废水产氢产酸/同型产乙酸耦合系统厌氧发酵产酸工艺及条件优化
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摘要
废水处理正由单纯的污染控制向污染控制和资源化利用并重方向发展，利用废水生产生物能和生化品的研究正日益受到人们的广泛关注。通过废水厌氧发酵产酸为进一步发酵生产高值生化品提供相对单一的溶解性碳源是实现废水资源化的一条新途径。厌氧发酵产生的乙酸，很容易通过食乙酸产甲烷菌的作用转变为甲烷而减少酸的累积，本研究采用加热法处理厌氧活性污泥杀灭产甲烷菌；同时构建了产氢产酸／同型产乙酸耦合系统，通过同型产乙酸菌的耗氢产乙酸作用解除产氢产酸过程中的氢抑制，强化乙酸生产。
对葡萄糖浓度为5g/L的模拟废水进行厌氧酸化，当初始pH为8．0时，采用加热种泥的乙酸产率比采用未加热种泥高3．1倍。利用产氢产酸／同型产乙酸耦合系统对含葡萄糖30g/L的模拟废水和COD为34g/L的污泥预处理液分别进行厌氧发酵产酸，得到的乙酸产率分别比一般产酸系统提高52-87％和29-39％。产氢产酸／同型产乙酸耦合系统对乙酸生产的强化可归因于同型产乙酸作用、产氢产酸相产物抑制的解除以及葡萄糖转化和乙醇氧化的强化。
利用单因素试验方法，研究了底物浓度、初始pH和种泥浓度等关键因素对耦合系统产酸的影响。发现产氢产酸相中的条件变化，不仅影响本相产酸，而且影响同型产乙酸相产酸。乙酸浓度一般随着底物浓度的增加而增加，但在发酵后期(192 h)，底物浓度为30 g／L时产生的乙酸浓度比底物为40g/L时高27％。pH为5时主要进行乙醇型发酵；pH为6和7时主要进行丁酸型发酵；而pH处于8-11时均以乙酸为主要产物，其中pH为10时，产乙酸最高。pH和底物浓度对产酸的影响归因于游离酸对各种微生物的抑制程度上的差异。在种泥浓度为2-8 g／L范围内，两相种泥浓度均为4g/L时产乙酸最高。乙酸产率随种泥浓度的变化归因于不同种泥浓度条件下代谢途径的改变以及耦合系统两相间产耗氢不平衡程度的差异。在上述单因素实验基础上，采用均匀设计方法，得出耦合系统厌氧产酸以高乙酸产量为主要目标导向同时兼顾高乙酸产率和高乙酸生产强度目标的优化条件是：发酵时间10 d，初始pH 11，底物浓度40g/L，种泥浓度6 g／L。
同型产乙酸相产生的乙酸占耦合系统总乙酸的比例只有5-9％，而根据厌氧产酸反应计量学理论计算，该比例应可达到33％。这可能是耦合系统中存在严重的产耗氢不平衡以及两相间物质传递不顺畅所致。论文采用分批补料技术防止发酵初期产氢产酸相的有机负荷过高，同时减轻产耗氢不平衡程度；采用气体循环策略强化H_2+CO_2由产氢产酸相向同型产乙酸相转移，提高同型产乙酸相的底物浓度，降低产氢产酸相的氢分压。结果表明：采取气体循环和分批补料两种策略后，耦合系统乙酸产率约提高44％。乙酸有三种直接来源：葡萄糖转化、H_2+CO_2同型乙酸化和乙醇氧化，其中葡萄糖的直接转化所占比例最高，达49-80％；在产氢产酸相真空度为28 kPa时，H_2+CO_2同型乙酸化对于分批补料试验产乙酸的贡献>30％；乙醇氧化对于分批试验产乙酸的贡献>40％。
为了解产氢产酸／同型产乙酸耦合系统长期运行的稳定性以及产酸条件下微生物的种群动态，论文采用半连续方式进行长期运行并利用分子生态学技术对产酸微生物的种群动态进行了分析。结果表明：产氢产酸／同型产乙酸耦合系统半连续运行100 d后达到稳定状态，即有机酸和乙醇浓度基本恒定，pH值波动较小。在底物负荷为4-6g/(L·d)、HRT为20 d时，乙酸产率为0．4-0．48g/g。随着底物负荷率由4 g／(L·d)上调到6g/(L·d)，耦合系统产氢产酸相的香农指数由0．71 dit下降到0．50 dit；当负荷率回调到4g/Ld时，香农指数增加到0．75 dit。生物多样性丰富度与乙酸产率呈正相关。同型产乙酸相的香农指数只有0．21 dit，远低于产氢产酸相。耦合系统两相始终均有Clostridium和Fusobacterium，而且占有较高的比例。定量PCR检测到耦合系统产氢产酸相同型乙酸菌为6．3×10~8个／mL，同型产乙酸相为8．9×10~8个／mL，比一般厌氧消化反应器高2-3个数量级。耦合系统污泥微生物产乙酸关键酶活性为：产氢产酸相中的乙酸激酶(AK)活性1．5-2．5 U／mg，磷酸转移乙酰酶(PTA)为0．17-0．33 U／mg：同型产乙酸相中的AK活性1．0-1．7 U／mg，PTA为0．11-0．22 U／mg。关键酶活性与产乙酸强度呈正相关。
Wastewater treatment is shifting from single pollution control to paying equal attention to both pollution control and waste resource utilization. Research about using wastewater to produce bioenergy and biochemical materials is increasingly attracting widespread attention. Volatile fatty acids can be produced by wastewater anaerobic fermentation and be supplied to produce highly valuable biochemical products by further fermentation, which provides a new way for achieving wastewater resource utilization. Acetate is produced by anaerobic fermentation and can be easily converted to methane, and carboxylate accumulation is reduced. This study treated the anaerobic activated sludge by heat to kill methanogen, and constructed a novel acidificationhomoacetogenesiscoupling system. In this case, hydrogen inhibition during wastewater acidification is relieved resulting from hydrogen consumption by homoacetogen, which enhances acetate production.
