B.R3菌株生物制氢系统发酵条件与化学增强技术研究
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摘要
厌氧发酵生物制氢技术可将高浓度的有机废水和生物质(如复合固体废弃物、阴沟底泥等)等作为原材料(底物)进行氢气制取,这既可以解决环境问题又能产生氢气,同时由于其反应简单、可持续、稳定产氢、产氢速率高、反应器的设计、操作、管理简单方便等特点而越来越受到世界的关注。
本文依据厌氧发酵生物制氢的产氢机理,研究了高效产氢菌种Biohydrogenbacterium R3sp.nov(以下简称B.R3)在间歇实验条件下的发酵产氢效能,以及不同发酵条件(包括起始pH值、温度、生物质底物等)、有机氮源、无机氮源、磷酸盐、以及各种金属离子及其浓度对其产氢发酵效能的影响作用,同时研究了在连续流实验条件下,高效产氢菌种B.R3以糖蜜废水为底物的连续发酵产氢效能。研究结果表明：
起始pH值和温度对细胞生长和产氢量具有较明显的影响,与国内外学者的研究结果相比,当起始pH值为5.5时,B.R3的细胞干重和产氢量分别达到最大值0.63g/L和34.2mmol/L。当温度为30℃时B.R3的细胞干重与比产氢率达到最大值0.66g/L和1.0145molH2/mol葡萄糖,此时乙醇和乙酸的产量分别为3067.95mg/L与2554.57mg/L,低于或高于这个温度时,产氢率和细胞生长都会受到抑制。
B.R3以葡萄糖、绵白糖、白砂糖、红糖、面粉、牛奶、玉米面、淀粉、糖蜜和琼脂分别为底物进行发酵产氢试验的结果表明,当以葡萄糖作为底物时,生物气体和氢气产量较高分别为187.5mol/L和89.28mol/L,氢气含量为47.62%,当以面粉、牛奶、玉米粉、淀粉和琼脂作为发酵底物时,B.R3的发酵产氢量极低,然而以牛奶、玉米粉作为底物时,B.R3的氢气含量却相对较高(36.92%和43.75%)。大部分学者的研究结果都显示糖类物质具有较高的产氢能力,而纤维类物质需要经过一定酸处理、碱处理等预处理方式才能被菌种利用产氢,因此不适宜作为生物制氢的底物选择,但是本文认为虽然目前这类物质作为发酵产氢底物时的产氢效率不高,但由于其含有较丰富的碳水化合物从而具有较高的产氢潜力,并且在我国农业大国的国情下,还是十分具有研究价值的,这样既可做到废弃物处理同时也满足了能源物质——-H2的生产。
纯菌种在连续流条件下进行发酵产氢的研究相对混合菌种而言比较少,且实验时体积相对较小,因此本文以B.R3进行连续流厌氧发酵生物制氢,实验时总体积为20L,实验证明通过控制适宜的实验条件,可实现CSTR反应器连续产氢。实验进行时以有机负荷为4kgCOD/L·d启动,运行至第23d时有机负荷降为3kgCOD/L·d,运行至第49d时有机负荷降低为2kgCOD/L·d,运行过程中氢气产量与含量、末端发酵产物以及有机等随着反应器时间的运行而呈现不同变化。反应器运行1-23d时氢气产量与含量呈现波动性上升趋势,在这个阶段内当反应器运行至第23d时气体产量达到最大值24.904L/d；当反应器运行至第23～48d时,反应器内的产气量与氢气含量都比较稳定且较高,当反应器运行至第28d时,产气量达到最大值26.39L/d；当反应器运行至第49～61d时,反应器内的产气量与氢气含量都开始下降,当反应器运行至第51d时氢气产量降低到最低值0.90L/d,随着反应器的继续运行以及NaHCO3的添加,B.R3的产气量与氢气含量缓慢上升趋势。
氮元素及对B.R3产氢发酵效能的影响研究结果显示有机氮源对B.R3的生长及产氢效能的促进作用大大高于无机氮源对其的促进作用。酵母粉是较有效的氮源提供者,能够更好的促进B.R3的细胞生长和发酵产氢。无机氮源对发酵产氢细菌的发酵产氢效能促进作用不明显,因此在培养基中加入有机氮源(尤其是酵母粉)对于提高B.R3的发酵产氢效能是十分必要的。
磷元素及对B.R3产氢发酵效能的影响研究结果显示磷酸氢二钾对B.R3的生长及产氢效能都具有良好的促进作用,在反应器内起始pH值为5.5,温度为30℃并且以酵母粉作为氮源时,当磷酸氢二钾浓度为1.5g/L时,B.R3的生物气体产量、氢气产量以及产氢率都达到最大值,分别为4960mL/L-培养基,2107.5mL/L·培养基和1.93mol H2/mol葡萄糖。磷酸氢二钾对培养基中的pH值具有较好的缓冲作用,当培养基内添加了磷酸氢二钾时其pH值始终维持在3.0～5.0范围内。
磷酸盐缓冲溶液对B.R3的发酵产氢效能以及缓冲培养基终pH值同样也具有较良好的促进作用,当磷酸盐缓冲溶液浓度为0.15mol时,B.R3的生物气体、氢气产量和平均产氢速率分别达到最大值3860mL/L-培养基,1832.7mL H2/L·培养基和2.6324mmol H2/g·cell-h。
综合对比NH4HCO3、NaHCO3、Na2CO3、K2HPO4、KH2PO4、NaH2PO4和Na2HPO4对B.R3产氢效能的影响,反应器内添加了Na2HPO4与K2HPO4的B.R3的氢气产量与氢气含量达到最高值,分别为1978.56mL/L培养基、44.1%与2160.9mL/L-培养基、45.8%。
金属离子对产氢菌种发酵产氢效能的影响实验表明,在反应器内起始pH值为5.5,温度为30℃并且以酵母粉作为氮源条件下,当CoCl2浓度为0.05mg/L,B.R3的氢气产量达到最大值为205mL/L·培养基,当添加Fe粉浓度为200mg/L时,B.R3的氢气产量达到最大值为315mL/L-培养基,当添加Fe2+浓度为40mg/L时,B.R3的氢气产量达到最大值为281mL/L·培养基,当添加Cu2+浓度为0.03mg/L时,B.R3产氢量达到最大值192mL/L·培养基,当添加Zn2+浓度为0.2mg/L时,B.R3产氢量达到最大值144.2mL/L·培养基。
