瘤胃真菌分离鉴定及酶学特性
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摘要
厌氧真菌(Neocallimastigomycota)具有很强的植物组织降解能力,是草食动物消化道内一类重要的降解植物细胞壁的功能菌。为筛选可提高粗饲料利用率的高植物细胞壁降解酶活性厌氧真菌,本研究采用亨盖特厌氧滚管技术从西农萨能奶山羊(Capra hircus)瘤胃中分离获得12株真菌菌株,通过形态学观察、核糖体内转录间隔区和28S rDNA D1/D2区基因序列分析确定其分类地位。测定12株真菌的植物细胞壁降解酶活性(木聚糖酶,羧甲基纤维素酶,微晶纤维素酶,乙酰酯酶和β-葡聚糖酶),并对活性最高菌株分泌的木聚糖酶和乙酰酯酶进行酶学特性分析。结果表明,12株厌氧真菌均鉴定为Piromyces属,分别命名为Piromyces sp.CN1~Piromyces sp.CN12,ITS基因序列Genbank登录号为KY368100~KY368111。其中,Piromyces sp.CN6的木聚糖酶、羧甲基纤维素酶和乙酰酯酶活性分别为1655.3、93.4和152.8 m U,显著高于其他菌株(P<0.05);Piromyces sp.CN3的微晶纤维素酶活性最高,但与Piromyces sp.CN6差异不显著(P>0.05);各菌株间β-葡聚糖酶活性差异不显著(P>0.05)。木聚糖酶与羧甲基纤维素酶、乙酰酯酶存在极显著正相关(P<0.01),与微晶纤维素酶呈显著正相关(P<0.05)。酶学特性表明,Piromyces sp.CN6分泌的木聚糖酶最适反应温度为50℃,最适pH为5.0,该酶在40℃和pH5.0~8.0下较稳定;K~+、Ca~(2+)和Co~(2+)对其有激活作用,Zn~(2+)、Cu~(2+)、Mg~(2+)、Fe~(2+)和Mn~(2+)抑制该酶活性。乙酰酯酶的最适反应温度为50℃,最适pH为9.0,该酶在40℃和pH 5.0~10.0下较稳定;Mg~(2+)、K+、Ca~(2+)对乙酰酯酶有激活作用,Zn~(2+)、Fe~(2+)、Co~(2+)和Mn~(2+)抑制该酶活性。本研究从奶山羊瘤胃内容物筛选获得的Piromyces sp.CN6具有较高的植物细胞壁降解酶活性,通过酶活测定和酶学特性分析,丰富和完善瘤胃真菌的酶系信息,为深入应用提供依据。
Anaerobic fungi(Neocallimastigomycota) are important fiber-degrading functional microorganisms in the herbivores' digestive tract.The purpose of the present study was to isolate anaerobic rumen fungi with high activities of plant cell wall degrading enzymes for practical applications to improve the utilization of roughage.Twelve anaerobic fungi strains were isolated from the ruminal contents of Xinong Saanen dairy goat(Capra hircus) using the Hungate roll tube technique.The fungi strains were identified using the morphological characteristics,nucleotide sequence analysis of internal transcribed spacer region and 28 S rDNA D1/D2 region.Meanwhile,activities of xylanase,carboxymethyl cellulase(CMCase),avicelase,acetyl esterase(AE) and β-glucanase of 12 fungi cultures were assayed,and the enzymatic characteristics of xylanase and AE produced by fungus with the highest enzyme activities were also analysed.The results showed that 12 anaerobic fungi strains belonged to Piromyces,named Piromyces sp.CN1~ Piromyces sp.CN12,respectively.The ITS gene Genbank number is KY368100~KY368111.The xylanase,CMCase and AE activities of Piromyces sp.CN6 were 1655.3,93.4 and 152.8 m U which were significantly higher than those of other fungi strains(P<0.05).The avicelase activity of Piromyces sp.CN3 was the highest among these fungi,but its activity showed no difference with that of Piromyces sp.CN6(P>0.05).There was no significant difference on β-glucanase activity produced by fungi strains(P>0.05).The xylanase activities had extremely significant positive correlation with CMCase and AE activities(P<0.01),and the xylanase activities had significant positive with avicelase activity(P<0.05).The optimum temperature of CMCase activity was 50 ℃,and the optimum pH was 5.0.The CMCase was stable at 40 ℃ and pH 5.0~8.0.K~+,Ca~(2+) and Co~(2+) had active effects on its enzyme activity,whereas Zn~(2+) ,Cu~(2+) ,Mg~(2+) ,Fe~(2+) and Mn~(2+) had inhibitory be promoted on it.The AE activity preformed optimally at 50 ℃ and pH 9.0,and it was stable at 40 ℃ and pH 5.0~10.0.The AE activity could be promoted by Mg~(2+),K~+ and Ca~(2+) and inhibited by Zn~(2+),F~(2+),Co~(2+) and Mn~(2+).Piromyces sp.CN6 isolated from rumen of Xinong Saanen dairy goats showed the highest plant cell wall degrading enzymes activity.Through enzyme activity assays and enzymological characterization,the enzyme system information of rumen fungi can be enriched and improved,which can provide basis for further application.
