3′)/NS8 (5′-TCCGC AGGTT CACCT Microorganism: Hohenbuehelia sp. ZW-16, stored at 4°C ACGGA-3′) (White et al. 1990). The amplifications were on a potato dextrose agar (PDA) slant containing potato performed in a ThermoHybaid PCR Sprint Thermal extract 20 % (w/v), glucose 20 g/L, and agar 20 g/L. Cycler (Thermo Electron, USA). The PCR reaction Lignocellulosic hydrolysate: Prepared from corn straw details were as follows: 4 min at 94 °C for initial and corncob in our laboratory, and the reducing sugar con- denaturation, 45 s at 94 °C for denaturation, 1 min at centrations were adjusted for the experiments utilization. 50 °C for annealing of 18S rDNA, 1 min 45 s at 72 °C for The main sugars (including glucose, xylose and arabinose) extension with total 30 cycles of amplification, and 10 min at of the hydrolysates were determined by high-performance final extension. The 18S rDNA were sequenced by Shanghai liquid chromatography (Agilent 1100 system; Agilent Shengon, China. Technologies, USA; Aminex HPX-87P analytical column, BLAST (http://www.ncbi.nlm.nih.gov/BLAST) searches 300×7.8 mm; BioRad, USA). were used to finde homologous sequences. The Culture: Mycelia of the strain Hohenbuehelia sp. ZW-16 corresponding sequences of representative species were se- on the slant were transferred and grown on the PDA plates lected for phylogenetic analyses. Preliminary multiple align- in 28 °C for 10 days, and then the mycelia were incubated in ments were conducted using Clustal W v.1.83 for the a 250-mL Erlenmeyer flask containing 50 mL of the datasets. Alignment adjustments were made by BioEdit fermentation medium composed by 20 g/L of glucose (or v.7.0.1. Distance trees were generated based on neighbor- other sugars), 10 g/L yeast extract, 10 g/L KH PO , joining methods using Mega .3.1, tree topologies were esti- 2 4 2g/L (NH ) SO , and 0.5 g/L MgSO ·7H O, which were mated by bootstrap analyses with 1,000 replicates. 4 2 4 4 2 Fig. 1 A neighbor-joining tree Laccocephalum mylittae (AY944218) analysis of 18S rDNA of the Laccocephalum mylittae (EU157728) strain Hohenbuehelia sp. ZW-16 Pleurotus ostreatus (FJ379284) Lentinus sajor-caju (FJ379278) Psilocybe cyanescens (AY705949) Pholiota squarrosa (DQ465337) Coprinus comatus (NG 016488) 88 Calvatia gigantea (AF026622) Hohenbuehelia tremula (DQ440645) Hohenbuehelia tristis (DQ851573) Hohenbuehelia (JQ926736) 0.001 Ann Microbiol (2013) 63:719–723 721 Analytical methods The sugar concentration was determined by high- performance liquid chromatography (Agilent 1100; Agilent Technologies), and the bioethanol content was measured by a SBA-40E biosensor (Biology Institute of Glucose Ethanol Shandong Acadamy of Sciences) after diluted to the optimal concentrations. Results and discussion 0 24 48 72 96 120 144 168 192 Main sugars in the lignocellulosic hydrolysate Time (h) The main sugars in the hydrolysate prepared in our 2.0 laboratory were determined and the results are shown in Table 1. For hydrolysate of corn straw, glucose content was higher than the other two kinds of sugars; 1.5 however, xylose was the main sugar in corncob hydro- lysate. Arabinose content was very low in the two kinds Xylose 1.0 Ethanol of hydrolysates in our study. 0.5 18S rDNA analyses 10 0.0 A 1,660-bp sequence was amplified from the genome DNA with the NS1/NS8 as primers, which was submit- ted to GenBank (JQ926736). Hohenbuehelia tristis 0 24 48 72 96 120 144 168 192 Time (h) (DQ851573) was very closely related to the strain ZW-16, which was on the 100 % bootstrap-supported branch with Hohenbuehelia sp. (JQ926736) (Fig. 1). 