CN109613136A - 利用碳稳定同位素分馏比率判断厌氧消化过程的方法 - Google Patents

利用碳稳定同位素分馏比率判断厌氧消化过程的方法 Download PDF

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CN109613136A
CN109613136A CN201811586254.0A CN201811586254A CN109613136A CN 109613136 A CN109613136 A CN 109613136A CN 201811586254 A CN201811586254 A CN 201811586254A CN 109613136 A CN109613136 A CN 109613136A
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吕作鹏
储云博
袁博
梁家卓
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Abstract

本发明涉及一种利用甲烷中碳稳定同位素分馏比率判断厌氧消化过程的方法,包括以下几个步骤:第一步、沼气的采集;第二步、δ13/12CH4和δ13/12CO2测定;第三步、计算δ13C值(‰);第四步、δ13/12CH4和δ13/12CO2的比率α值计算。本发明针对厌氧消化过程产甲烷过程监控难这一难题,结合前期大量的分子生物数据的支持,找到微生物特群落组成变化别是产甲烷古菌群落波动与δ值和α值的统计关系,得出利用δ值和α值快速反映产甲烷古菌代谢的方法。该方法突破了传统的分子生物技术的耗时、费力和花费高以及应用潜力若等技术障碍,实现快速反映厌氧消化过程中产甲烷代谢途径。

