秸秆发酵制取生化腐殖酸过程中的理化特性及微生物学特性研究
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摘要
我国的秸秆资源十分丰富,常规方法是将秸秆发酵作为有机肥,利用秸秆发酵制取生化腐殖酸,既可减少环境污染,又可得到高附加值的生化腐殖酸产品,从而提高秸秆资源化利用率。本研究探讨了稻草段+尿素、稻草粉+尿素、稻草段+牛粪、稻草粉+牛粪四种处理在发酵过程中的理化性质、秸秆组分化、腐殖质形成和微生物类群特征,取得了以下结果。
发酵过程中,4种处理的理化性质发生了系列变化。堆料颜色逐渐由黄色或黄棕色转化成棕黑色；发酵最高温度达74℃,高温阶段(≥55℃)持续了25d；发酵料pH呈现出先降低再升高的趋势,发酵结束后物料pH为7.92-8.08。
各处理中温和高温微生物数量细菌>放线菌>真菌。发酵结束后,中温细菌数量介于1.10×109～1.32×109cfu·g-1,中温放线菌数量介于1.10×106～1.22×106cfu·g-1;中温真菌数量介于4.33×103～5.64×103cfu·g-1之间。各处理的高温细菌数量介于2.99×108～4.6×108cfu·g-1,高温放线菌数量介于7.81×105～8.21×105cfu·g-1。而发酵初期,物料中未能检测到高温真菌；至第30d,各处理高温真菌数量达到最高,介于1.25×104～1.52×104cfu·g-1,至发酵结束(60d),高温真菌数量介于2.47×103～5.00×103cfu·g-1。
不同处理的纤维素、半纤维素和木质素含量随发酵时间延长而降低。其中,纤维素含量由初始值29.61%-31.99%降至1.24～1.96%,半纤维素含量从初期的24.16%～27.74%降至后期的5.97%-6.42%。木质素含量略有减少,由初始的25.28%～25.93%降低至24.76%-25.11%。
水溶性组分含量表现为先增加后降低,由初期4.3%～9.8%增加至17.5%-18.1%,随后降至9.8%-10.9%。发酵料的C/N由初始的31.7～32.8下降至13.9-14.7。腐殖质含量由初始的55.6g·kg-1～81.3g·kg-1增加到117.3g·kg-1～125.8g·kg-1。富里酸含量由初期的38.2g·kg-1～41.3g·kg-1降低至29.9g·kg-1～33.4g·kg-1,而胡敏酸含量由12.7g.kg-1～34.4g·kg-1增加至81.3g·kg-1～85.8g·kg-1,发酵料的H/F由0.31-0.83增加到2.49-2.70。
对4个处理的腐熟期样品细菌群落进行了DGGE-PCR分析,结果表明,处理Ⅰ和Ⅱ的条带数为22,处理Ⅲ和Ⅳ的条带数为20；对DGGE图谱进行多样性指数分析,结果显示,处理Ⅰ和Ⅱ细菌丰度和多样性指数高于处理Ⅲ和Ⅳ。对DGGE条带克隆测序,结果表明,样品中的细菌主要有苍白杆菌属(Ochrobactrum sp.)、不动杆菌属(Acinetobacter sp.).假单胞菌属(Pseudomonas sp.).未可培养屈挠杆菌科(UnculturedFlexibacteraceae bacterium),其余为未培养菌(Uncultured bacterium)。这些菌属于γ变形菌门(γ-proteobacteria),α变形菌门(α-proteobacteria)和拟杆菌门(Bacteroidetes)。
采用454焦磷酸测序对添加尿素和牛粪的两种秸秆发酵样品(处理Ⅰ对应S1；处理Ⅳ对应S2)的细菌类群进行了分析,结果表明,在门、纲、目、科、属的分类水平上,两个样品的细菌群落结构和优势菌群不同。主要集中在变形杆菌门(Proteobacteria)和拟杆菌门(Bacteroidetes);S1和S2的变形杆菌门类群比例分别为31.62%和35.81%,拟杆菌门比例分别占27.41%和35.29%。此外,S1中厚壁菌门(Firmicutes)占18.15%,unclassified Bacteria占8.80%,柔膜菌门(Tenericutes)占7.28%,其他门占6.74%；而在S2中,厚壁菌门(Firmicutes)占3.43%,unclassified Bacteria数量占9.63%,浮霉菌门(Planctomycetes)占7.06%,其他门的细菌占8.78%。
在属的分类水平,在≥1%比例上,S1中有17个属,S2有13个属。样品S1主要有假单胞菌属(Pseudomonas)、无胆甾原体属(Acholeplasma)、Alkaliflexus、Sulfurimonas、 Petrimonas、Serprns、Tissierella、Sedimentibacter、密螺旋体属(Treponema)、纤维杆菌属(Fibrobacter)、Sphingopyxis、Saccharofermentans、梭菌属(Clostridium)、螺旋体属(Spirochaeta)、脱硫单胞菌属(Desulfuromonas)、Luteimonas、Pusillimonas,S2中主要有Acholeplasma、Alkaliflexus、Sphingopyxis、Devosia、Methylobacillus、Steroidobacter、 Aequorivita、Arenibacter、Rhodopirellula、Vitellibacter、Lysobacter、Crocinitomix、 Blastopirellua。在≥1%比例上,仅Acholeplasma、 Alkaliflexus、Sphingopyxis三个属为两个样品共有的类群。在两处理样品中,top10的OTUs中没有共同的单元,说明发酵料添加牛粪和尿素对细菌类群影响大,导致优势类群完全不同。