Anaerobic fungi(Neocallimastigomycota) are important fiber-degrading functional microorganisms in the herbivores' digestive tract.The purpose of the present study was to isolate anaerobic rumen fungi with high activities of plant cell wall degrading enzymes for practical applications to improve the utilization of roughage.Twelve anaerobic fungi strains were isolated from the ruminal contents of Xinong Saanen dairy goat(Capra hircus) using the Hungate roll tube technique.The fungi strains were identified using the morphological characteristics,nucleotide sequence analysis of internal transcribed spacer region and 28 S rDNA D1/D2 region.Meanwhile,activities of xylanase,carboxymethyl cellulase(CMCase),avicelase,acetyl esterase(AE) and β-glucanase of 12 fungi cultures were assayed,and the enzymatic characteristics of xylanase and AE produced by fungus with the highest enzyme activities were also analysed.The results showed that 12 anaerobic fungi strains belonged to Piromyces,named Piromyces sp.CN1~ Piromyces sp.CN12,respectively.The ITS gene Genbank number is KY368100~KY368111.The xylanase,CMCase and AE activities of Piromyces sp.CN6 were 1655.3,93.4 and 152.8 m U which were significantly higher than those of other fungi strains(P<0.05).The avicelase activity of Piromyces sp.CN3 was the highest among these fungi,but its activity showed no difference with that of Piromyces sp.CN6(P>0.05).There was no significant difference on β-glucanase activity produced by fungi strains(P>0.05).The xylanase activities had extremely significant positive correlation with CMCase and AE activities(P<0.01),and the xylanase activities had significant positive with avicelase activity(P<0.05).The optimum temperature of CMCase activity was 50 ℃,and the optimum pH was 5.0.The CMCase was stable at 40 ℃ and pH 5.0~8.0.K~+,Ca~(2+) and Co~(2+) had active effects on its enzyme activity,whereas Zn~(2+) ,Cu~(2+) ,Mg~(2+) ,Fe~(2+) and Mn~(2+) had inhibitory be promoted on it.The AE activity preformed optimally at 50 ℃ and pH 9.0,and it was stable at 40 ℃ and pH 5.0~10.0.The AE activity could be promoted by Mg~(2+),K~+ and Ca~(2+) and inhibited by Zn~(2+),F~(2+),Co~(2+) and Mn~(2+).Piromyces sp.CN6 isolated from rumen of Xinong Saanen dairy goats showed the highest plant cell wall degrading enzymes activity.Through enzyme activity assays and enzymological characterization,the enzyme system information of rumen fungi can be enriched and improved,which can provide basis for further application.
引文
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曹阳春,杨红建,沈博通.2010.高产纤维降解酶牦牛瘤胃厌氧真菌分离株的筛选与鉴定[J].中国农业大学学报,15(3):70-74.(Cao Y C,Yang H J,Shen B T.2010.Screening and identification of high activity cellulolytic anaerobic fungi from rumen of yak[J].Journal of China Agricultural University,3(120):571-587.)