The anaerobic fermentation biohydrogen production can make use of the high concentration organic waste and biomass (such as complex solid waste, sewage sludge and so on) as raw material to produce hydrogen. This technology has caught more and more focus since it can operate easily, produce hydrogen efficiency and sustainly, but the most important is that this technology can not only solve the environment problem but also produce hydrogen.
This paper investigated the hydrogen production efficiency of Biohydrogenbacterium R3sp.nov (B.R3in single) in batch condition and the promotion of different fermentation condition (including initial pH, temperature, biomass etc.), organic nitrogen, inorganic nitrogen, phosphate and metal ions on hydrogen production efficiency and cell growth to it according to the principle of anaerobic fermentation biohydrogen production. We also investigated the efficiency of B.R3in continuous stirred tank reactor (CSTR) which makes molasses as substrate. The results are as followed in simply.
The results indicated that pH and temperature had a noticeable effect on the cell growth and hydrogen production. The dry cell weight and hydrogen production yield got the maximum of0.63g/L and34.2mmol/L respectively when the initial pH was5.5. The final pHs in the culture were always kept at3.0-4.0. Temperature affected the maximum cell growth and specific hydrogen production ration (SHPR) and they got the maximum of0.66g/L and1.01mol H2/mol glucose respectively at the temperature of30℃. Whether the pH and temperature are lower or higher, hydrogen production and biomass will be inhibited gradually.
The biogas, hydrogen production and the hydrogen concentrations were187.5mol/L,89.28mol/L and47.62%respectively when was used the glucose as subtract. The hydrogen concentrations were36.92%and43.75%while the biogas and hydrogen production yield were relatively lower than those when the milk and maize flour were used as subtract. This indicated that the milk and maize flour have the capacity of hydrogen production although the hydrogen production rations were low since they have the rich carbonate. Many reachers' investigation shows that the carbohydrate has high hydrogen producing ablitity. While the cellulose must be pretreated by acid or alkali so they thought it's not suit for hydrogen production. But China waste huge amount of cellulose every so we think it is valuable because this can treat the waste and produce hydrogen at the same time.