本研究结果表明,应用不同外加氮源,秸秆发酵形成生化腐殖酸过程中,尽管发酵温度等参数、秸秆降解率、腐解物中的腐殖质含量及其组分不存在显著差异,但微生物类群显著不同,且添加尿素的秸秆发酵物料细菌类群多样性高于添加牛粪的处理,说明在不同环境中,参与腐殖酸形成与转化的微生物群落呈现多样化。
Straw is one of the richest renewable agricultural resource in China, and are usually used as organic fertilizer after fermentation. In the recent years, the straw has been used to produce the biochemical humic acid by fermentation, and which can reduce environmental pollution, and improve the straw resource utilization, and product had high added-value. In this paper, four treatments, i.e. straw section+urea (treatment I), straw powder+urea (treatment II), straw section+cow dung (treatment III), straw powder+cow dung (treatment IV), were used to ferment and produce biochemical humic acid, and the physical and chemical properties, degradation of straw component, humus formation and the microbial community was studied during the fermentation process, and the results were as follows.
During the fermentation, the physical and chemical property of4treatments was studied. The results showed that the color of compost material changed from yellow or yellow brown into dark brown gradually; The maximum fermentation temperature reached to74℃, and the high temperature stage (≥55℃) was sustained for25d; The pH value of fermentation material decreased at the beginning of fermentation, and then followed by rising, and the final pH value was7.92-8.08at the end of fermentation.
The mesophilic and thermophilic microbial quantities in defferent compost materials were as the seriation:bacteria＞actinomycete＞fungi. At late fermentation stage, the quantity of mesophilic bacteria was between1.10×109～1.32×109cfu·g-1; and the quantity of mesophilic actinomycetes was between1.10×106～1.22×106cfu·g-1; the amount of mesophilic fungi was4.33×103～5.64×103cfu·g-1.The amount of thermophilic bacteria was between2.99×108～4.6×108cfu·g-1; the amount of thermophilic actinomycete was7.81×105～8.21×105cfu·g-1. At the early fermentation stage, there was no fungus in the compost straw detected. On the30th day, fungi quantity reached the highest, and was1.25×104～1.52×104cfu·g-1, and the quantity was2.47×103～5.00×103cfu·g-1when fermentation finished.
The cellulose, hemicellulose, and lignin content of different treatment materials decreased with the extension of fermentation time. The cellulose content decreased from29.61%～31.99%at beginning to1.24～1.96%in the end; and the hemicellulose content varied from24.16%～27.74%at early to5.97%～6.42%in the end; the lignin content reduced slightly, and changed from25.28%～25.93%at early to24.76%～25.11%.