0.35 According to the results of morphology and 18S rDNA analyses, the strain ZW-16 should belong to the 0.30 genus Hohenbuehelia. For the Basidiomycetes, to iden- 0.25 tify a species usually requires the fruit-body of the Arobinose 0.20 strain. However, the fruit-body of the strain ZW-16 was not Ethanol 0.15 obtained in the laboratory conditions, so the specific taxonom- ic status of the strain needs further investigation. 0.10 0.05 Effect of different carbon sources on ethanol production 0.00 Three kinds of sugars, including glucose, xylose and 0 24 48 72 96 120 144 168 192 arabinose, were investigated in the study. The results Time (h) showed that glucose was the best carbon source for Fig. 2 a Time course of ethanol production by the strain Hohenbue- ethanol production by Hohenbuehelia sp. ZW-16 helia sp. ZW-16 with glucose. b Time course of ethanol production by (Fig. 2a), then xylose and arabinose (Fig. 2b, c). The the strain Hohenbuehelia sp. ZW-16 with xylose. c Time course of highest ethanol yield reached 3.9 g/L after 8 days of ethanol production by the strain Hohenbuehelia sp. ZW-16 with arobinose fermentation under oxygen-limited condition (the flasks were sealed tightly with plastic film). It was also found that, when the oxygen was rich, the strain could not produce ethanol at all; however, when the plastic film a shaker. However, Okamoto et al. (2011)used T. was changed by a rubber plug, the strain could not hirsuta for bioethanol production under stationary fer- grow. When the strain was cultivated stationary, the mentation, and the yield reached about 10 g/L in 6 days highest ethanol yield was lower than that cultivated in of cultivation when glucose was used as carbon source. Arabinose (g/L) Xylose (g/L) Glucose (g/L) Ethanol (g/L) Ehtanol (g/L) Ethanol (g/L) 722 Ann Microbiol (2013) 63:719–723 3.5 22 4 3.0 20 Reducing sugar Ethanol 2.5 2.0 1.5 1.0 0.5 -1 0.0 0 24 48 72 96 120 144 168 192 123 4 56 Time (h) Nitrogen sources (g/L) Fig. 5 Effect of nitrogen sources on on ethanol production by the 2.0 strain Hohenbuehelia sp. ZW-16 (1 yeast extract; 2 urea; 3 (NH ) SO , 4 2 4 4 NaNO ; 5 soybean meals; 6 peptone) Reducing sugar 1.5 Ethanol Ethanol production from hydrolysate 1.0 Corn straw hydrolysate and corncob hydrolysate were both used for ethanol production in this study, and the results are 0.5 12 shown in Fig. 3a, b. It can be concluded that, after 8 days of fementation, the ethanol production reached 2.9 g/L and 1.3 g/L, respectively. The corncob hydrolysate produced a 0.0 lower level of ethanol than that of the corn straw hydroly- 0 24 48 72 96 120 144 168 192 sate, which might be because of the higher concentration of Time (h) xylose in the latter. So the corn straw hydrolysate should be Fig. 3 a Time course of ethanol production by the strain Hohenbue- better for bioethanol production in the experiments. helia sp. ZW-16 with corn straw hydrolysate. b Time course of ethanol In Fig. 4, it can be concluded that the sugar concentration production by the strain Hohenbuehelia sp. ZW-16 with corncob of the corn straw hydrolysate influenced the bioethanol hydrolysate production. When the sugar concentration reached 50 g/L, the bioethanol yield was 4.1 g/L, and then declined. High The bioethanol yield of Hohenbuehelia sp. ZW-16 was lower sugar concentration could produce more bioethanol gener- than T. hirsuta due to slow growth of the strain ZW-16. ally, which might be because more biomass was yielded when the reducing sugars were at a high concentration 4.5 during the fermentation period. 4.0 Nitrogen sources can significantly affect the bioethanol production of the strain Hohenbuehelia sp. ZW-16 (Fig. 5). 