Description

利用碳稳定同位素分馏比率判断厌氧消化过程的方法
技术领域
本发明涉及一种利用甲烷中碳稳定同位素分馏比率判断厌氧消化过程的方法,更具体的是涉及一种利用沼气中甲烷稳定性碳同位素分馏比率判断厌氧消化产甲烷的过程,属于厌氧消化过程监测技术领域。
背景技术
厌氧消化技术利用生物质资源产生沼气是实现清洁利用的有效方法。厌氧消化过程中已知各类型监测参数不能直接地反映出发酵微生物动态波动,群落结构组成、功能基因转录水平和多样性难以对厌氧消化过程进行深度诊断和优化。
产甲烷古菌代谢作为整个厌氧消化过程的末端,对过程反应敏感从而牵动其它代谢途径波动而影响整个厌氧消化过程。产甲烷古菌代谢需紧密依靠产乙酸代谢和产氢产乙酸代谢途径提供乙酸和氢以及代谢所需能量,而这些代谢不平衡是导致过程不稳定的重要因素。产氢产乙酸菌依赖产甲烷古菌来转移氢,从而将两类微生物的活性联系起来,并控制整个反应过程。协同乙酸氧化菌群在厌氧消化微生物中比重虽小,却是连接乙酸型产甲烷代谢和嗜氢型产甲烷代谢途径最为重要的桥梁。
产甲烷过程引发甲烷中稳定性碳同位素迁移转化(δ13/12CH4)发生相应变化,被广泛应用到环境和厌氧消化系统评价中。但是上述评价体系忽略了其他代谢途径对δ13/12CH4影响导致监测不准确。因此,明确δ13/12CH4和厌氧微生物动态波动的响应关系,揭示关键功能基因转录水平与δ13/12CH4波动的统计关系是深度诊断产甲烷过程的关键,有助于优化厌氧消化过程,提高有机废弃物利用率。
发明内容
本发明所要解决的技术问题是,克服现有技术的缺点,提供一种快速监控产甲烷代谢过程的方法。
为了解决以上技术问题,本发明提供一种利用甲烷中碳稳定同位素分馏比率判断厌氧消化过程的方法,其特征在于包括以下几个步骤:
第一步、沼气的采集
第1.1步骤、在发酵罐出气口连接软管,软管有止气阀,软管内径和20ml注射器口大小一致,有助于密闭和采集气体;
第1.2步骤、用20ml注射器抽取20ml沼气并打入20ml真空瓶中;
第二步、δ13/12CH4和δ13/12CO2测定
将50uL的上述沼气用气密针打入气相色谱,载气为高纯He 99.999%,柱流速1.5mL/min;进样口温度120℃,不分流进样;燃烧炉温度为960℃,还原炉温度640℃;IRMS离子化能量80eV,发射电流1.5mA,加速电压3kV;真空度为2.0×10-8kPa;
第三步、计算δ13C值(‰)
δ13C-CH4和δ13C-CO2值以PDB国际标准作为参考标准,其值按以下公式计算:
式中,R(13C/12CVPDB)为国际标准物VPDB的碳同位素丰度比值;公式里13C/12Csample代表沼气中测量得到的δ13C-CH4和δ13C-CO2值,13C/12CVPDB代表国际标准下δ13C-CH4和δ13C-CO2的值,其分析精度为±0.2‰;
上述δ13/12C的值可通过气相同位素色谱质谱联用仪进行分析得出,数值得出δ13C-CH4和δ13C-CO2的值,通过计算得到α值用于判断产甲烷过程代谢波动。
第四步、δ13/12CH4和δ13/12CO2的比率α值计算
其计算公式为:αA,B≈(δA+103)/(δB+103),其中δA为δ13/12CH4,δB为δ13/12CO2
若α>1.065,则产甲烷过程为嗜氢型产甲烷代谢途径;若α<1.025则产甲烷过程为乙酸型产甲烷代谢途径;若1.025≤α≤1.065,则甲烷来自上述两个途径。
本发明进一步限定的技术方案是:所述δ13/12CH4为CH413C和12C分馏比率。
进一步的,所述的δ13/12CO2为CO213C和12C分馏比率。
本发明的有益效果是:本发明针对厌氧消化过程产甲烷过程监控难这一难题,结合前期大量的分子生物数据的支持,找到微生物特群落组成变化别是产甲烷古菌群落波动与δ值和α值的统计关系,得出利用δ值和α值快速反映产甲烷古菌代谢的方法。该方法突破了传统的分子生物技术的耗时、费力和花费高以及应用潜力若等技术障碍,实现快速反映厌氧消化过程中产甲烷代谢途径。厌氧消化其他运行参数如pH、沼气产量、甲烷产量、有机酸浓度等是厌氧发酵微生物代谢累积的数值,有一定的迟滞性不能实时反映厌氧消化微生物的动态变化。而沼气中的δ值和计算得到的α值是产甲烷古菌直接代谢得到的,因此可以直接地反映产甲烷古菌代谢过程。本发明可以直接快速在线的反映出厌氧消化过程中产甲烷过程,以此评价厌氧消化过程的好坏,有很大的应用潜力。
附图说明
图1为同位素质谱仪原理模式图。
图2为δ13/12C值在产甲烷过程的差异。
图3为δ13/12C值预测产甲烷古菌群落组成的应用。
图4为δ13/12C印证尿素和氨氮抑制类似对产甲烷过程具有显著的抑制作用。
图5产甲烷潜力实验(BMP)设计思路。
具体实施方式
下面结合附图和具体实施方式对本发明做进一步的说明。
实施例1
实施案例1
本实施例提供一种预测沼气中δ13/12C厌氧消化过程中填料对产甲烷古菌代谢途径的影响,同位素质谱仪原理模式图如图1所示,乙酸CH3COOH中CH3+在乙酸型产甲烷古菌的利用下产生CH4,-COOH在乙酸型产甲烷古菌代谢下产生CO2。自然界中,CH3+中12C和13C占比相对固定,99.9%12C和0.01%13C,在乙酸型产甲烷古菌利用下,12C和13C代谢到CH4中,CH4通过气相色谱转变为CO2,12CO213CO2分子量分别为44和45,CO2分子在离子源的驱动下通过高压磁场实现12CO213CO2分馏,分馏比率通过信号放大和接收器反馈到计算机中,通过不同的色谱峰进行输出。
本实施例是利用沼气中甲烷δ13/12CH4变化趋势,判断厌氧消化产甲烷过程;通过计算分析δ13/12CH4,明晰产甲烷两个过程乙酸型产甲烷代谢和嗜氢型产甲烷代谢途径对甲烷产量的贡献,实现精准调控甲烷产量。另外,通过分析δ13/12CH4和δ13/12CO2的比率α值,判断主导产甲烷代谢的代谢途径,实现产甲烷过程的监控。具体方法如下:
第一步、沼气的采集
第1.1步骤、在发酵罐出气口连接软管,软管有止气阀,软管内径和20ml注射器口大小一致,有助于密闭和采集气体;
第1.2步骤、用20ml注射器抽取20ml沼气并打入20ml真空瓶中;
第二步、δ13/12CH4和δ13/12CO2测定
将50uL的上述沼气用气密针打入气相色谱,载气为高纯He 99.999%,柱流速1.5mL/min;进样口温度120℃,不分流进样;燃烧炉温度为960℃,还原炉温度640℃;IRMS离子化能量80eV,发射电流1.5mA,加速电压3kV;真空度为2.0×10-8kPa;
第三步、计算δ13C值(‰)
其计算公式为:δ13C=[(13C/12C)样品-(13C/12C)标准品]/(13C/12C)标准品×103(‰),其中碳稳定性同位素标准品代表国际稳定性同位素计量标准,碳的同位素为Peedee Belemnite。
第四步、δ13/12CH4和δ13/12CO2的比率α值计算
其计算公式为:αA,B≈(δA+103)/(δB+103),其中δA为δ13/12CH4,δB为δ13/12CO2
若α>1.065,则产甲烷过程为嗜氢型产甲烷代谢途径;若α<1.025则产甲烷过程为乙酸型产甲烷代谢途径;若1.025≤α≤1.065,则甲烷来自上述两个途径。
填料对厌氧消化产沼气的影响很大,这与填料前后产甲烷微生物群落组成变化有关系,利用传统的分子生物技术如末端限制性片段长度多态分析(T-RFLP)及高通量测序分析出产甲烷古菌群落的波动通常需要从发酵底物中①提取DNA或者RNA;②产甲烷古菌功能引物(mcrA)PCR扩增;③PCR产物纯化;④上机测序,高通量测序或者T-RFLP;⑤数据分析。通常完成上述步骤需要4天,而沼气中碳稳定同位素分馏比率δ13/12C的测定只需要取气体样品然后在同位素质谱仪上面进行操作,一个样品的时间通常为10分钟,因此极大的缩短了时间并,具有很大的应用潜力。图3清楚的表明δ13/12CH4趋势与填料后Methanosarcina比重增大是显著相关,p<0.01。
本实施例的方法中的δ13/12CH4为CH413C和12C分馏比率,适合判断产甲烷代谢过程。
本实施例的方法中的δ13/12CO2为CO213C和12C分馏比率,适合判断嗜氢型产甲烷代谢途径。
图2为δ13/12C值在产甲烷过程的代谢途径的差异。其中乙酸型产甲烷代谢途径为CH3COOH→CH4+CO2。嗜氢型产甲烷的代谢途径为4H2+CO2→CH4+H2O。
图3所示,填料前嗜氢型产甲烷代谢途径是产甲烷主要途径,填料之后三小时乙酸型产甲烷代谢Methanosaeta相对丰度逐渐增加,说明填料后乙酸型产甲烷代谢对甲烷产量的贡献逐渐增加,而乙酸型产甲烷代谢比重的增加伴随着沼气组分中甲烷比例的增加。
实施案例2
本实施例的评估方法和实施例1基本类似,具体如图5所示,本实施例是通过沼气潜力实验评估物料厌氧消化过程重要的试验方法。本实施例是利用δ13/12C来评估该方法,准确预测纤维素厌氧消化过程中尿素与氨氮一样对于参与产甲烷过程的微生物有抑制效应,如图4中图4a~4d所示。
根据图4中δ13/12CH4的趋势,我们判断出:与阴性和阳性对照比较,在尿素和纤维素以及氨氮与纤维素厌氧消化过程中,沼气中δ13/12C的剧烈降低,δ13/12C的降低引起了α值的降低,说明嗜氢型产甲烷代谢途径主导这两个系统,乙酸型产甲烷代谢产生的δ13/12CH4值高于嗜氢型产甲烷代谢途径产生的δ13/12CH4值。据此判断,尿素与氨氮都对纤维素厌氧消化有抑制作用。
除上述实施例外,本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案,均落在本发明要求的保护范围。