成艳芬,朱伟云.2009.ARISA方法研究产甲烷菌共存及去除条件下瘤胃真菌多样性变化[J].微生物学报,49(4):504-511.(Cheng Y F,Zhu W Y.2009.Diversity analysis of anaerobic fungi in the co-cultures with or without methanogens by amplified ribosomal intergenic spacer analysis[J].Acta Microbiologica Sinica,49(4):504-511.)
崔蜜蜜,蒋琳莉,颜廷武.2016.基于资源密度的作物秸秆资源化利用潜力测算与市场评估[J].中国农业大学学报,21(6):117-131.(Cui M M,Jiang L L,Yan T W,et al.2017.Potential evaluation and market assessment on crop straw resource utilization based on resource density[J].Journal of China Agricultural University,21(6):117-131.)
公维丽,王禄山,张怀强.2015.植物细胞壁多糖合成酶系及真菌降解酶系[J].生物技术通报,31(4):149-165.(Gong W L,Wang L S,Zhang H Q.et al.2015.Diverse synthetases and fungi degradation enzymes for the polysaccharides of plant cell walls[J].Biotechnology Bulletin31(4):149-165.)
杨红建,岳群,曹阳春.2011.荷斯坦阉牛瘤胃液中阿魏酸酯酶和乙酰酯酶的酶学特性研究[J].中国农业大学学报,16(1):73-78.(Yang H J,Yue Q,Cao Y C.2011.Enzymological characterization of feruloyl and acetyl esterases in the rumen fluids of Holstein steers[J].Journal of China Agricultural University,16(1):73-78.)
岳群.2009.瘤胃厌氧真菌阿魏酸酯酶酶学性质及其对木质纤维素性饲料的降解功效研究[D].博士学位论文,中国农业大学,导师:杨红建,pp.48-67.(Yue Q.2009.Enzymological characteristics of feruloyl esterase of anaerobic rumen fungi and its fibrolytic potential to dergde lignocellulosic feedstuffs.[D].Thesis for Ph.D.,China Agricultural University,Supervisor:Yang H J,pp.48-67.)
Aydin S,Y?ld?r?m E,Ince O,et al.2017.Rumen anaerobic fungi create new opportunities for enhanced methane production from microalgae biomass[J].Algal Research,23:150-160.
Bauchop T.1979.Rumen anaerobic fungi of cattle and sheep[J].Applied and Environmental Microbiology,38(1):148-158.
Brunecky R,Alahuhta M,Xu Q,et al.2013.Revealing nature's cellulase diversity:The digestion mechanism of Caldicellulosiruptor bescii Cel A[J].Science,342(6165):1513-1516.
Callaghan T M,Podmirseg S M,Hohlweck D,et al.2015.Buwchfawromyces eastonii gen.nov.,sp.nov.:A new anaerobic fungus(Neocallimastigomycota)isolated from buffalo faeces[J].Myco Keys,9:11.
Cao Y C,Yang H J,Zhang D F.2013.Enzymatic characteristics of crude feruloyl and acetyl esterases of rumen fungus Neocallimastix sp.YAK11 isolated from yak(Bos grunniens)[J].Journal of Animal Physiology and Animal Nutrition,97(2):363-373.
Cao Y C,Yang H J.2011.Effect of roughage fibre content on fibrolytic activities and volatile fatty acid profiles of Neocallimastix sp.YAK11 isolated from rumen fluids of yak(Bos grunniens)[J].Animal Feed Science and Technology,170(3):284-290.
Dagar S S,Kumar S,Mudgil P,et al.2011.D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp.[J].Applied and Environmental Microbiology,77(18):6722-6725.
Dijkerman R,Vervuren M B,Hj O D C,et al.1996.Adsorption characteristics of cellulolytic enzymes from the anaerobic fungus Piromyces sp.strain E2 on microcrystalline cellulose[J].Applied&Environmental Microbiology,62(1):20.
Dore J,Stahl D A.1991.Phylogeny of anaerobic rumen Chytridiomycetes inferred from small subunit ribosomal RNA sequence comparisons[J].Canadian Journal of Botany,69(9):1964-1971.