3.5 When the soybean meal was used as the sole nitrogen 3.0 source, the highest ethanol production was observed. 2.5 Generally, organic nitrogen sources were better than inor- 2.0 ganic nitrogen sources in bioethanol production. 1.5 1.0 Conclusions 0.5 0.0 In this paper, the white rot fungus strain Hohenbuehelia sp. 10 20 30 40 50 60 ZW-16 was used for bioethanol production for the first time. Sugar concentration (g/L) It was found that this strain showed fermentability to glu- cose, xylose and arabinose, which are the main sugar com- Fig. 4 Effect of sugar concentration of the corn straw hydrolysate on ethanol production by the strain Hohenbuehelia sp. ZW-16 ponents in corn straw and other agricultural residues. Reducing sugar (g/L) Reducing sugar (g/L) Ethanol (g/L) Ethanol (g/L) Ethanol (g/L) Ethanol production (g/L) Ann Microbiol (2013) 63:719–723 723 xylose, and wood hydrolyzates. Enzyme Microb Technol Hohenbuehelia sp. ZW-16 can produce bioethanol from 36:294–300 lignocellulosic materials hydrolysate of corn straw and corn- Okamoto K, Imashiro K, Akizawa Y, Onimura A, Yoneda M, Nitta Y, cob. Corn straw hydrolysate showed higher bioethanol yield Maekawa N, Yanase H (2010) Production of ethanol by the white- in the experiment. The highest bioethanol production rot basidiomycetes Peniophora cinerea and Trametes suaveolens. Biotechnol Lett 32:909–913 reached 4.6 g/L when the corn straw hydrolysate and soy- Okamoto K, Nitta Y, Maekawa N, Yanase H (2011) Direct ethanol bean meal concentration were 50 g/L and 10 g/L, respec- production from starch, wheat bran and rice straw by the white tively. Further studies should improve the bioethanol rot fungus Trametes hirsuta. Enzyme Microb Technol 48:273– production of the strain, and the co-culture or fermentation 277 Olsson L, Hahn-agerdal B (1996) Fermentaion of lignocellulosic of two or more microorganisms including the strain hydrolysates for ethanol production. Enzyme Microb Technol Hohenbuehelia sp. ZW-16 should be investigated. 18:312–331 Paavanen-Huhtala S, Hyvönen J, Bulat SA, Yli-mattila T (1999) Acknowledgments This study was supported by Twelfth Five-Year RAPD-PCR, isozyme, rDNA RFLP and rDNA sequence analyses Plan of National Science and Technology (No. 2011BAD14B03), State in identification of Finnish Fusarium oxysporum isolates. Mycol “863” projects (No. 2012AA101803), Young Scientists’ Program of Res 103:625–634 National Natural Science Foundation of China (Grant No. 31100030), Skory CD, Freer SN, Bothast RJ (1997) Screening for ethanol- the Excellent Middle-Aged and Youth Scientist Award Foundation of producing filamentous fungi. Biotechnol Lett 19(6):203–206 Shandong Province (Grant No. BS2011SW033) and State Key Labo- Stevenson DM, Weimer PJ (2002) Isolation and characterization of a ratory of Microbial Technology of Shandong University (M2011-17). Trichoderma strain capable of fermenting cellulose to ethanol. Appl Microbiol Biotechnol 59(6):721–726 Sues A, Millati R, Edebo L, Taherzadeh MJ (2005) Ethanol production from hexoses, pentoses, and dilute-acid hydrolyzate by Mucor References indicus. FEMS Yeast Res 5:659–676 Tang YQ, An MZ, Liu K (2006) Ethanol production from acid Christakopoulos P, Kekos D, Macris BJ (1989) Direct fermentation of hydrolysate of wood biomass using the flocculating yeast cellulose to ethanol by Fusarium oxysporum. Enzyme Microb Saccharomyces cerevisiae strain KF-7. Process Biochem Technol 11(39):236–239 41:909–914 Kenealy WR, Dietrich DM (2004) Growth and fermentation responses White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct of Phanerochaete chrysosporium to O limitation. Enzym Microb sequencing of fungal ribosomal RNA genes for phylogenetics. In: Technol 34:490–498 Innis MA, Gelfland DH, Sninsky JJ, White TJ (eds) PCR proto- Lin CW, Tran DT, Lai CY, I CY, Wu CH (2010) Response surface cols: a guide to methods and applications., . Academic, San optimization for ethanol production from Pennisetum Alopecoider Diego, pp. 315–322 by Klebsiella oxytoca THLC0409. Biomass Bioener 34:1922–1929 Zhang X, Zhu DQ, Wang D, Lin JQ, Qu YB, Yu SY (2003) Study on Millati R, Edebo L, Taherzadeh MJ (2005) Performance of Rhizopus xylose fermentation by Neurospora crassa. Acta Microbiol Sin Rhizomucor,and Mucor in ethanol production from glucose, 43(4):466–472 (in Chinese) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Production of bioethanol using lignocellulosic hydrolysate by the white rot fungus Hohenbuehelia sp. ZW-16

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Springer Journals
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Copyright © 2012 by Springer-Verlag and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
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10.1007/s13213-012-0524-6
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Abstract

Ann Microbiol (2013) 63:719–723 DOI 10.1007/s13213-012-0524-6 ORIGINAL ARTICLE Production of bioethanol using lignocellulosic hydrolysate by the white rot fungus Hohenbuehelia sp. ZW-16 Xiaohui Liang & Dongliang Hua & Zhixin Wang & Jie Zhang & Yuxiao Zhao & Haipeng Xu & Yan Li & Mintian Gao & Xiaodong Zhang Received: 13 May 2012 /Accepted: 30 July 2012 /Published online: 17 August 2012 Springer-Verlag and the University of Milan 2012 Abstract A novel white rot fungus strain Hohenbue- Introduction helia sp. ZW-16 was identified and first used for bio- ethanol production in this study. It was found that the Bioethanol is a type of promising alternative new energy, strain could produce bioethanol with glucose, xylose which has recently attracted more and more attention, and is and arabinose under limited oxygen condition. Then, mainly produced from starch of corn which competes with corn straw hydrolysate and corncob hydrolysate (mainly food resources. As a kind of sustainable and renewable composed of glucose, xylose, and arabinose) were used resources, lignocellulose was recently used for bioethanol for bioethanol production; the former substrate could production (Lin et al. 2010). After pretreatment, these produce more bioethanol in the experiment. The opti- resources could be used for ethanol production by yeasts mal sugar concentration and nitrogen sources were se- (Tang et al. 2006). The hydrolysate of lignocellulose was lected (50 g/L corn straw hydrolysate and 10 g/L soybean composed not only of glucose but also of pentoses such as meals, respectively) and the maximum yield of bioethanol xylose, and arabinose (Olsson and Hahn-agerdal 1996), reached 4.6 g/L after 8 days of fermentation. which were not fermented by Saccharomyces cerevisiae. So it is very necessary to find new microorganism strains for ethanol production with the pentoses mentioned above. . . . Except for some species of yeasts (for example, Pachysolen Keywords Bioethanol Production Hydrolysate Hohenbuehelia sp. tannophilus, Pichia stipitis), filamentous fungi (including the genus Aspergillus, Rhizopus, Monilia, Neurospora, Fusarium, Trichoderma and Mucor) have also been used for ethanol production with biomass (Skory et al. 1997; Millati et al. 2005; Zhang et al. 2003; Christakopoulos et al. 1989; Stevenson and Weimer 2002; Sues