发酵产氢菌的分离及其产氢研究
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摘要
随着环境污染和能源枯竭的日益恶化,寻找一种清洁可再生能源是人类当务之急。氢气以其燃烧值高、清洁、可再生等为人类首选。在众多制氢方法中,暗发酵法制取氢气在利用有机废液和农作物秸秆同时,起到了清洁环境和获得能源的双重作用。发酵产氢细菌的分离在产氢机理的研究、菌种改造和耦合制氢有中很大的意义。
本文主要是对厌氧发酵产氢过程进行初步研究,研究内容主要包括：产氢菌种的筛选、鉴定；接种量和接种时间对累积产氢量和平均产氢速率的影响；初始pH值对累积产氢量和平均产氢速率的影响；调查三种无机氮源NH4C、CH3COONH4和(NH2)2CO在葡萄糖和果渣为碳源的条件下,对产氢量的影响；纯菌种(CP)与混合菌种(MB)比较,以果渣、玉米秆和葡萄糖为碳源发酵产氢随时间的影响；不同浓度(0.5、1.0、2.0、3.0、4.0、5.0 g·L-1)下葡萄糖中累积产气量和平均产氢速率随时间的变化；初始pH值为7,发酵过程中控制pH值在4.7-5.0、5.5-5.8、6.3-6.6的范围内累积产氢量(mL·L-1-glu.和mL·g-1-glu.)随时间的影响；初始pH值为7,用NaOH溶液和氨水调节pH值及无调节下,累积产氢量(m1·L-1-glu.和m1·g-1-glu.)随浓度(0.5、1.0、2.0、3.0、4.0、5.0 g·L-1)的变化；把纯菌种还原到原污泥环境中的产氢实验(灭菌)和纯菌种与污泥(处理过的产氢污泥)耦合产氢实验。
实验结果表明：经16S rRNA基因特征基因测序得到,与Clostridium perfringens ATCC 13124(CP)的16S rRNA基因相似性达99%,可以认为分离得到的菌种为该菌,菌种静态培养得到,在培养8小时菌种达到最佳浓度,动态培养时可以看出菌数量1～9小时增加,9～11小时下降,产氢实验证明CP为高效产氢菌；最佳的接种量和接种时间为1/50(v/v)和8h；当初始pH值为7时得到了最佳的为最适产氢初始pH;CH3COONH4为无机氮源,与果渣为碳源时能有效降低产氢成本,分析可知产氢菌CP对纤维素有一定的分解能力；产氢菌CP分解生物质产氢能力没有混菌能力强,但利用单糖能力却比混菌强,充分说明在利用单糖产氢时应该选用纯菌；产氢菌CP利用葡萄糖产氢时,底物浓度越低时底物利用率越高,但单位体积累积产氢量为2 g·L-1时为最大,所以在选着产氢底物浓度是应该按运行成本核算选择最低产氢成本；初始pH值为7,发酵过程中控制pH为4.7-5.0时为最佳控制范围,底物浓度越高过程中控制pH产氢量增加越大。以3g··L-1的葡萄糖为发酵底物,把产氢菌还原到原污泥环境中时：污泥为无菌时,污泥在发酵液中的重量和驯化时间会影响累积产氢量的大小,最佳污泥重量为15 g,最佳的驯化时间为12h；产氢菌CP与混合菌耦合时,随着驯化时间的增加累积产氢量先增加后减少,最佳的驯化时间为3 h。
The increasing crisis of global fossil fuels shortage, climate change, environmental pollution and associated health problems are all driving the need of a clean and alternative energy. Hydrogen, a high combustion value, clean, safe and renewable energy carrier, is considered as a preferred resource. Nowadays, people concentrate their attentions on the development of hydrogen energy, and more and more researches have focused on this field. Among the numerous hydrogen production methods, dark fermentation appears to be the most attractive method because it can utilize a variety of cheap carbon sources such as organic wastewater and crops straw,and this method can clean environment and obtain energy. The isolation of fermented bacteria can contribute to study the mechanism of producing hydrogen, spawning transformation and co-culturing hydrogen production.
In this work, bio-hydrogen technological skills have been researched, such as screening and identifying the hydrogen producing strains; the influence of inoculation quantity and vaccination time on cumulative hydrogen yield and the average production rate of hydrogen; effect of initial pH on the cumulative hydrogen yield and the average hydrogen producing rate. Surveying the influence of inorganic nitrogen source on hydrogen production, with three kinds of inorganic nitrogen source NH4Cl, CH3COONH4 and (NH2)2CO in glucose and apple pomace for carbon sources; influence of pure strains (CP) and the mixed bacteria (MB) hydrogen production on fermentative time, with apple pomace, cornstalk and glucose as carbon sources; change of accumulate hydrogen production and average hydrogen production rate from glucose on time in different concentration (0.5,1.0,2.0 and 3.0,4.0 and 5.0 g·L-1); change of accumulate hydrogen production(ml·L-1-glu and ml·g-1-glu) on time with initial pH value as 7 and controlling fermentative process pH in 4.7-5.0,5.5-5.8 and 6.3-6.6 range; change of accumulate hydrogen production (ml·L-1-glu and ml·g-1-glu) on the concentrations (0.5,1.0,2.0 and 3.0,4.0 and 5.0 g·L-1), with initial pH value as 7 and comparison of no adjustment and adjusting the pH by NaOH as well as ammonia solution; putting the strains isolated from the sludge back to pure hydrogen production environment (sterilization) and pure strains as well as sludge (processed produce hydrogen sludge produced hydrogen experiment) co-culturing to investigate the hydrogen productive effect.