Simulated wastewater containing 5 g/L glucose was acidified by heat-treated sludge and non-heat-treated sludge, respectively. It is indicated that acetate yield of the former is 3.1-fold higher than the latter at an initial pH 8.0. Then, the acidification-homoacetogenesis coupling system was used to produce acetate from simulated wastewater containing 30 g/L glucose and pretreated sludge liquor containing 34 g/L COD. Acetate yields in the coupling system are 52-87% and 29-39% higher than that in ordinary acidification systems, respectively. Enhancement of acetate production is attributed principally to homoacetogenesis, relief of the products (H_2 and CO_2) inhibition to acidification and syntrophic acetogenesis, enhancement of the conversion of substrate and the oxidation of ethanol.
The single-factor experiments were conducted to study the effects of substrate concentrations, initial pH and inoculum concentrations on acetate production in the acidification and homoacetogenesis coupling system. The results show the variation of a condition in the acidification phase affects acetate production not only in the acidification phase itself but also in the homoacetogenesis phase. With the increase of substrate concentration, acetate concentrations usually increase. But at 192 h, the acetate concentration at 30 g/L of substrate is 27% higher than that at 40 g/L of substrate. Ethanol-type fermentation mainly takes place at initial pH 5 while butyrate-type fermentation at initial pH 6 and 7. But acetate is the dominant product at initial pH 8-11. The optimal initial pH is 10 for acetate production in the coupling system. Such an effect of initial pH and substrate concentration can be attributed principally to the difference of inhibition degree of non-dissociated acids on different microbes. At 2-8 g/L of inoculum sludge, the optimum sludge inoculum size in the syntrophic acetogenesis phase and the homoacetogenesis phase is both 4 g/L. At that sludge inoculum size, the maximum acetate production and yield can be achieved. The variation of acetate yield may be attributed to different metabolic pathways and the imbalance between hydrogen production and hydrogen consumption in the coupling system with different inoculum concentrations. Based on above single factor tests, uniformity design test was used to obtain the optimization conditions as follows: fermentation time 10 d, initial pH 11, substrate concentration 40 g/L and inoculum sludge 6 g/L. At these conditions, the maximum acetate production, high acetate yield and high acetate production rate could be achieved.
