光合细菌利用低分子有机酸产氢的试验研究
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摘要
氢能由于高效、可再生以及无污染的特性,被认为是最具发展潜力的未来能源。同传统产氢方法相比,生物制氢方法对发展清洁高效的可再生能源和减少环境污染具有重要意义,符合可持续发展战略。目前,生物产氢的研究主要分为发酵和光合两种方法。由于发酵产氢过程中产生的低分子有机酸(VFAs)不能进一步有效的分解,而且这些低分子有机酸的累积在一定程度上阻碍了产氢底物的充分有效地利用。光合细菌能够利用这些低分子有机酸产氢。因而将光合细菌和发酵细菌耦合产氢是理想的产氢选择。其中,光合细菌利用低分子有机酸产氢是耦合法产氢的关键步骤。
本文主要研究光合细菌沼泽红假单胞菌Rh.Palustris Z02利用这些低分子有机酸,如乙酸、丙酸、丁酸和乳酸等,进行光合产氢的试验研究。由于菌体是生物产氢过程中的关键因素,因而本文对光合细菌利用低分子有机酸的生长特性进行了研究。通过对比确定了修正的Gompertz模型作为光合细菌利用低分子有机酸生长的动力学模型。并对影响光合细菌的生长因素进行了试验研究。结果表明在pH7.0、温度30℃和光强2300 lux是光合细菌Rh.Palustris Z02最适宜的生长条件。
微生物反应动力学是研究各种环境因素与微生物代谢活动之间相互作用随时间变化的规律。因而反应过程的参数对产氢研究是很重要的。本文建立了利用VFAs产氢过程中的菌体生长、产物形成、有机酸消耗、反应液pH变化以及有机酸降解等反应动力学模型,并进行了试验验证。同时通过试验发现有机酸为产氢抑制性底物,所以在Monod方程基础上进行修正,得到有机酸抑制的反应动力学方程(Andrew底物抑制模型),并通过试验验证了模型的可靠性。
利用响应曲面法确定出适合光合细菌利用低分子有机酸的产氢条件。结果得到最佳产氢条件为pH7.0、温度30℃和光强6700 lux,菌体有最大的产氢率1.90 mol H_2/mol乙酸、2.38 mol H_2/mol丙酸、3.02 mol H_2/mol丁酸和1.75 mol H_2/mol乳酸。同时通过ANOVA分析发现,温度和pH具有相关性,而光强与pH和温度不偶联,所以光强可以作为产氢的独立影响因子进行单独研究。由于碳酸盐的缺乏会限制有机酸的利用和产氢,所以针对不同浓度NaHCO_3对Rh.Palustris Z02利用有机酸产氢的影响进行了研究。结果发现光合细菌Rh.Palustris Z02利用丙酸和丁酸产氢时需要NaHCO_3作为电子受体。本文还研究了抑制光合细菌产氢的两大因素:氧以及氨氮,结果表明NH_4~+古对于光合细菌利用低分子有机酸产氢的抑制是一贯的;大量O_2的存在抑制光合产氢,而微量的O_2可以促进产氢。当以氩气和微量的氧混合时达到最大产氢率。
进一步研究了有机酸浓度对光合细菌Rh.Palustris Z02产氢的影响,主要对累积产氢量、产氢率和氢转化率几个指标进行探讨,并对这几个指标进行了试验研究和分析,分别建立以有机酸浓度为变量的累积产氢量、产氢率和氢转化率的数学模型,同时通过数学模型和试验数据的分析确定了氢转化率适合作为衡量不同有机酸不同浓度时菌体产氢能力的指标。结果表明试验范围内有机酸浓度对光合产氢的影响很大,过高浓度的有机酸对产氢有明显的抑制作用,产氢过程中这几种低分子有机酸均各自存在着一个最佳的产氢浓度值。并通过对数学模型的优化,发现三种低分子有机酸达到各自产氢时最佳有机酸浓度值的大小顺序分别为:乙酸＞丙酸＞丁酸。这说明丁酸浓度对产氢的影响作用要大于乙酸和丙酸。本文除对单一有机酸产氢的研究外,同时还进行了混合酸产氢的研究。由于乙酸和丁酸是发酵法产氢能力的指标也是发酵产氢的主要液相产物,所以本文对混合有机酸产氢的研究主要是集中在乙酸和丁酸混合的光合细菌产氢。而乙酸和丁酸的浓度比直接影响着光合产氢效果,通过响应曲面法(RSM)确定最佳的乙酸和丁酸浓度比为3.792,乙酸浓度为0.0383 mol/L时,达到最大的氢转化率43.88%。
为了提高光合细菌利用低分子有机酸产氢以及抗环境因素干扰的能力,对这些低分子有机酸进行了固定化菌体光合产氢特性的试验研究。结果表明固定化能提高产氢率,以海藻酸钠为固定化载体的产氢效果最佳。同时发现固定化菌体利用有机酸产氢也存在最佳有机酸浓度,固定化菌体产氢的最佳有机酸浓度要高于游离态的最佳有机酸浓度。对于乙酸、丙酸和丁酸这三种小分子羧酸,其最佳有机酸浓度的大小同样随着有机酸碳原子数的增加而减小,即乙酸(0.043 mol/1)＞丙酸(0.029 mol/1)＞丁酸(0.022 mol/1)。其中乙酸的氢转化率最高,达到65.3%。在利用低分子有机酸所产生气相成分中,氢气含量因有机酸类型的不同而不同,并不受有机酸浓度,以及菌体固定化与否的影响。对于乙酸、丙酸和丁酸,氢气含量随着有机酸碳原子数的增加而增大。
本文最后基于光合细菌利用VFAs产氢特性的研究结果,通过三种耦合方式产氢试验的对比,确定了两级串联生物产氢反应系统为最佳的耦合方式。