et al. 2005; : : : : : : : X. Liang D. Hua J. Zhang Y. Zhao H. Xu Y. Li M. Gao Okamoto et al. 2010). White rot fungi, such as Peniophora X. Zhang Energy Research Institute of Shandong Academy of Sciences, cinerea, Trametes suaveolens, and Phanerochaete chryso- Jinan 250014, China sporium, can produce ethanol directly from biomass (Okamoto et al. 2010; Kenealy and Dietrich 2004), and the : : : : : : X. Liang (*) D. Hua J. Zhang Y. Zhao H. Xu Y. Li production can reach over 5 g/L in 2 weeks or more. M. Gao X. Zhang (*) Key Laboratory for Biomass Gasification Technology Okamotoetal(2011) also reported that the white rot of Shandong Province, fungus Trametes hirsuta can produce ethanol directly Jinan 250014, China from biomass such as wheat bran and rice straw, and e-mail: liang_xiaohui2002@yahoo.com.cn that the maximum yield can reach 3.4 g ethanol/20 g e-mail: xd_zhang77@yahoo.com.cn ball-milled rice straw. However, there has been no re- Z. Wang port on bioethanol production from lignocellulosic hy- College of Bioscience and Bioengineering, drolysate, which has been mainly used for biofuels Hebei University of Science and Technology, production. In this study, a novel white rot fungus Shijiazhuang 050018, China 720 Ann Microbiol (2013) 63:719–723 Table 1 Sugars in the lignocellulosic hydrolysate describedbyOkamoto et al (2011)The strain was incubated in 30 °C for 8 days on a reciprocal shaker at Material Glucose (g/L) Xylose (g/L) Arabinose (g/L) Total 100 rpm. In order to produce bioethanol, the the flasks (g/L) were sealed tightly with plastic film to obtain oxygen- Corn straw 4.9 (73.1 %) 1.6 (23.9 %) 0.2 (3.0 %) 6.7 limited condition after 24 h of incubation. Corncob 0.6 (12.5 %) 3.9 (81.3 %) 0.3 (6.3 %) 4.8 Genomic DNA extraction and 18S rDNA analysis strain, Hohenbuehelia sp. ZW-16, was identified and Samples used for DNA extraction were collected by liquid first used for bioethanol production from glucose, xy- state fermentation in the shake flask, and the medium com- lose and arabinose. Then, lignocellulosic hydrolysate position was: glucose 20 g/L, potato extract 20 % (w/v), pH (from corn straw and corncob) was used for bioethanol 6.0. Strain ZW-16 was incubated in a 250-mL Erlenmeyer production and the highest yield reached 4.6 g/L in flask containing of 50 mL medium at 28 °C on a reciprocal 8 days of fermentation when the corn straw hydrolysate shaker at 200 rpm for 72 h. The fresh mycelia were precip- wasusedascarbonsource. itated by centrifugation at 10,000 g,4°C for15min. Genomic DNA was isolated as described by Paavanen- Huhtala et al. (1999). The 18S rDNA was amplified using Materials and methods the universal primer pairs of NS1 (5′-GTAGT CATAT GCTTG TCTC-3′)/NS8 (5′-TCCGC AGGTT CACCT Microorganism: Hohenbuehelia sp. ZW-16, stored at 4°C ACGGA-3′) (White et al. 1990). The amplifications were on a potato dextrose agar (PDA) slant containing potato performed in a ThermoHybaid PCR Sprint Thermal extract 20 % (w/v), glucose 20 g/L, and agar 20 g/L. Cycler (Thermo Electron, USA). The PCR reaction Lignocellulosic hydrolysate: Prepared from corn straw details were as follows: 4 min at 94 °C for initial and corncob in our laboratory, and the reducing sugar con- denaturation, 45 s at 94 °C for denaturation, 1 min at centrations were adjusted for the experiments utilization. 