Gordon G L,Phillips M W.1989.Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen[J].Applied and Environmental Microbiology,55(7):1703-1710.
Gruninger R J,Puniya A K,Callaghan T M,et al.2014.Anaerobic fungi(phylum Neocallimastigomycota):Advances in understanding their taxonomy,life cycle,ecology,role and biotechnological potential[J].FEMS Microbiology Ecology,90(1):1-17.
Ho Y W,Barr D J S.1995.Classification of anaerobic gut fungi from herbivores with emphasis on rumen fungi from Malaysia[J].Mycologia,87(5):655-677.
Hungate R E,Macy J.1973.The roll-tube method for cultivation of strict anaerobes[J].Bulletins from the Ecological Research Committee,(17):123-126.
Haitjema C H,Solomon K V,Henske J K,et al.2014.Anaerobic gut fungi:Advances in isolation,culture,and cellulolytic enzyme discovery for biofuel production[J].Biotechnology and Bioengineering,111(8):1471-1482.
Jiang D,Zhuang D,Fu J,et al.2012.Bioenergy potential from crop residues in China:Availability and distribution[J].Renewable&Sustainable Energy Reviews,16(3):1377-1382.
Koetschan C,Kittelmann S,Lu J,et al.2014.Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi(Neocallimastigomycota)[J].Plo S One,9(3):e91928.
Lee S M,Guan L L,Eun J S,et al.2015.The effect of anaerobic fungal inoculation on the fermentation characteristics of rice straw silages[J].Journal of Applied Microbiology,118(3):565.
Leis S,Dresch P,Peintner U,et al.2014.Finding a robust strain for biomethanation:Anaerobic fungi(Neocallimastigomycota)from the Alpine ibex(Capra ibex)and their associated methanogens[J].Anaerobe,29:34-43.
Lockhart R J,Van Dyke M I,Beadle I R,et al.2006.Molecular biological detection of anaerobic gut fungi(Neocallimastigales)from landfill sites[J].Applied and Environmental Microbiology,72(8):5659-5661.
Nkemka V N,Gilroyed B,Yanke J,et al.2015.Bioaugmentation with an anaerobic fungus in a two-stage process for biohydrogen and biogas production using corn silage and cattail[J].Bioresource Technology,185(6):79-88.
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Orpin C G.1977.The occurrence of chitin in the cell walls of the rumen organisms Neocallimastix frontalis,Piromonas communis and Sphaeromonas communis[J].Microbiology,99(1):215-218.
Procházka J,Mrázek J,?trosováL,et al.2012.Enhanced biogas yield from energy crops with rumen anaerobic fungi[J].Engineering in Life Sciences,12(3):343-351.
Rezaeian M,Beakes G W,Parker D S.2004.Methods for the isolation,culture and assessment of the status of anaerobic rumen chytrids in both in vitro and in vivo systems[J].Mycological Research,108(10):1215-1226.
Schoch C L,Seifert K A,Huhndorf S,et al.2012.Nuclear ribosomal internal transcribed spacer(ITS)region as a universal DNA barcode marker for fungi[J].Proceedings of the National Academy of Sciences of the USA,109(16):6241-6246.
Solomon K V,Henske J K,Theodorou M K,et al.2015.Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi[J].Anaerobe,38:39-46.
Solomon K V,Haitjema C H,Henske J K,et al.2016.Earlybranching gut fungi possess a large,comprehensive array of biomass-degrading enzymes[J].Science,351(6278):1192.
Tan H,Cao L.2014.Fungal diversity in sheep(Ovis aries)and cattle(Bos taurus)feces assessed by comparison of 18S,28S and ITS ribosomal regions[J].Annals of Microbiology,64(3):1423-1427.
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Wei Y Q,Yang H J,Luan Y,et al.2016b.Isolation,identification and fibrolytic characteristics of rumen fungi grown with indigenous methanogen from yaks(Bos grunniens)grazing on the Qinghai Tibetan Plateau[J].Journal of Applied Microbiology,3(120):571-587.