Hydrogen is the fuel of the future mainly due to its high conversion efficiency, recyclability and nonpolluting nature. Biological hydrogen production processes are found to be more environment friendly and less energy intensive as compared to traditional hydrogen production processes. It can be divided into two main categories, photosynthetic and fermentation process. The fermentative bacteria ferment organic wastes to H_2 and organic acids, but cannot utilize the organic acids as electron donors. Accumulation of these organic acids restrained hydrogen production and substrate adequate utilization. However, the photosynthetic bacteria can use these small-molecular organic acids as electron donors for the hydrogen production at the expense of light energy. Therefore, a more promising method of hydrogen production was combining the fermentative and phototrophic hydrogen production. And phototrophic hydrogen production from volatile fatty acids (VFAs) using phototrophic bacteria was the key approach of the hybrid system.
In this paper, hydrogen production by photosynthetic bacteria (PSB) Rh.Palustris Z02 from individual VFAs, i.e. acetate, propionate, butyrate, lactic acid, which were the main effluent from the fermentative H_2 production reactor, was investigated. Bacteria is a key factor of biohydrogen production process, so the growth feature of PSB Rh.Palustris Z02 using VFAs were investigated. The modified Gompertz model was determined to describe the growth kinetics of Rh.Palustris Z02 by models comparison. And the growth factor was investigated also. The result showed that pH7.0, temperature 30℃and light intensity 2300 lux was the most suitable condition for the growth of Rh.Palustris Z02.
Reaction kinetics of microorganism is research on relationship of all kinds of environmental factors and microorganism metabolism. Reaction parameters was vital for hydrogen production. In this paper, several reaction kinetic models were used to describe the growth of hydrogen producing microorganisms, consumption of VFAs, formation of product ,pH change and VFAs degradation in this work. The experimental data were subjected to numerical simulation to estimate the unknown kinetic constants for the substrate utilization, microbial growth and product formation in this hydrogen-producing process. Based on monad equation, Andrew substrate inhibitory model was used as the reaction kinetics equation of VFAs inhibition because higher concentration of VFAs was detrimental to hydrogen production. The results showed that the experimental data could be described by the proposed kinetic models with good agreements.