The reacher of bacteria in continuous condition is less than that of mixed culture relatively. The B.R3can produce hydrogen continuously in the right condition. The CSTR was set up at the initial COD of4kgCOD/L-d and it dropped to3kgCOD/L-d at the23th day while it dropped to2kgCOD/L·d at the49th day. The hydrogen production, concentration and end liquid fermentation fluctuated with the operation of CSTR. The hydrogen production and concentration increased when the CSTR operated at1th～23th day and the hydrogen production yield got the maximum of24.904L/d. The hydrogen production yield and concentration kept stable and got the maximum of26.39L/d at the28th day. The hydrogen production yield and concentration dropped to the minimum of0.90L/d at the51th day and they were increasing slowly with the NaHCO3and the operation of CSTR.
The performance of organic nitrogen resource to B.R3on cell growth and hydrogen production was better than that of inorganic nitrogen resource. The yeast powder was most beneficial to promote cell growth and hydrogen production among all those nitrogen resources. So it's essential for B.R3to dose organic nitrogen (epically yeast powder) to promote the hydrogen production.
K2HPO4can promote the hydrogen production and maintain the pH in medium of B.R3well. When the initial pH was5.5, the temperature was30℃and the yeast power as nitrogen provider, the biogas production yield, hydrogen production yield and hydrogen production ration got the maximum of4960mL/L·culture,2107.5mL/L·culture,1.93mol H2/mol glucose when the concentration of K2HPO4was1.5g/L. The pH in medium always maintained3.0-5.0since the K2HPO4can buffer the medium well.
The phosphate buffer solution (PBS) can promote the hydrogen production and buffer the pH in the medium efficiency to B.R3The biogas and hydrogen production yield and average hydrogen production ration got the maximum of3860mL/L·culture,1832.7mLH2/L·culture and2.6324mmol H2/g·cell·h when the concentration of PBS was0.15mol.
Compare the promotion to B.R3of NH4HCO3, NaHCO3, Na2CO3, K2HPO4, KH2PO4, NaH2PO4and Na2HPO4comprehensive, the hydrogen production yield and hydrogen concentration got the maximum of1978.56mL/L·culture,44.1%,2160.9mL/L·culture and45.8%when Na2HPO4and K2HPO4was dosed in medium.
The hydrogen production yield got the maximum of205mL/L·culture,315mL/L·culture,281mL/L·culture,192mL/L·culture,144.2mL/L·culture respectively at the concentration of CoCl2at0.05mg/L, Fe粉at200mg/L, Fe2+at40mg/L, Cu2+at0.03mg/L, Zn2+at0.2mg/L.