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Yue Q,Yang H J,Cao Y C,et al.2009.Feruloyl and acetyl esterase production of an anaerobic rumen fungus Neocallimastix sp.YQ2 effected by glucose and soluble nitrogen supplementations and its potential in the hydrolysis of fibrous feedstuffs[J].Animal Feed Science and Technology,153(3):263-277.
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曹阳春,杨红建,沈博通.2010.高产纤维降解酶牦牛瘤胃厌氧真菌分离株的筛选与鉴定[J].中国农业大学学报,15(3):70-74.(Cao Y C,Yang H J,Shen B T.2010.Screening and identification of high activity cellulolytic anaerobic fungi from rumen of yak[J].Journal of China Agricultural University,3(120):571-587.)
成艳芬,朱伟云.2009.ARISA方法研究产甲烷菌共存及去除条件下瘤胃真菌多样性变化[J].微生物学报,49(4):504-511.(Cheng Y F,Zhu W Y.2009.Diversity analysis of anaerobic fungi in the co-cultures with or without methanogens by amplified ribosomal intergenic spacer analysis[J].Acta Microbiologica Sinica,49(4):504-511.)
崔蜜蜜,蒋琳莉,颜廷武.2016.基于资源密度的作物秸秆资源化利用潜力测算与市场评估[J].中国农业大学学报,21(6):117-131.(Cui M M,Jiang L L,Yan T W,et al.2017.Potential evaluation and market assessment on crop straw resource utilization based on resource density[J].Journal of China Agricultural University,21(6):117-131.)
公维丽,王禄山,张怀强.2015.植物细胞壁多糖合成酶系及真菌降解酶系[J].生物技术通报,31(4):149-165.(Gong W L,Wang L S,Zhang H Q.et al.2015.Diverse synthetases and fungi degradation enzymes for the polysaccharides of plant cell walls[J].Biotechnology Bulletin31(4):149-165.)
杨红建,岳群,曹阳春.2011.荷斯坦阉牛瘤胃液中阿魏酸酯酶和乙酰酯酶的酶学特性研究[J].中国农业大学学报,16(1):73-78.(Yang H J,Yue Q,Cao Y C.2011.Enzymological characterization of feruloyl and acetyl esterases in the rumen fluids of Holstein steers[J].Journal of China Agricultural University,16(1):73-78.)
岳群.2009.瘤胃厌氧真菌阿魏酸酯酶酶学性质及其对木质纤维素性饲料的降解功效研究[D].博士学位论文,中国农业大学,导师:杨红建,pp.48-67.(Yue Q.2009.Enzymological characteristics of feruloyl esterase of anaerobic rumen fungi and its fibrolytic potential to dergde lignocellulosic feedstuffs.[D].Thesis for Ph.D.,China Agricultural University,Supervisor:Yang H J,pp.48-67.)
Aydin S,Y?ld?r?m E,Ince O,et al.2017.Rumen anaerobic fungi create new opportunities for enhanced methane production from microalgae biomass[J].Algal Research,23:150-160.
Bauchop T.1979.Rumen anaerobic fungi of cattle and sheep[J].Applied and Environmental Microbiology,38(1):148-158.
Brunecky R,Alahuhta M,Xu Q,et al.2013.Revealing nature's cellulase diversity:The digestion mechanism of Caldicellulosiruptor bescii Cel A[J].Science,342(6165):1513-1516.
Callaghan T M,Podmirseg S M,Hohlweck D,et al.2015.Buwchfawromyces eastonii gen.nov.,sp.nov.:A new anaerobic fungus(Neocallimastigomycota)isolated from buffalo faeces[J].Myco Keys,9:11.
Cao Y C,Yang H J,Zhang D F.2013.Enzymatic characteristics of crude feruloyl and acetyl esterases of rumen fungus Neocallimastix sp.YAK11 isolated from yak(Bos grunniens)[J].Journal of Animal Physiology and Animal Nutrition,97(2):363-373.
Cao Y C,Yang H J.2011.Effect of roughage fibre content on fibrolytic activities and volatile fatty acid profiles of Neocallimastix sp.YAK11 isolated from rumen fluids of yak(Bos grunniens)[J].Animal Feed Science and Technology,170(3):284-290.