The effects of pH, temperature and light intensity on the maximum hydrogen yield (mol/mol) were evaluated using a response-surface methodology (RSM). Experimental results showed that pH, temperature and light intensity all had an influence on hydrogen production. The maximum hydrogen yield of 1.90 mol H_2/mol acetate, 2.38 mol H_2/mol propionate, 3.02 mol H_2/mol butyrate, 1.75 mol H_2/mol lactic acid was estimated under the optimum conditions of pH 7, temperature 30℃and light intensity 6700 lux. The effect of pH and temperature were significant, but light intensity was insignificant with pH and temperature by ANOVA analysis. Since the absence of carbonate limits VFAs utilization and hydrogen production, the effect of various NaHCO_3 concentrations on hydrogen production from VFAs were evaluated. The results obtained in this research showed that the phototrophic hydrogen production from propionate and butyrate required carbonate as an electron acceptor. Furthermore, two main inhibitory factor of phototrophic hydrogen production (oxygen and NH_4~+-N_2) were investigated. the results showed that the inhibitory effect of NH_4~+ for phototrophic hydrogen production from VFAs was obvious. A large amount of O_2 in gas phase was toxic for phototrophic hydrogen production. However, a small quantity of O_2 can promote hydrogen production. When Argon gas and a small quantity of O_2 mixed together, the highest hydrogen yield was achieved.
Effects of individual initial VFAs concentration (acetate and propionate ranging from 0.015 mol/l to 0.090 mol/l, and butyrate ranging from 0.005 mol/l to 0.090 mol/l) on phototrophic hydrogen production were evaluated by using PSB Rh.Palustris Z02. Experimental results indicated that VFAs concentration had a substantial effect on phototrophic hydrogen production. Hydrogen yield, cumulative hydrogen production volume and hydrogen conversion efficiency were evaluated at various initial VFAs concentration. The quadratic model was calculated for hydrogen yield and hydrogen conversion efficiency when initial VFAs concentration was variable. A modified cubic model was calculated for accumulative hydrogen production. The results demonstrated that H_2 conversion efficiency as a good indicator is more effective than the other two indices, hydrogen yield and cumulative hydrogen production volume. The observation demonstrated that higher VFAs concentration was detrimental to phototrophic hydrogen production. There was an optimal VFAs concentration within trial stretch for three VFAs individually. The optimal VFAs concentration of H_2 conversion efficiency followed the order of acetate > propionate > butyrate. Furthermore, hydrogen production from a mixture of the butyrate and acetate, which were the main effluent from the fermentative H_2 production reactor, was investigated. The ratio of acetate to butyrate (HAc/HBu ratio) had a substantial effect on phototrophic hydrogen production from mixture VFAs. HAc/HBu ratio on the maximum hydrogen conversion efficiency was evaluated using RSM. Experimental results showed that acetate concentration and butyrate concentration had an influence on hydrogen conversion efficiency. The effect of acetate concentration and butyrate concentration on hydrogen conversion efficiency were significant. A maximum hydrogen conversion efficiency of 43.88% was estimated at HAc/HBu ratio of 3.792, when acetate concentration was 0.0383 mol/L.
In order to enhance hydrogen yield from VFAs, phototrophic hydrogen production from VFAs by immobilized Rh.Palustris Z02 were investigated. The results indicated that hydrogen yield of immobilized cells is higher than that of free cells, and sodium alginate is a suitable immobilized carrier for hydrogen production. Furthermore, pH change, hydrogen conversion efficiency and the effect of initial concentration of VFAs on hydrogen production were analyzed. The results demonstrated that higher concentration of VFAs was still detrimental to hydrogen production. However, the optimal VFAs concentration of immobilized cells was higher than that of free cells, the optimal initial concentration decreasing with carbon atomicity of VFAs increasing also, and following the order of acetate(0.043 mol/l)>propionate(0.029 mol/l)> butyrate(0.022 mol/l). The highest hydrogen conversion efficiency of 65.3% was achieved from acetate by immobilized cells. For acetate, propionate and butyrate, hydrogen content of biogas is increasing with carbon atomicity of VFAs increasing. But, initial VFAs concentration and immobilization of cells has no effect on hydrogen content.