Water-soluble component content of compost material increased at first and then decreased, and the value increased from4.3%～9.8%to17.5%～18.1%, and decreased to9.8%～10.9%. The C/N ratio of compost matter decreased from31.7～32.8to13.9～14.7; and humus content increased from55.6g·kg-1～81.3g·kg-1to117.3g·kg-1～125.8g·kg-1; humic acid content increased from12.7g·kg-1～34.4g·kg-1to81.3g·kg-1～85.8g·kg-1; however, the fulvic acid content decreased from38.2g·kg-1～41.3g·kg-1to29.9g·kg-1～33.4g·kg-1; the H/F ratio increased from0.31～0.83to2.49～2.70.
The DGGE bands were cloned and sequenced; the results revealed that the dominant bacteria were Ochrobactrum sp., Acinetobacter sp., Pseudomonas sp., and uncultured Flexibacteraceae bacterium; the others belonged to uncultured bacterium. These bacteria mainly belong to γ-proteobacteria, α-proteobacteria and bacteroidetes at the classification level of phylum.
In late fermentation stage, the microbial community in compost matter of the straw section+urea (treatment I corresponds to S1) and straw section+cow-dungs (treatment Ⅲ corresponds to S2) was analyzed using the454pyrosequencing method. The results suggested that the bacterial groups were very different at the classification level of phylum, class, order, and family and genus. At the level of phylum, the predominant bacteria groups of the two samples mainly belonged to Proteobacteria and Bacteroidetes. The ratio of Proteobacteria in sample S1and S2was31.62%and35.81%, and the ratio of Bacteroidetes in S1and S2was27.41%and35.29%. Furthermore, the proportion of bacteria belong to Firmicutes, unclassified Bacteria, Tenericutes in S1was18.15%,8.80%and7.28%; but the proportion of bacteria belong to Firmicutes, unclassified Bacteria and Planctomycetes in S2was3.43%,9.63%and7.06%.
Analysis of454pyrosequencing suggested that there were no common bacteria groups existed in the top10OTUs in S1and S2, which revealed that, adding cow manure and urea into straw, the bacteria groups involved in the fermentation were influenced heavily, and the dominant bacteria groups were completely different.
The results indicated that, when applying the different nitrogen source (cow dung and urea) in the straw, though the fermentation parameters such as fermentation temperature etc., degradation rate of straw, decomposing and the humus content and humic acid components had not significant differences, the microbial groups were quite different, and the bacteria diversity index of fermentation materials adding urea was higher than that adding cow manure during the processing of fermentation. The results revealed that the microbial communities participating in humic acid formation and transformation were diverse in different environments.
发酵过程中,4种处理的理化性质发生了系列变化。堆料颜色逐渐由黄色或黄棕色转化成棕黑色；发酵最高温度达74℃,高温阶段(≥55℃)持续了25d；发酵料pH呈现出先降低再升高的趋势,发酵结束后物料pH为7.92-8.08。