The contribution of homoacetogenesis to acetate production should reach up to 33% by calculation according to acidification reaction metrology. But the acetate production in the homoacetogenesis phase accounted for only 5-9% in the coupling system. This may be resulted from the serious imbalance between hydrogen production and hydrogen consumption, and mass transfer obstruct between two phases. Gas circulation and fed-batch fermentation were applied for enhancing acetate production. The fed-batch method helps to balance hydrogen production in the acidification phase and hydrogen consumption in the homoacetogenesis phase of the coupling system, and helps to reduce the shock loading of organics at the beginning of the fermentation. Gas circulation enhances mass transfer from acidification phase to homoacetogenesis phase, hence resulting in increasing substrate concentrations in homoacetogenesis phase and decreasing the hydrogen partial pressure in the acidification phase. The results show that the acetate yield in the fed-batch test with gas circulation is about 44% higher than that in the batch test without gas circulation. Acetate can be directly from glucose conversion, H_2+CO_2 homoacetogenesis and ethanol oxidation. Acetate from glucose conversion accounted for 49-80%. When vacuum degree in the acidification phase was 28 kPa, H_2+CO_2 homoacetogenesis contributed more than 30% of acetate production for fed-batch tests, and ethanol oxidation contributed more than 40% of acetate production for batch tests.
To investigate the operation stability of the acidification-homoacetogenesis coupling system and the microbial population dynamics under acidification condition, semi-continuous long term run was conducted and molecular ecology techniques were used to analyze acidification microbial population dynamics. The stable status of the semi-continuous run test can be attained after 100 d, at which organic acids and ethanol concentrations and pH are almost constant. When substrate loading rate is 4-6 g/(L·d) and HRT is 20 d, acetate yield in the coupling system is 0.4-0.48 g/g. When the loading rate increases from 4 g/(L·d) to 6 g/(L·d), Shannon-Weiner index decreases from 0.71 dit to 0.50 dit in the acidogenesis phase. When the loading rate is adjusted back to 4 g/(L·d), the Shannon-Weiner index then increases to 0.75 dit. There is a positive correlation between biology diversity and acetate yield. The Shannon-Weiner index in the homoacetogenesis phase is only 0.21 dit, which is much lower than acidogenesis phase. There are Clostridium and Fusobacterium in two phases of the coupling system all the time, and they represent a higher proportion. Through quantitative PCR, homoacetogens in samples of the acidification phase and homoacetogenesis phase are 6.3×10~8 cells/mL and 8.9×10~8 cells/mL, respectively. These are 2-3 orders of magnitude higher than the ordinary anaerobic digestor. In the coupling system, the activities of key enzymes are as follows: the activities of acetate kinase (AK) and phosphotransacetylase (PTA) in the acidification phase are 1.5-2.5 U/mg and 0.17-0.33 U/mg, respectively; the activities of AK and PTA in the homoacetogenesis phase are 1.0-1.7 U/mg and 0.11-0.22 U/mg, respectively. There is a positive correlation between key enzyme activity and acetate production rate.