本文依据厌氧发酵生物制氢的产氢机理,研究了高效产氢菌种Biohydrogenbacterium R3sp.nov(以下简称B.R3)在间歇实验条件下的发酵产氢效能,以及不同发酵条件(包括起始pH值、温度、生物质底物等)、有机氮源、无机氮源、磷酸盐、以及各种金属离子及其浓度对其产氢发酵效能的影响作用,同时研究了在连续流实验条件下,高效产氢菌种B.R3以糖蜜废水为底物的连续发酵产氢效能。研究结果表明：
起始pH值和温度对细胞生长和产氢量具有较明显的影响,与国内外学者的研究结果相比,当起始pH值为5.5时,B.R3的细胞干重和产氢量分别达到最大值0.63g/L和34.2mmol/L。当温度为30℃时B.R3的细胞干重与比产氢率达到最大值0.66g/L和1.0145molH2/mol葡萄糖,此时乙醇和乙酸的产量分别为3067.95mg/L与2554.57mg/L,低于或高于这个温度时,产氢率和细胞生长都会受到抑制。
B.R3以葡萄糖、绵白糖、白砂糖、红糖、面粉、牛奶、玉米面、淀粉、糖蜜和琼脂分别为底物进行发酵产氢试验的结果表明,当以葡萄糖作为底物时,生物气体和氢气产量较高分别为187.5mol/L和89.28mol/L,氢气含量为47.62%,当以面粉、牛奶、玉米粉、淀粉和琼脂作为发酵底物时,B.R3的发酵产氢量极低,然而以牛奶、玉米粉作为底物时,B.R3的氢气含量却相对较高(36.92%和43.75%)。大部分学者的研究结果都显示糖类物质具有较高的产氢能力,而纤维类物质需要经过一定酸处理、碱处理等预处理方式才能被菌种利用产氢,因此不适宜作为生物制氢的底物选择,但是本文认为虽然目前这类物质作为发酵产氢底物时的产氢效率不高,但由于其含有较丰富的碳水化合物从而具有较高的产氢潜力,并且在我国农业大国的国情下,还是十分具有研究价值的,这样既可做到废弃物处理同时也满足了能源物质——-H2的生产。
纯菌种在连续流条件下进行发酵产氢的研究相对混合菌种而言比较少,且实验时体积相对较小,因此本文以B.R3进行连续流厌氧发酵生物制氢,实验时总体积为20L,实验证明通过控制适宜的实验条件,可实现CSTR反应器连续产氢。实验进行时以有机负荷为4kgCOD/L·d启动,运行至第23d时有机负荷降为3kgCOD/L·d,运行至第49d时有机负荷降低为2kgCOD/L·d,运行过程中氢气产量与含量、末端发酵产物以及有机等随着反应器时间的运行而呈现不同变化。反应器运行1-23d时氢气产量与含量呈现波动性上升趋势,在这个阶段内当反应器运行至第23d时气体产量达到最大值24.904L/d；当反应器运行至第23～48d时,反应器内的产气量与氢气含量都比较稳定且较高,当反应器运行至第28d时,产气量达到最大值26.39L/d；当反应器运行至第49～61d时,反应器内的产气量与氢气含量都开始下降,当反应器运行至第51d时氢气产量降低到最低值0.90L/d,随着反应器的继续运行以及NaHCO3的添加,B.R3的产气量与氢气含量缓慢上升趋势。
氮元素及对B.R3产氢发酵效能的影响研究结果显示有机氮源对B.R3的生长及产氢效能的促进作用大大高于无机氮源对其的促进作用。酵母粉是较有效的氮源提供者,能够更好的促进B.R3的细胞生长和发酵产氢。无机氮源对发酵产氢细菌的发酵产氢效能促进作用不明显,因此在培养基中加入有机氮源(尤其是酵母粉)对于提高B.R3的发酵产氢效能是十分必要的。
磷元素及对B.R3产氢发酵效能的影响研究结果显示磷酸氢二钾对B.R3的生长及产氢效能都具有良好的促进作用,在反应器内起始pH值为5.5,温度为30℃并且以酵母粉作为氮源时,当磷酸氢二钾浓度为1.5g/L时,B.R3的生物气体产量、氢气产量以及产氢率都达到最大值,分别为4960mL/L-培养基,2107.5mL/L·培养基和1.93mol H2/mol葡萄糖。磷酸氢二钾对培养基中的pH值具有较好的缓冲作用,当培养基内添加了磷酸氢二钾时其pH值始终维持在3.0～5.0范围内。
磷酸盐缓冲溶液对B.R3的发酵产氢效能以及缓冲培养基终pH值同样也具有较良好的促进作用,当磷酸盐缓冲溶液浓度为0.15mol时,B.R3的生物气体、氢气产量和平均产氢速率分别达到最大值3860mL/L-培养基,1832.7mL H2/L·培养基和2.6324mmol H2/g·cell-h。
综合对比NH4HCO3、NaHCO3、Na2CO3、K2HPO4、KH2PO4、NaH2PO4和Na2HPO4对B.R3产氢效能的影响,反应器内添加了Na2HPO4与K2HPO4的B.R3的氢气产量与氢气含量达到最高值,分别为1978.56mL/L培养基、44.1%与2160.9mL/L-培养基、45.8%。
金属离子对产氢菌种发酵产氢效能的影响实验表明,在反应器内起始pH值为5.5,温度为30℃并且以酵母粉作为氮源条件下,当CoCl2浓度为0.05mg/L,B.R3的氢气产量达到最大值为205mL/L·培养基,当添加Fe粉浓度为200mg/L时,B.R3的氢气产量达到最大值为315mL/L-培养基,当添加Fe2+浓度为40mg/L时,B.R3的氢气产量达到最大值为281mL/L·培养基,当添加Cu2+浓度为0.03mg/L时,B.R3产氢量达到最大值192mL/L·培养基,当添加Zn2+浓度为0.2mg/L时,B.R3产氢量达到最大值144.2mL/L·培养基。
The anaerobic fermentation biohydrogen production can make use of the high concentration organic waste and biomass (such as complex solid waste, sewage sludge and so on) as raw material to produce hydrogen. This technology has caught more and more focus since it can operate easily, produce hydrogen efficiency and sustainly, but the most important is that this technology can not only solve the environment problem but also produce hydrogen.