各处理中温和高温微生物数量细菌>放线菌>真菌。发酵结束后,中温细菌数量介于1.10×109～1.32×109cfu·g-1,中温放线菌数量介于1.10×106～1.22×106cfu·g-1;中温真菌数量介于4.33×103～5.64×103cfu·g-1之间。各处理的高温细菌数量介于2.99×108～4.6×108cfu·g-1,高温放线菌数量介于7.81×105～8.21×105cfu·g-1。而发酵初期,物料中未能检测到高温真菌；至第30d,各处理高温真菌数量达到最高,介于1.25×104～1.52×104cfu·g-1,至发酵结束(60d),高温真菌数量介于2.47×103～5.00×103cfu·g-1。
不同处理的纤维素、半纤维素和木质素含量随发酵时间延长而降低。其中,纤维素含量由初始值29.61%-31.99%降至1.24～1.96%,半纤维素含量从初期的24.16%～27.74%降至后期的5.97%-6.42%。木质素含量略有减少,由初始的25.28%～25.93%降低至24.76%-25.11%。
水溶性组分含量表现为先增加后降低,由初期4.3%～9.8%增加至17.5%-18.1%,随后降至9.8%-10.9%。发酵料的C/N由初始的31.7～32.8下降至13.9-14.7。腐殖质含量由初始的55.6g·kg-1～81.3g·kg-1增加到117.3g·kg-1～125.8g·kg-1。富里酸含量由初期的38.2g·kg-1～41.3g·kg-1降低至29.9g·kg-1～33.4g·kg-1,而胡敏酸含量由12.7g.kg-1～34.4g·kg-1增加至81.3g·kg-1～85.8g·kg-1,发酵料的H/F由0.31-0.83增加到2.49-2.70。
对4个处理的腐熟期样品细菌群落进行了DGGE-PCR分析,结果表明,处理Ⅰ和Ⅱ的条带数为22,处理Ⅲ和Ⅳ的条带数为20；对DGGE图谱进行多样性指数分析,结果显示,处理Ⅰ和Ⅱ细菌丰度和多样性指数高于处理Ⅲ和Ⅳ。对DGGE条带克隆测序,结果表明,样品中的细菌主要有苍白杆菌属(Ochrobactrum sp.)、不动杆菌属(Acinetobacter sp.).假单胞菌属(Pseudomonas sp.).未可培养屈挠杆菌科(UnculturedFlexibacteraceae bacterium),其余为未培养菌(Uncultured bacterium)。这些菌属于γ变形菌门(γ-proteobacteria),α变形菌门(α-proteobacteria)和拟杆菌门(Bacteroidetes)。
采用454焦磷酸测序对添加尿素和牛粪的两种秸秆发酵样品(处理Ⅰ对应S1；处理Ⅳ对应S2)的细菌类群进行了分析,结果表明,在门、纲、目、科、属的分类水平上,两个样品的细菌群落结构和优势菌群不同。主要集中在变形杆菌门(Proteobacteria)和拟杆菌门(Bacteroidetes);S1和S2的变形杆菌门类群比例分别为31.62%和35.81%,拟杆菌门比例分别占27.41%和35.29%。此外,S1中厚壁菌门(Firmicutes)占18.15%,unclassified Bacteria占8.80%,柔膜菌门(Tenericutes)占7.28%,其他门占6.74%；而在S2中,厚壁菌门(Firmicutes)占3.43%,unclassified Bacteria数量占9.63%,浮霉菌门(Planctomycetes)占7.06%,其他门的细菌占8.78%。
在属的分类水平,在≥1%比例上,S1中有17个属,S2有13个属。样品S1主要有假单胞菌属(Pseudomonas)、无胆甾原体属(Acholeplasma)、Alkaliflexus、Sulfurimonas、 Petrimonas、Serprns、Tissierella、Sedimentibacter、密螺旋体属(Treponema)、纤维杆菌属(Fibrobacter)、Sphingopyxis、Saccharofermentans、梭菌属(Clostridium)、螺旋体属(Spirochaeta)、脱硫单胞菌属(Desulfuromonas)、Luteimonas、Pusillimonas,S2中主要有Acholeplasma、Alkaliflexus、Sphingopyxis、Devosia、Methylobacillus、Steroidobacter、 Aequorivita、Arenibacter、Rhodopirellula、Vitellibacter、Lysobacter、Crocinitomix、 Blastopirellua。在≥1%比例上,仅Acholeplasma、 Alkaliflexus、Sphingopyxis三个属为两个样品共有的类群。在两处理样品中,top10的OTUs中没有共同的单元,说明发酵料添加牛粪和尿素对细菌类群影响大,导致优势类群完全不同。
本研究结果表明,应用不同外加氮源,秸秆发酵形成生化腐殖酸过程中,尽管发酵温度等参数、秸秆降解率、腐解物中的腐殖质含量及其组分不存在显著差异,但微生物类群显著不同,且添加尿素的秸秆发酵物料细菌类群多样性高于添加牛粪的处理,说明在不同环境中,参与腐殖酸形成与转化的微生物群落呈现多样化。
Straw is one of the richest renewable agricultural resource in China, and are usually used as organic fertilizer after fermentation. In the recent years, the straw has been used to produce the biochemical humic acid by fermentation, and which can reduce environmental pollution, and improve the straw resource utilization, and product had high added-value. In this paper, four treatments, i.e. straw section+urea (treatment I), straw powder+urea (treatment II), straw section+cow dung (treatment III), straw powder+cow dung (treatment IV), were used to ferment and produce biochemical humic acid, and the physical and chemical properties, degradation of straw component, humus formation and the microbial community was studied during the fermentation process, and the results were as follows.