对葡萄糖浓度为5g/L的模拟废水进行厌氧酸化，当初始pH为8．0时，采用加热种泥的乙酸产率比采用未加热种泥高3．1倍。利用产氢产酸／同型产乙酸耦合系统对含葡萄糖30g/L的模拟废水和COD为34g/L的污泥预处理液分别进行厌氧发酵产酸，得到的乙酸产率分别比一般产酸系统提高52-87％和29-39％。产氢产酸／同型产乙酸耦合系统对乙酸生产的强化可归因于同型产乙酸作用、产氢产酸相产物抑制的解除以及葡萄糖转化和乙醇氧化的强化。
利用单因素试验方法，研究了底物浓度、初始pH和种泥浓度等关键因素对耦合系统产酸的影响。发现产氢产酸相中的条件变化，不仅影响本相产酸，而且影响同型产乙酸相产酸。乙酸浓度一般随着底物浓度的增加而增加，但在发酵后期(192 h)，底物浓度为30 g／L时产生的乙酸浓度比底物为40g/L时高27％。pH为5时主要进行乙醇型发酵；pH为6和7时主要进行丁酸型发酵；而pH处于8-11时均以乙酸为主要产物，其中pH为10时，产乙酸最高。pH和底物浓度对产酸的影响归因于游离酸对各种微生物的抑制程度上的差异。在种泥浓度为2-8 g／L范围内，两相种泥浓度均为4g/L时产乙酸最高。乙酸产率随种泥浓度的变化归因于不同种泥浓度条件下代谢途径的改变以及耦合系统两相间产耗氢不平衡程度的差异。在上述单因素实验基础上，采用均匀设计方法，得出耦合系统厌氧产酸以高乙酸产量为主要目标导向同时兼顾高乙酸产率和高乙酸生产强度目标的优化条件是：发酵时间10 d，初始pH 11，底物浓度40g/L，种泥浓度6 g／L。
同型产乙酸相产生的乙酸占耦合系统总乙酸的比例只有5-9％，而根据厌氧产酸反应计量学理论计算，该比例应可达到33％。这可能是耦合系统中存在严重的产耗氢不平衡以及两相间物质传递不顺畅所致。论文采用分批补料技术防止发酵初期产氢产酸相的有机负荷过高，同时减轻产耗氢不平衡程度；采用气体循环策略强化H_2+CO_2由产氢产酸相向同型产乙酸相转移，提高同型产乙酸相的底物浓度，降低产氢产酸相的氢分压。结果表明：采取气体循环和分批补料两种策略后，耦合系统乙酸产率约提高44％。乙酸有三种直接来源：葡萄糖转化、H_2+CO_2同型乙酸化和乙醇氧化，其中葡萄糖的直接转化所占比例最高，达49-80％；在产氢产酸相真空度为28 kPa时，H_2+CO_2同型乙酸化对于分批补料试验产乙酸的贡献>30％；乙醇氧化对于分批试验产乙酸的贡献>40％。
为了解产氢产酸／同型产乙酸耦合系统长期运行的稳定性以及产酸条件下微生物的种群动态，论文采用半连续方式进行长期运行并利用分子生态学技术对产酸微生物的种群动态进行了分析。结果表明：产氢产酸／同型产乙酸耦合系统半连续运行100 d后达到稳定状态，即有机酸和乙醇浓度基本恒定，pH值波动较小。在底物负荷为4-6g/(L·d)、HRT为20 d时，乙酸产率为0．4-0．48g/g。随着底物负荷率由4 g／(L·d)上调到6g/(L·d)，耦合系统产氢产酸相的香农指数由0．71 dit下降到0．50 dit；当负荷率回调到4g/Ld时，香农指数增加到0．75 dit。生物多样性丰富度与乙酸产率呈正相关。同型产乙酸相的香农指数只有0．21 dit，远低于产氢产酸相。耦合系统两相始终均有Clostridium和Fusobacterium，而且占有较高的比例。定量PCR检测到耦合系统产氢产酸相同型乙酸菌为6．3×10~8个／mL，同型产乙酸相为8．9×10~8个／mL，比一般厌氧消化反应器高2-3个数量级。耦合系统污泥微生物产乙酸关键酶活性为：产氢产酸相中的乙酸激酶(AK)活性1．5-2．5 U／mg，磷酸转移乙酰酶(PTA)为0．17-0．33 U／mg：同型产乙酸相中的AK活性1．0-1．7 U／mg，PTA为0．11-0．22 U／mg。关键酶活性与产乙酸强度呈正相关。
Wastewater treatment is shifting from single pollution control to paying equal attention to both pollution control and waste resource utilization. Research about using wastewater to produce bioenergy and biochemical materials is increasingly attracting widespread attention. Volatile fatty acids can be produced by wastewater anaerobic fermentation and be supplied to produce highly valuable biochemical products by further fermentation, which provides a new way for achieving wastewater resource utilization. Acetate is produced by anaerobic fermentation and can be easily converted to methane, and carboxylate accumulation is reduced. This study treated the anaerobic activated sludge by heat to kill methanogen, and constructed a novel acidificationhomoacetogenesiscoupling system. In this case, hydrogen inhibition during wastewater acidification is relieved resulting from hydrogen consumption by homoacetogen, which enhances acetate production.
Simulated wastewater containing 5 g/L glucose was acidified by heat-treated sludge and non-heat-treated sludge, respectively. It is indicated that acetate yield of the former is 3.1-fold higher than the latter at an initial pH 8.0. Then, the acidification-homoacetogenesis coupling system was used to produce acetate from simulated wastewater containing 30 g/L glucose and pretreated sludge liquor containing 34 g/L COD. Acetate yields in the coupling system are 52-87% and 29-39% higher than that in ordinary acidification systems, respectively. Enhancement of acetate production is attributed principally to homoacetogenesis, relief of the products (H_2 and CO_2) inhibition to acidification and syntrophic acetogenesis, enhancement of the conversion of substrate and the oxidation of ethanol.