50 °C for annealing of 18S rDNA, 1 min 45 s at 72 °C for The main sugars (including glucose, xylose and arabinose) extension with total 30 cycles of amplification, and 10 min at of the hydrolysates were determined by high-performance final extension. The 18S rDNA were sequenced by Shanghai liquid chromatography (Agilent 1100 system; Agilent Shengon, China. Technologies, USA; Aminex HPX-87P analytical column, BLAST (http://www.ncbi.nlm.nih.gov/BLAST) searches 300×7.8 mm; BioRad, USA). were used to finde homologous sequences. The Culture: Mycelia of the strain Hohenbuehelia sp. ZW-16 corresponding sequences of representative species were se- on the slant were transferred and grown on the PDA plates lected for phylogenetic analyses. Preliminary multiple align- in 28 °C for 10 days, and then the mycelia were incubated in ments were conducted using Clustal W v.1.83 for the a 250-mL Erlenmeyer flask containing 50 mL of the datasets. Alignment adjustments were made by BioEdit fermentation medium composed by 20 g/L of glucose (or v.7.0.1. Distance trees were generated based on neighbor- other sugars), 10 g/L yeast extract, 10 g/L KH PO , joining methods using Mega .3.1, tree topologies were esti- 2 4 2g/L (NH ) SO , and 0.5 g/L MgSO ·7H O, which were mated by bootstrap analyses with 1,000 replicates. 4 2 4 4 2 Fig. 1 A neighbor-joining tree Laccocephalum mylittae (AY944218) analysis of 18S rDNA of the Laccocephalum mylittae (EU157728) strain Hohenbuehelia sp. ZW-16 Pleurotus ostreatus (FJ379284) Lentinus sajor-caju (FJ379278) Psilocybe cyanescens (AY705949) Pholiota squarrosa (DQ465337) Coprinus comatus (NG 016488) 88 Calvatia gigantea (AF026622) Hohenbuehelia tremula (DQ440645) Hohenbuehelia tristis (DQ851573) Hohenbuehelia (JQ926736) 0.001 Ann Microbiol (2013) 63:719–723 721 Analytical methods The sugar concentration was determined by high- performance liquid chromatography (Agilent 1100; Agilent Technologies), and the bioethanol content was measured by a SBA-40E biosensor (Biology Institute of Glucose Ethanol Shandong Acadamy of Sciences) after diluted to the optimal concentrations. Results and discussion 0 24 48 72 96 120 144 168 192 Main sugars in the lignocellulosic hydrolysate Time (h) The main sugars in the hydrolysate prepared in our 2.0 laboratory were determined and the results are shown in Table 1. For hydrolysate of corn straw, glucose content was higher than the other two kinds of sugars; 1.5 however, xylose was the main sugar in corncob hydro- lysate. Arabinose content was very low in the two kinds Xylose 1.0 Ethanol of hydrolysates in our study. 0.5 18S rDNA analyses 10 0.0 A 1,660-bp sequence was amplified from the genome DNA with the NS1/NS8 as primers, which was submit- ted to GenBank (JQ926736). Hohenbuehelia tristis 0 24 48 72 96 120 144 168 192 Time (h) (DQ851573) was very closely related to the strain ZW-16, which was on the 100 % bootstrap-supported branch with Hohenbuehelia sp. (JQ926736) (Fig. 1). 0.35 According to the results of morphology and 18S rDNA analyses, the strain ZW-16 should belong to the 0.30 genus Hohenbuehelia. For the Basidiomycetes, to iden- 0.25 tify a species usually requires the fruit-body of the Arobinose 0.20 strain. However, the fruit-body of the strain ZW-16 was not Ethanol 0.15 obtained in the laboratory conditions, so the specific taxonom- ic status of the strain needs further investigation. 