Finally, the mechanism and the feasibility of the hybrid biohydrogen production system of using fermentative and photosynthetic bacteria was analyzed and verified individually. Three hybrid approaches were tested, two-stage tandem connection biohydrogen production system was the best among three methods.
本文主要研究光合细菌沼泽红假单胞菌Rh.Palustris Z02利用这些低分子有机酸,如乙酸、丙酸、丁酸和乳酸等,进行光合产氢的试验研究。由于菌体是生物产氢过程中的关键因素,因而本文对光合细菌利用低分子有机酸的生长特性进行了研究。通过对比确定了修正的Gompertz模型作为光合细菌利用低分子有机酸生长的动力学模型。并对影响光合细菌的生长因素进行了试验研究。结果表明在pH7.0、温度30℃和光强2300 lux是光合细菌Rh.Palustris Z02最适宜的生长条件。
微生物反应动力学是研究各种环境因素与微生物代谢活动之间相互作用随时间变化的规律。因而反应过程的参数对产氢研究是很重要的。本文建立了利用VFAs产氢过程中的菌体生长、产物形成、有机酸消耗、反应液pH变化以及有机酸降解等反应动力学模型,并进行了试验验证。同时通过试验发现有机酸为产氢抑制性底物,所以在Monod方程基础上进行修正,得到有机酸抑制的反应动力学方程(Andrew底物抑制模型),并通过试验验证了模型的可靠性。
利用响应曲面法确定出适合光合细菌利用低分子有机酸的产氢条件。结果得到最佳产氢条件为pH7.0、温度30℃和光强6700 lux,菌体有最大的产氢率1.90 mol H_2/mol乙酸、2.38 mol H_2/mol丙酸、3.02 mol H_2/mol丁酸和1.75 mol H_2/mol乳酸。同时通过ANOVA分析发现,温度和pH具有相关性,而光强与pH和温度不偶联,所以光强可以作为产氢的独立影响因子进行单独研究。由于碳酸盐的缺乏会限制有机酸的利用和产氢,所以针对不同浓度NaHCO_3对Rh.Palustris Z02利用有机酸产氢的影响进行了研究。结果发现光合细菌Rh.Palustris Z02利用丙酸和丁酸产氢时需要NaHCO_3作为电子受体。本文还研究了抑制光合细菌产氢的两大因素:氧以及氨氮,结果表明NH_4~+古对于光合细菌利用低分子有机酸产氢的抑制是一贯的;大量O_2的存在抑制光合产氢,而微量的O_2可以促进产氢。当以氩气和微量的氧混合时达到最大产氢率。
进一步研究了有机酸浓度对光合细菌Rh.Palustris Z02产氢的影响,主要对累积产氢量、产氢率和氢转化率几个指标进行探讨,并对这几个指标进行了试验研究和分析,分别建立以有机酸浓度为变量的累积产氢量、产氢率和氢转化率的数学模型,同时通过数学模型和试验数据的分析确定了氢转化率适合作为衡量不同有机酸不同浓度时菌体产氢能力的指标。结果表明试验范围内有机酸浓度对光合产氢的影响很大,过高浓度的有机酸对产氢有明显的抑制作用,产氢过程中这几种低分子有机酸均各自存在着一个最佳的产氢浓度值。并通过对数学模型的优化,发现三种低分子有机酸达到各自产氢时最佳有机酸浓度值的大小顺序分别为:乙酸＞丙酸＞丁酸。这说明丁酸浓度对产氢的影响作用要大于乙酸和丙酸。本文除对单一有机酸产氢的研究外,同时还进行了混合酸产氢的研究。由于乙酸和丁酸是发酵法产氢能力的指标也是发酵产氢的主要液相产物,所以本文对混合有机酸产氢的研究主要是集中在乙酸和丁酸混合的光合细菌产氢。而乙酸和丁酸的浓度比直接影响着光合产氢效果,通过响应曲面法(RSM)确定最佳的乙酸和丁酸浓度比为3.792,乙酸浓度为0.0383 mol/L时,达到最大的氢转化率43.88%。
为了提高光合细菌利用低分子有机酸产氢以及抗环境因素干扰的能力,对这些低分子有机酸进行了固定化菌体光合产氢特性的试验研究。结果表明固定化能提高产氢率,以海藻酸钠为固定化载体的产氢效果最佳。同时发现固定化菌体利用有机酸产氢也存在最佳有机酸浓度,固定化菌体产氢的最佳有机酸浓度要高于游离态的最佳有机酸浓度。对于乙酸、丙酸和丁酸这三种小分子羧酸,其最佳有机酸浓度的大小同样随着有机酸碳原子数的增加而减小,即乙酸(0.043 mol/1)＞丙酸(0.029 mol/1)＞丁酸(0.022 mol/1)。其中乙酸的氢转化率最高,达到65.3%。在利用低分子有机酸所产生气相成分中,氢气含量因有机酸类型的不同而不同,并不受有机酸浓度,以及菌体固定化与否的影响。对于乙酸、丙酸和丁酸,氢气含量随着有机酸碳原子数的增加而增大。
本文最后基于光合细菌利用VFAs产氢特性的研究结果,通过三种耦合方式产氢试验的对比,确定了两级串联生物产氢反应系统为最佳的耦合方式。