The single-factor experiments were conducted to study the effects of substrate concentrations, initial pH and inoculum concentrations on acetate production in the acidification and homoacetogenesis coupling system. The results show the variation of a condition in the acidification phase affects acetate production not only in the acidification phase itself but also in the homoacetogenesis phase. With the increase of substrate concentration, acetate concentrations usually increase. But at 192 h, the acetate concentration at 30 g/L of substrate is 27% higher than that at 40 g/L of substrate. Ethanol-type fermentation mainly takes place at initial pH 5 while butyrate-type fermentation at initial pH 6 and 7. But acetate is the dominant product at initial pH 8-11. The optimal initial pH is 10 for acetate production in the coupling system. Such an effect of initial pH and substrate concentration can be attributed principally to the difference of inhibition degree of non-dissociated acids on different microbes. At 2-8 g/L of inoculum sludge, the optimum sludge inoculum size in the syntrophic acetogenesis phase and the homoacetogenesis phase is both 4 g/L. At that sludge inoculum size, the maximum acetate production and yield can be achieved. The variation of acetate yield may be attributed to different metabolic pathways and the imbalance between hydrogen production and hydrogen consumption in the coupling system with different inoculum concentrations. Based on above single factor tests, uniformity design test was used to obtain the optimization conditions as follows: fermentation time 10 d, initial pH 11, substrate concentration 40 g/L and inoculum sludge 6 g/L. At these conditions, the maximum acetate production, high acetate yield and high acetate production rate could be achieved.
The contribution of homoacetogenesis to acetate production should reach up to 33% by calculation according to acidification reaction metrology. But the acetate production in the homoacetogenesis phase accounted for only 5-9% in the coupling system. This may be resulted from the serious imbalance between hydrogen production and hydrogen consumption, and mass transfer obstruct between two phases. Gas circulation and fed-batch fermentation were applied for enhancing acetate production. The fed-batch method helps to balance hydrogen production in the acidification phase and hydrogen consumption in the homoacetogenesis phase of the coupling system, and helps to reduce the shock loading of organics at the beginning of the fermentation. Gas circulation enhances mass transfer from acidification phase to homoacetogenesis phase, hence resulting in increasing substrate concentrations in homoacetogenesis phase and decreasing the hydrogen partial pressure in the acidification phase. The results show that the acetate yield in the fed-batch test with gas circulation is about 44% higher than that in the batch test without gas circulation. Acetate can be directly from glucose conversion, H_2+CO_2 homoacetogenesis and ethanol oxidation. Acetate from glucose conversion accounted for 49-80%. When vacuum degree in the acidification phase was 28 kPa, H_2+CO_2 homoacetogenesis contributed more than 30% of acetate production for fed-batch tests, and ethanol oxidation contributed more than 40% of acetate production for batch tests.
To investigate the operation stability of the acidification-homoacetogenesis coupling system and the microbial population dynamics under acidification condition, semi-continuous long term run was conducted and molecular ecology techniques were used to analyze acidification microbial population dynamics. The stable status of the semi-continuous run test can be attained after 100 d, at which organic acids and ethanol concentrations and pH are almost constant. When substrate loading rate is 4-6 g/(L·d) and HRT is 20 d, acetate yield in the coupling system is 0.4-0.48 g/g. When the loading rate increases from 4 g/(L·d) to 6 g/(L·d), Shannon-Weiner index decreases from 0.71 dit to 0.50 dit in the acidogenesis phase. When the loading rate is adjusted back to 4 g/(L·d), the Shannon-Weiner index then increases to 0.75 dit. There is a positive correlation between biology diversity and acetate yield. The Shannon-Weiner index in the homoacetogenesis phase is only 0.21 dit, which is much lower than acidogenesis phase. There are Clostridium and Fusobacterium in two phases of the coupling system all the time, and they represent a higher proportion. Through quantitative PCR, homoacetogens in samples of the acidification phase and homoacetogenesis phase are 6.3×10~8 cells/mL and 8.9×10~8 cells/mL, respectively. These are 2-3 orders of magnitude higher than the ordinary anaerobic digestor. In the coupling system, the activities of key enzymes are as follows: the activities of acetate kinase (AK) and phosphotransacetylase (PTA) in the acidification phase are 1.5-2.5 U/mg and 0.17-0.33 U/mg, respectively; the activities of AK and PTA in the homoacetogenesis phase are 1.0-1.7 U/mg and 0.11-0.22 U/mg, respectively. There is a positive correlation between key enzyme activity and acetate production rate.
引文
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