The the genetic traits 16 SrRNA gene sequencing experimental results show that the isolated bacteria gets the 16SrRNA genetic similarity as 99% with Clostridium perfringens ATCC13124 (CP), which recognize the strains isolated from sludge. The strains static training achieved the best concentration when cultivate strains for 8h., In dynamic training, bacteria number increased 1～9 hours, and declined in 9～11 hours. The hydrogen production experiments substantiate that CP appears to be the high efficient hydrogen production bacteria. The best quantity and vaccination time prove to be 1/50 (v/v) and 8 h respectively,and the optimal initial pH for hydrogen producing appears to be 7; The production cost can be effectively reduced when utilizing CH3COONH4 as inorganic nitrogen source and apple pomace as carbon source. In addition, the analysis of experiment results suggest that CP has the ability to utilize cellulose. And CP utilizes biomass to producing hydrogen is no better than mixed bacteria, but it favors to use simple sugars, which suggest that pure bacteria should be chosen to utilize simple sugars. The hydrogen production by CP from glucose suggests that the lower the substrate concentration is, the higher the substrate utilizing effect. And unit volume cumulative yield as 2g·L-1 is the largest, so it is chosen to be the optimal hydrogen production substrate concentration considering the productive cost. When the initial pH value is 7, fermentation process controlling pH in 4.7-5.0 proves to be the optimal control range, and the higher the substrate concentration is, the more hydrogen production increases in the process of on-line controlling pH.
With using 3g·L-1 glucose as the fermentation substrate, replacing the CP to sludge environment results in two different phenomenon. If the sewage sludge are sterilized, the weight and domestication in fermented liquid hydrogen production time affects the quantity of accumulation, and the best sludge weight is 15 g, the best domesticated time is 12 h. If CP is co-cultured with the mixed bacteria in sewage sludge, the accumulated hydrogen production elevated at first and then decreased with the increase of domesticated time, and the best domesticated time appeared to be 3 h.
本文主要是对厌氧发酵产氢过程进行初步研究,研究内容主要包括：产氢菌种的筛选、鉴定；接种量和接种时间对累积产氢量和平均产氢速率的影响；初始pH值对累积产氢量和平均产氢速率的影响；调查三种无机氮源NH4C、CH3COONH4和(NH2)2CO在葡萄糖和果渣为碳源的条件下,对产氢量的影响；纯菌种(CP)与混合菌种(MB)比较,以果渣、玉米秆和葡萄糖为碳源发酵产氢随时间的影响；不同浓度(0.5、1.0、2.0、3.0、4.0、5.0 g·L-1)下葡萄糖中累积产气量和平均产氢速率随时间的变化；初始pH值为7,发酵过程中控制pH值在4.7-5.0、5.5-5.8、6.3-6.6的范围内累积产氢量(mL·L-1-glu.和mL·g-1-glu.)随时间的影响；初始pH值为7,用NaOH溶液和氨水调节pH值及无调节下,累积产氢量(m1·L-1-glu.和m1·g-1-glu.)随浓度(0.5、1.0、2.0、3.0、4.0、5.0 g·L-1)的变化；把纯菌种还原到原污泥环境中的产氢实验(灭菌)和纯菌种与污泥(处理过的产氢污泥)耦合产氢实验。
实验结果表明：经16S rRNA基因特征基因测序得到,与Clostridium perfringens ATCC 13124(CP)的16S rRNA基因相似性达99%,可以认为分离得到的菌种为该菌,菌种静态培养得到,在培养8小时菌种达到最佳浓度,动态培养时可以看出菌数量1～9小时增加,9～11小时下降,产氢实验证明CP为高效产氢菌；最佳的接种量和接种时间为1/50(v/v)和8h；当初始pH值为7时得到了最佳的为最适产氢初始pH;CH3COONH4为无机氮源,与果渣为碳源时能有效降低产氢成本,分析可知产氢菌CP对纤维素有一定的分解能力；产氢菌CP分解生物质产氢能力没有混菌能力强,但利用单糖能力却比混菌强,充分说明在利用单糖产氢时应该选用纯菌；产氢菌CP利用葡萄糖产氢时,底物浓度越低时底物利用率越高,但单位体积累积产氢量为2 g·L-1时为最大,所以在选着产氢底物浓度是应该按运行成本核算选择最低产氢成本；初始pH值为7,发酵过程中控制pH为4.7-5.0时为最佳控制范围,底物浓度越高过程中控制pH产氢量增加越大。以3g··L-1的葡萄糖为发酵底物,把产氢菌还原到原污泥环境中时：污泥为无菌时,污泥在发酵液中的重量和驯化时间会影响累积产氢量的大小,最佳污泥重量为15 g,最佳的驯化时间为12h；产氢菌CP与混合菌耦合时,随着驯化时间的增加累积产氢量先增加后减少,最佳的驯化时间为3 h。
The increasing crisis of global fossil fuels shortage, climate change, environmental pollution and associated health problems are all driving the need of a clean and alternative energy. Hydrogen, a high combustion value, clean, safe and renewable energy carrier, is considered as a preferred resource. Nowadays, people concentrate their attentions on the development of hydrogen energy, and more and more researches have focused on this field. Among the numerous hydrogen production methods, dark fermentation appears to be the most attractive method because it can utilize a variety of cheap carbon sources such as organic wastewater and crops straw,and this method can clean environment and obtain energy. The isolation of fermented bacteria can contribute to study the mechanism of producing hydrogen, spawning transformation and co-culturing hydrogen production.