During the fermentation, the physical and chemical property of4treatments was studied. The results showed that the color of compost material changed from yellow or yellow brown into dark brown gradually; The maximum fermentation temperature reached to74℃, and the high temperature stage (≥55℃) was sustained for25d; The pH value of fermentation material decreased at the beginning of fermentation, and then followed by rising, and the final pH value was7.92-8.08at the end of fermentation.
The mesophilic and thermophilic microbial quantities in defferent compost materials were as the seriation:bacteria＞actinomycete＞fungi. At late fermentation stage, the quantity of mesophilic bacteria was between1.10×109～1.32×109cfu·g-1; and the quantity of mesophilic actinomycetes was between1.10×106～1.22×106cfu·g-1; the amount of mesophilic fungi was4.33×103～5.64×103cfu·g-1.The amount of thermophilic bacteria was between2.99×108～4.6×108cfu·g-1; the amount of thermophilic actinomycete was7.81×105～8.21×105cfu·g-1. At the early fermentation stage, there was no fungus in the compost straw detected. On the30th day, fungi quantity reached the highest, and was1.25×104～1.52×104cfu·g-1, and the quantity was2.47×103～5.00×103cfu·g-1when fermentation finished.
The cellulose, hemicellulose, and lignin content of different treatment materials decreased with the extension of fermentation time. The cellulose content decreased from29.61%～31.99%at beginning to1.24～1.96%in the end; and the hemicellulose content varied from24.16%～27.74%at early to5.97%～6.42%in the end; the lignin content reduced slightly, and changed from25.28%～25.93%at early to24.76%～25.11%.
Water-soluble component content of compost material increased at first and then decreased, and the value increased from4.3%～9.8%to17.5%～18.1%, and decreased to9.8%～10.9%. The C/N ratio of compost matter decreased from31.7～32.8to13.9～14.7; and humus content increased from55.6g·kg-1～81.3g·kg-1to117.3g·kg-1～125.8g·kg-1; humic acid content increased from12.7g·kg-1～34.4g·kg-1to81.3g·kg-1～85.8g·kg-1; however, the fulvic acid content decreased from38.2g·kg-1～41.3g·kg-1to29.9g·kg-1～33.4g·kg-1; the H/F ratio increased from0.31～0.83to2.49～2.70.
The DGGE bands were cloned and sequenced; the results revealed that the dominant bacteria were Ochrobactrum sp., Acinetobacter sp., Pseudomonas sp., and uncultured Flexibacteraceae bacterium; the others belonged to uncultured bacterium. These bacteria mainly belong to γ-proteobacteria, α-proteobacteria and bacteroidetes at the classification level of phylum.
In late fermentation stage, the microbial community in compost matter of the straw section+urea (treatment I corresponds to S1) and straw section+cow-dungs (treatment Ⅲ corresponds to S2) was analyzed using the454pyrosequencing method. The results suggested that the bacterial groups were very different at the classification level of phylum, class, order, and family and genus. At the level of phylum, the predominant bacteria groups of the two samples mainly belonged to Proteobacteria and Bacteroidetes. The ratio of Proteobacteria in sample S1and S2was31.62%and35.81%, and the ratio of Bacteroidetes in S1and S2was27.41%and35.29%. Furthermore, the proportion of bacteria belong to Firmicutes, unclassified Bacteria, Tenericutes in S1was18.15%,8.80%and7.28%; but the proportion of bacteria belong to Firmicutes, unclassified Bacteria and Planctomycetes in S2was3.43%,9.63%and7.06%.
Analysis of454pyrosequencing suggested that there were no common bacteria groups existed in the top10OTUs in S1and S2, which revealed that, adding cow manure and urea into straw, the bacteria groups involved in the fermentation were influenced heavily, and the dominant bacteria groups were completely different.
The results indicated that, when applying the different nitrogen source (cow dung and urea) in the straw, though the fermentation parameters such as fermentation temperature etc., degradation rate of straw, decomposing and the humus content and humic acid components had not significant differences, the microbial groups were quite different, and the bacteria diversity index of fermentation materials adding urea was higher than that adding cow manure during the processing of fermentation. The results revealed that the microbial communities participating in humic acid formation and transformation were diverse in different environments.
引文
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