Hydrogen is the fuel of the future mainly due to its high conversion efficiency, recyclability and nonpolluting nature. Biological hydrogen production processes are found to be more environment friendly and less energy intensive as compared to traditional hydrogen production processes. It can be divided into two main categories, photosynthetic and fermentation process. The fermentative bacteria ferment organic wastes to H_2 and organic acids, but cannot utilize the organic acids as electron donors. Accumulation of these organic acids restrained hydrogen production and substrate adequate utilization. However, the photosynthetic bacteria can use these small-molecular organic acids as electron donors for the hydrogen production at the expense of light energy. Therefore, a more promising method of hydrogen production was combining the fermentative and phototrophic hydrogen production. And phototrophic hydrogen production from volatile fatty acids (VFAs) using phototrophic bacteria was the key approach of the hybrid system.
In this paper, hydrogen production by photosynthetic bacteria (PSB) Rh.Palustris Z02 from individual VFAs, i.e. acetate, propionate, butyrate, lactic acid, which were the main effluent from the fermentative H_2 production reactor, was investigated. Bacteria is a key factor of biohydrogen production process, so the growth feature of PSB Rh.Palustris Z02 using VFAs were investigated. The modified Gompertz model was determined to describe the growth kinetics of Rh.Palustris Z02 by models comparison. And the growth factor was investigated also. The result showed that pH7.0, temperature 30℃and light intensity 2300 lux was the most suitable condition for the growth of Rh.Palustris Z02.
Reaction kinetics of microorganism is research on relationship of all kinds of environmental factors and microorganism metabolism. Reaction parameters was vital for hydrogen production. In this paper, several reaction kinetic models were used to describe the growth of hydrogen producing microorganisms, consumption of VFAs, formation of product ,pH change and VFAs degradation in this work. The experimental data were subjected to numerical simulation to estimate the unknown kinetic constants for the substrate utilization, microbial growth and product formation in this hydrogen-producing process. Based on monad equation, Andrew substrate inhibitory model was used as the reaction kinetics equation of VFAs inhibition because higher concentration of VFAs was detrimental to hydrogen production. The results showed that the experimental data could be described by the proposed kinetic models with good agreements.