In this work, bio-hydrogen technological skills have been researched, such as screening and identifying the hydrogen producing strains; the influence of inoculation quantity and vaccination time on cumulative hydrogen yield and the average production rate of hydrogen; effect of initial pH on the cumulative hydrogen yield and the average hydrogen producing rate. Surveying the influence of inorganic nitrogen source on hydrogen production, with three kinds of inorganic nitrogen source NH4Cl, CH3COONH4 and (NH2)2CO in glucose and apple pomace for carbon sources; influence of pure strains (CP) and the mixed bacteria (MB) hydrogen production on fermentative time, with apple pomace, cornstalk and glucose as carbon sources; change of accumulate hydrogen production and average hydrogen production rate from glucose on time in different concentration (0.5,1.0,2.0 and 3.0,4.0 and 5.0 g·L-1); change of accumulate hydrogen production(ml·L-1-glu and ml·g-1-glu) on time with initial pH value as 7 and controlling fermentative process pH in 4.7-5.0,5.5-5.8 and 6.3-6.6 range; change of accumulate hydrogen production (ml·L-1-glu and ml·g-1-glu) on the concentrations (0.5,1.0,2.0 and 3.0,4.0 and 5.0 g·L-1), with initial pH value as 7 and comparison of no adjustment and adjusting the pH by NaOH as well as ammonia solution; putting the strains isolated from the sludge back to pure hydrogen production environment (sterilization) and pure strains as well as sludge (processed produce hydrogen sludge produced hydrogen experiment) co-culturing to investigate the hydrogen productive effect.
The the genetic traits 16 SrRNA gene sequencing experimental results show that the isolated bacteria gets the 16SrRNA genetic similarity as 99% with Clostridium perfringens ATCC13124 (CP), which recognize the strains isolated from sludge. The strains static training achieved the best concentration when cultivate strains for 8h., In dynamic training, bacteria number increased 1～9 hours, and declined in 9～11 hours. The hydrogen production experiments substantiate that CP appears to be the high efficient hydrogen production bacteria. The best quantity and vaccination time prove to be 1/50 (v/v) and 8 h respectively,and the optimal initial pH for hydrogen producing appears to be 7; The production cost can be effectively reduced when utilizing CH3COONH4 as inorganic nitrogen source and apple pomace as carbon source. In addition, the analysis of experiment results suggest that CP has the ability to utilize cellulose. And CP utilizes biomass to producing hydrogen is no better than mixed bacteria, but it favors to use simple sugars, which suggest that pure bacteria should be chosen to utilize simple sugars. The hydrogen production by CP from glucose suggests that the lower the substrate concentration is, the higher the substrate utilizing effect. And unit volume cumulative yield as 2g·L-1 is the largest, so it is chosen to be the optimal hydrogen production substrate concentration considering the productive cost. When the initial pH value is 7, fermentation process controlling pH in 4.7-5.0 proves to be the optimal control range, and the higher the substrate concentration is, the more hydrogen production increases in the process of on-line controlling pH.
With using 3g·L-1 glucose as the fermentation substrate, replacing the CP to sludge environment results in two different phenomenon. If the sewage sludge are sterilized, the weight and domestication in fermented liquid hydrogen production time affects the quantity of accumulation, and the best sludge weight is 15 g, the best domesticated time is 12 h. If CP is co-cultured with the mixed bacteria in sewage sludge, the accumulated hydrogen production elevated at first and then decreased with the increase of domesticated time, and the best domesticated time appeared to be 3 h.
引文
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