Claims (3)

1.利用碳稳定同位素分馏比率判断厌氧消化过程的方法,其特征在于包括以下几个步骤:
第一步、沼气的采集
第1.1步骤、在发酵罐出气口连接软管,软管有止气阀,软管内径和20ml注射器口大小一致,有助于密闭和采集气体;
第1.2步骤、用20ml注射器抽取20ml沼气并打入20ml真空瓶中;
第二步、δ13/12CH4和δ13/12CO2测定
将50uL的上述沼气用气密针打入气相色谱,载气为高纯He 99.999%,柱流速1.5mL/min;进样口温度120℃,不分流进样;燃烧炉温度为960℃,还原炉温度640℃;IRMS离子化能量80eV,发射电流1.5mA,加速电压3kV;真空度为2.0×10-8kPa;
第三步、计算δ13C值(‰)
δ13C-CH4和δ13C-CO2值以PDB国际标准作为参考标准,其值按以下公式计算:
式中,R(13C/12CVPDB)为国际标准物VPDB的碳同位素丰度比值;公式里13C/12Csample代表沼气中测量得到的δ13C-CH4和δ13C-CO2值,13C/12CVPDB代表国际标准下δ13C-CH4和δ13C-CO2的值,其分析精度为±0.2‰;
第四步、δ13/12CH4和δ13/12CO2的比率α值计算
其计算公式为:αA,B≈(δA+103)/(δB+103),其中δA为δ13/12CH4,δB为δ13/12CO2
若α>1.065,则产甲烷过程为嗜氢型产甲烷代谢途径;若α<1.025则产甲烷过程为乙酸型产甲烷代谢途径;若1.025≤α≤1.065,则甲烷来自上述两个途径。
2.根据权利要求1所述的利用碳稳定同位素分馏比率判断厌氧消化过程的方法,其特征在于:所述δ13/12CH4为CH413C和12C分馏比率。
3.根据权利要求2所述的利用碳稳定同位素分馏比率判断厌氧消化过程的方法,其特征在于:所述的δ13/12CO2为CO213C和12C分馏比率。
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