0.10 0.05 Effect of different carbon sources on ethanol production 0.00 Three kinds of sugars, including glucose, xylose and 0 24 48 72 96 120 144 168 192 arabinose, were investigated in the study. The results Time (h) showed that glucose was the best carbon source for Fig. 2 a Time course of ethanol production by the strain Hohenbue- ethanol production by Hohenbuehelia sp. ZW-16 helia sp. ZW-16 with glucose. b Time course of ethanol production by (Fig. 2a), then xylose and arabinose (Fig. 2b, c). The the strain Hohenbuehelia sp. ZW-16 with xylose. c Time course of highest ethanol yield reached 3.9 g/L after 8 days of ethanol production by the strain Hohenbuehelia sp. ZW-16 with arobinose fermentation under oxygen-limited condition (the flasks were sealed tightly with plastic film). It was also found that, when the oxygen was rich, the strain could not produce ethanol at all; however, when the plastic film a shaker. However, Okamoto et al. (2011)used T. was changed by a rubber plug, the strain could not hirsuta for bioethanol production under stationary fer- grow. When the strain was cultivated stationary, the mentation, and the yield reached about 10 g/L in 6 days highest ethanol yield was lower than that cultivated in of cultivation when glucose was used as carbon source. Arabinose (g/L) Xylose (g/L) Glucose (g/L) Ethanol (g/L) Ehtanol (g/L) Ethanol (g/L) 722 Ann Microbiol (2013) 63:719–723 3.5 22 4 3.0 20 Reducing sugar Ethanol 2.5 2.0 1.5 1.0 0.5 -1 0.0 0 24 48 72 96 120 144 168 192 123 4 56 Time (h) Nitrogen sources (g/L) Fig. 5 Effect of nitrogen sources on on ethanol production by the 2.0 strain Hohenbuehelia sp. ZW-16 (1 yeast extract; 2 urea; 3 (NH ) SO , 4 2 4 4 NaNO ; 5 soybean meals; 6 peptone) Reducing sugar 1.5 Ethanol Ethanol production from hydrolysate 1.0 Corn straw hydrolysate and corncob hydrolysate were both used for ethanol production in this study, and the results are 0.5 12 shown in Fig. 3a, b. It can be concluded that, after 8 days of fementation, the ethanol production reached 2.9 g/L and 1.3 g/L, respectively. The corncob hydrolysate produced a 0.0 lower level of ethanol than that of the corn straw hydroly- 0 24 48 72 96 120 144 168 192 sate, which might be because of the higher concentration of Time (h) xylose in the latter. So the corn straw hydrolysate should be Fig. 3 a Time course of ethanol production by the strain Hohenbue- better for bioethanol production in the experiments. helia sp. ZW-16 with corn straw hydrolysate. b Time course of ethanol In Fig. 4, it can be concluded that the sugar concentration production by the strain Hohenbuehelia sp. ZW-16 with corncob of the corn straw hydrolysate influenced the bioethanol hydrolysate production. When the sugar concentration reached 50 g/L, the bioethanol yield was 4.1 g/L, and then declined. High The bioethanol yield of Hohenbuehelia sp. ZW-16 was lower sugar concentration could produce more bioethanol gener- than T. hirsuta due to slow growth of the strain ZW-16. ally, which might be because more biomass was yielded when the reducing sugars were at a high concentration 4.5 during the fermentation period. 4.0 Nitrogen sources can significantly affect the bioethanol production of the strain Hohenbuehelia sp. ZW-16 (Fig. 5). 3.5 When the soybean meal was used as the sole nitrogen 3.0 source, the highest ethanol production was observed. 2.5 Generally, organic nitrogen sources were better than inor- 2.0 ganic nitrogen sources in bioethanol production. 1.5 1.0 Conclusions 0.5 0.0 In this paper, the white rot fungus strain Hohenbuehelia sp. 10 20 30 40 50 60 ZW-16 was used for bioethanol production for the first time. Sugar concentration (g/L) It was found that this strain showed fermentability to glu- cose, xylose and arabinose, which are the main sugar com- Fig. 4 Effect of sugar concentration of the corn straw hydrolysate on ethanol production by the strain Hohenbuehelia sp. ZW-16 ponents in corn straw and other agricultural residues. Reducing sugar (g/L) Reducing sugar (g/L) Ethanol (g/L) Ethanol (g/L) Ethanol (g/L) Ethanol production (g/L) Ann Microbiol (2013) 63:719–723 723 xylose, and wood hydrolyzates. Enzyme Microb Technol Hohenbuehelia sp. ZW-16 can produce bioethanol from 36:294–300 lignocellulosic materials hydrolysate of corn straw and corn- Okamoto K, Imashiro K, Akizawa Y, Onimura A, Yoneda M, Nitta Y, cob. Corn straw hydrolysate showed higher bioethanol yield Maekawa N, Yanase H (2010) Production of ethanol by the white- in the experiment. The highest bioethanol production rot basidiomycetes Peniophora cinerea and Trametes suaveolens. Biotechnol Lett 32:909–913 reached 4.6 g/L when the corn straw hydrolysate and soy- Okamoto K, Nitta Y, Maekawa N, Yanase H (2011) Direct ethanol bean meal concentration were 50 g/L and 10 g/L, respec- production from starch, wheat bran and rice straw by the white tively. Further studies should improve the bioethanol rot fungus Trametes hirsuta. Enzyme Microb Technol 48:273– production of the strain, and the co-culture or fermentation 277 Olsson L, Hahn-agerdal B (1996) Fermentaion of lignocellulosic of two or more microorganisms including the strain hydrolysates for ethanol production. Enzyme Microb Technol Hohenbuehelia sp. ZW-16 should be investigated. 18:312–331 Paavanen-Huhtala S, Hyvönen J, Bulat SA, Yli-mattila T (1999) Acknowledgments This study was supported by Twelfth Five-Year RAPD-PCR, isozyme, rDNA RFLP and rDNA sequence analyses Plan of National Science and Technology (No. 2011BAD14B03), State in identification of Finnish Fusarium oxysporum isolates. Mycol “863” projects (No. 2012AA101803), Young Scientists’ Program of Res 103:625–634 National Natural Science Foundation of China (Grant No. 31100030), Skory CD, Freer SN, Bothast RJ (1997) Screening for ethanol- the Excellent Middle-Aged and Youth Scientist Award Foundation of producing filamentous fungi. Biotechnol Lett 19(6):203–206 Shandong Province (Grant No. BS2011SW033) and State Key Labo- Stevenson DM, Weimer PJ (2002) Isolation and characterization of a ratory of Microbial Technology of Shandong University (M2011-17). Trichoderma strain capable of fermenting cellulose to ethanol. Appl Microbiol Biotechnol 59(6):721–726 Sues A, Millati R, Edebo L, Taherzadeh MJ (2005) Ethanol production from hexoses, pentoses, and dilute-acid hydrolyzate by Mucor References indicus. FEMS Yeast Res 5:659–676 Tang YQ, An MZ, Liu K (2006) Ethanol production from acid Christakopoulos P, Kekos D, Macris BJ (1989) Direct fermentation of hydrolysate of wood biomass using the flocculating yeast cellulose to ethanol by Fusarium oxysporum. Enzyme Microb Saccharomyces cerevisiae strain KF-7. Process Biochem Technol 11(39):236–239 41:909–914 Kenealy WR, Dietrich DM (2004) Growth and fermentation responses White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct of Phanerochaete chrysosporium to O limitation. Enzym Microb sequencing of fungal ribosomal RNA genes for phylogenetics. In: Technol 34:490–498 Innis MA, Gelfland DH, Sninsky JJ, White TJ (eds) PCR proto- Lin CW, Tran DT, Lai CY, I CY, Wu CH (2010) Response surface cols: a guide to methods and applications., . Academic, San optimization for ethanol production from Pennisetum Alopecoider Diego, pp. 315–322 by Klebsiella oxytoca THLC0409. Biomass Bioener 34:1922–1929 Zhang X, Zhu DQ, Wang D, Lin JQ, Qu YB, Yu SY (2003) Study on Millati R, Edebo L, Taherzadeh MJ (2005) Performance of Rhizopus xylose fermentation by Neurospora crassa. Acta Microbiol Sin Rhizomucor,and Mucor in ethanol production from glucose, 43(4):466–472 (in Chinese)

Journal

Annals of Microbiology – Springer Journals

Published: Aug 17, 2012

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