This paper investigated the hydrogen production efficiency of Biohydrogenbacterium R3sp.nov (B.R3in single) in batch condition and the promotion of different fermentation condition (including initial pH, temperature, biomass etc.), organic nitrogen, inorganic nitrogen, phosphate and metal ions on hydrogen production efficiency and cell growth to it according to the principle of anaerobic fermentation biohydrogen production. We also investigated the efficiency of B.R3in continuous stirred tank reactor (CSTR) which makes molasses as substrate. The results are as followed in simply.
The results indicated that pH and temperature had a noticeable effect on the cell growth and hydrogen production. The dry cell weight and hydrogen production yield got the maximum of0.63g/L and34.2mmol/L respectively when the initial pH was5.5. The final pHs in the culture were always kept at3.0-4.0. Temperature affected the maximum cell growth and specific hydrogen production ration (SHPR) and they got the maximum of0.66g/L and1.01mol H2/mol glucose respectively at the temperature of30℃. Whether the pH and temperature are lower or higher, hydrogen production and biomass will be inhibited gradually.
The biogas, hydrogen production and the hydrogen concentrations were187.5mol/L,89.28mol/L and47.62%respectively when was used the glucose as subtract. The hydrogen concentrations were36.92%and43.75%while the biogas and hydrogen production yield were relatively lower than those when the milk and maize flour were used as subtract. This indicated that the milk and maize flour have the capacity of hydrogen production although the hydrogen production rations were low since they have the rich carbonate. Many reachers' investigation shows that the carbohydrate has high hydrogen producing ablitity. While the cellulose must be pretreated by acid or alkali so they thought it's not suit for hydrogen production. But China waste huge amount of cellulose every so we think it is valuable because this can treat the waste and produce hydrogen at the same time.
The reacher of bacteria in continuous condition is less than that of mixed culture relatively. The B.R3can produce hydrogen continuously in the right condition. The CSTR was set up at the initial COD of4kgCOD/L-d and it dropped to3kgCOD/L-d at the23th day while it dropped to2kgCOD/L·d at the49th day. The hydrogen production, concentration and end liquid fermentation fluctuated with the operation of CSTR. The hydrogen production and concentration increased when the CSTR operated at1th～23th day and the hydrogen production yield got the maximum of24.904L/d. The hydrogen production yield and concentration kept stable and got the maximum of26.39L/d at the28th day. The hydrogen production yield and concentration dropped to the minimum of0.90L/d at the51th day and they were increasing slowly with the NaHCO3and the operation of CSTR.
The performance of organic nitrogen resource to B.R3on cell growth and hydrogen production was better than that of inorganic nitrogen resource. The yeast powder was most beneficial to promote cell growth and hydrogen production among all those nitrogen resources. So it's essential for B.R3to dose organic nitrogen (epically yeast powder) to promote the hydrogen production.
K2HPO4can promote the hydrogen production and maintain the pH in medium of B.R3well. When the initial pH was5.5, the temperature was30℃and the yeast power as nitrogen provider, the biogas production yield, hydrogen production yield and hydrogen production ration got the maximum of4960mL/L·culture,2107.5mL/L·culture,1.93mol H2/mol glucose when the concentration of K2HPO4was1.5g/L. The pH in medium always maintained3.0-5.0since the K2HPO4can buffer the medium well.
The phosphate buffer solution (PBS) can promote the hydrogen production and buffer the pH in the medium efficiency to B.R3The biogas and hydrogen production yield and average hydrogen production ration got the maximum of3860mL/L·culture,1832.7mLH2/L·culture and2.6324mmol H2/g·cell·h when the concentration of PBS was0.15mol.
Compare the promotion to B.R3of NH4HCO3, NaHCO3, Na2CO3, K2HPO4, KH2PO4, NaH2PO4and Na2HPO4comprehensive, the hydrogen production yield and hydrogen concentration got the maximum of1978.56mL/L·culture,44.1%,2160.9mL/L·culture and45.8%when Na2HPO4and K2HPO4was dosed in medium.
The hydrogen production yield got the maximum of205mL/L·culture,315mL/L·culture,281mL/L·culture,192mL/L·culture,144.2mL/L·culture respectively at the concentration of CoCl2at0.05mg/L, Fe粉at200mg/L, Fe2+at40mg/L, Cu2+at0.03mg/L, Zn2+at0.2mg/L.
引文
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