Dagar S S,Kumar S,Mudgil P,et al.2011.D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp.[J].Applied and Environmental Microbiology,77(18):6722-6725.
Dijkerman R,Vervuren M B,Hj O D C,et al.1996.Adsorption characteristics of cellulolytic enzymes from the anaerobic fungus Piromyces sp.strain E2 on microcrystalline cellulose[J].Applied&Environmental Microbiology,62(1):20.
Dore J,Stahl D A.1991.Phylogeny of anaerobic rumen Chytridiomycetes inferred from small subunit ribosomal RNA sequence comparisons[J].Canadian Journal of Botany,69(9):1964-1971.
Gordon G L,Phillips M W.1989.Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen[J].Applied and Environmental Microbiology,55(7):1703-1710.
Gruninger R J,Puniya A K,Callaghan T M,et al.2014.Anaerobic fungi(phylum Neocallimastigomycota):Advances in understanding their taxonomy,life cycle,ecology,role and biotechnological potential[J].FEMS Microbiology Ecology,90(1):1-17.
Ho Y W,Barr D J S.1995.Classification of anaerobic gut fungi from herbivores with emphasis on rumen fungi from Malaysia[J].Mycologia,87(5):655-677.
Hungate R E,Macy J.1973.The roll-tube method for cultivation of strict anaerobes[J].Bulletins from the Ecological Research Committee,(17):123-126.
Haitjema C H,Solomon K V,Henske J K,et al.2014.Anaerobic gut fungi:Advances in isolation,culture,and cellulolytic enzyme discovery for biofuel production[J].Biotechnology and Bioengineering,111(8):1471-1482.
Jiang D,Zhuang D,Fu J,et al.2012.Bioenergy potential from crop residues in China:Availability and distribution[J].Renewable&Sustainable Energy Reviews,16(3):1377-1382.
Koetschan C,Kittelmann S,Lu J,et al.2014.Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi(Neocallimastigomycota)[J].Plo S One,9(3):e91928.
Lee S M,Guan L L,Eun J S,et al.2015.The effect of anaerobic fungal inoculation on the fermentation characteristics of rice straw silages[J].Journal of Applied Microbiology,118(3):565.
Leis S,Dresch P,Peintner U,et al.2014.Finding a robust strain for biomethanation:Anaerobic fungi(Neocallimastigomycota)from the Alpine ibex(Capra ibex)and their associated methanogens[J].Anaerobe,29:34-43.
Lockhart R J,Van Dyke M I,Beadle I R,et al.2006.Molecular biological detection of anaerobic gut fungi(Neocallimastigales)from landfill sites[J].Applied and Environmental Microbiology,72(8):5659-5661.
Nkemka V N,Gilroyed B,Yanke J,et al.2015.Bioaugmentation with an anaerobic fungus in a two-stage process for biohydrogen and biogas production using corn silage and cattail[J].Bioresource Technology,185(6):79-88.
Orpin C G.1975.Studies on the rumen flagellate Neocallimastix frontalis.[J].Journal of General Microbiology,91(2):249-62.
Orpin C G.1977.The occurrence of chitin in the cell walls of the rumen organisms Neocallimastix frontalis,Piromonas communis and Sphaeromonas communis[J].Microbiology,99(1):215-218.
Procházka J,Mrázek J,?trosováL,et al.2012.Enhanced biogas yield from energy crops with rumen anaerobic fungi[J].Engineering in Life Sciences,12(3):343-351.
Rezaeian M,Beakes G W,Parker D S.2004.Methods for the isolation,culture and assessment of the status of anaerobic rumen chytrids in both in vitro and in vivo systems[J].Mycological Research,108(10):1215-1226.
Schoch C L,Seifert K A,Huhndorf S,et al.2012.Nuclear ribosomal internal transcribed spacer(ITS)region as a universal DNA barcode marker for fungi[J].Proceedings of the National Academy of Sciences of the USA,109(16):6241-6246.
Solomon K V,Henske J K,Theodorou M K,et al.2015.Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi[J].Anaerobe,38:39-46.
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