The effects of pH, temperature and light intensity on the maximum hydrogen yield (mol/mol) were evaluated using a response-surface methodology (RSM). Experimental results showed that pH, temperature and light intensity all had an influence on hydrogen production. The maximum hydrogen yield of 1.90 mol H_2/mol acetate, 2.38 mol H_2/mol propionate, 3.02 mol H_2/mol butyrate, 1.75 mol H_2/mol lactic acid was estimated under the optimum conditions of pH 7, temperature 30℃and light intensity 6700 lux. The effect of pH and temperature were significant, but light intensity was insignificant with pH and temperature by ANOVA analysis. Since the absence of carbonate limits VFAs utilization and hydrogen production, the effect of various NaHCO_3 concentrations on hydrogen production from VFAs were evaluated. The results obtained in this research showed that the phototrophic hydrogen production from propionate and butyrate required carbonate as an electron acceptor. Furthermore, two main inhibitory factor of phototrophic hydrogen production (oxygen and NH_4~+-N_2) were investigated. the results showed that the inhibitory effect of NH_4~+ for phototrophic hydrogen production from VFAs was obvious. A large amount of O_2 in gas phase was toxic for phototrophic hydrogen production. However, a small quantity of O_2 can promote hydrogen production. When Argon gas and a small quantity of O_2 mixed together, the highest hydrogen yield was achieved.
Effects of individual initial VFAs concentration (acetate and propionate ranging from 0.015 mol/l to 0.090 mol/l, and butyrate ranging from 0.005 mol/l to 0.090 mol/l) on phototrophic hydrogen production were evaluated by using PSB Rh.Palustris Z02. Experimental results indicated that VFAs concentration had a substantial effect on phototrophic hydrogen production. Hydrogen yield, cumulative hydrogen production volume and hydrogen conversion efficiency were evaluated at various initial VFAs concentration. The quadratic model was calculated for hydrogen yield and hydrogen conversion efficiency when initial VFAs concentration was variable. A modified cubic model was calculated for accumulative hydrogen production. The results demonstrated that H_2 conversion efficiency as a good indicator is more effective than the other two indices, hydrogen yield and cumulative hydrogen production volume. The observation demonstrated that higher VFAs concentration was detrimental to phototrophic hydrogen production. There was an optimal VFAs concentration within trial stretch for three VFAs individually. The optimal VFAs concentration of H_2 conversion efficiency followed the order of acetate > propionate > butyrate. Furthermore, hydrogen production from a mixture of the butyrate and acetate, which were the main effluent from the fermentative H_2 production reactor, was investigated. The ratio of acetate to butyrate (HAc/HBu ratio) had a substantial effect on phototrophic hydrogen production from mixture VFAs. HAc/HBu ratio on the maximum hydrogen conversion efficiency was evaluated using RSM. Experimental results showed that acetate concentration and butyrate concentration had an influence on hydrogen conversion efficiency. The effect of acetate concentration and butyrate concentration on hydrogen conversion efficiency were significant. A maximum hydrogen conversion efficiency of 43.88% was estimated at HAc/HBu ratio of 3.792, when acetate concentration was 0.0383 mol/L.
In order to enhance hydrogen yield from VFAs, phototrophic hydrogen production from VFAs by immobilized Rh.Palustris Z02 were investigated. The results indicated that hydrogen yield of immobilized cells is higher than that of free cells, and sodium alginate is a suitable immobilized carrier for hydrogen production. Furthermore, pH change, hydrogen conversion efficiency and the effect of initial concentration of VFAs on hydrogen production were analyzed. The results demonstrated that higher concentration of VFAs was still detrimental to hydrogen production. However, the optimal VFAs concentration of immobilized cells was higher than that of free cells, the optimal initial concentration decreasing with carbon atomicity of VFAs increasing also, and following the order of acetate(0.043 mol/l)>propionate(0.029 mol/l)> butyrate(0.022 mol/l). The highest hydrogen conversion efficiency of 65.3% was achieved from acetate by immobilized cells. For acetate, propionate and butyrate, hydrogen content of biogas is increasing with carbon atomicity of VFAs increasing. But, initial VFAs concentration and immobilization of cells has no effect on hydrogen content.
Finally, the mechanism and the feasibility of the hybrid biohydrogen production system of using fermentative and photosynthetic bacteria was analyzed and verified individually. Three hybrid approaches were tested, two-stage tandem connection biohydrogen production system was the best among three methods.
引文
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