餐厨垃圾同步糖化发酵产乳酸与双水相分离
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摘要
餐厨垃圾含水率与挥发性有机质含量高,极易腐败,成为恶臭的主要来源,并且其含有大量的致病菌,容易造成疾病的传播。另一方面,餐厨垃圾富含碳水化合物及丰富的营养,包括脂肪、蛋白质和多种糖类,可作为生物质能源,具有广阔的利用前景。目前厌氧产甲烷技术是餐厨垃圾资源化的主流技术,但其存在发酵时间长,沼气产率低等问题。本课题组以往研究表明餐厨垃圾厌氧消化过程中大量产生的乳酸容易抑制产甲烷菌的活性,并造成餐厨垃圾利用率低等问题。而乳酸作为一种工业原料广泛运用于食品、医药、化工等领域;并且由乳酸还可合成生物可降解性塑料,成为石油化工生产通用塑料的替代品。相比于厌氧产甲烷,生物发酵产乳酸具有成本低,生物质利用率高,副产物少,工艺简单,对环境污染小等特点。因此,开发高效的餐厨垃圾厌氧发酵产乳酸技术可望成为一种有效实现餐厨垃圾减量化、无害化和资源化的新途径。
为提高餐厨垃圾中生物质的降解率、最大程度地实现餐厨垃圾减量化与资源化,本论文对餐厨垃圾同步糖化发酵产乳酸和双水相技术提取乳酸进行了研究,着重对影响这两种过程的限制因素及其改善措施进行系统研究:针对餐厨垃圾极易腐化,容易在运输过程中产生恶臭,滋生腐败菌的问题,本文研究了添加同型乳杆菌快速保存餐厨垃圾的方法;针对传统乳酸发酵工艺流程长,操作复杂,乳酸产率低的问题,本文将同步糖化发酵技术运用到餐厨垃圾发酵中,分析α-淀粉酶、蛋白酶、Yeast extract、温度及CaCO3对餐厨垃圾同步糖化发酵的影响规律,并提出提高餐厨垃圾发酵速率的措施;针对传统的乳酸提取工艺流程长,消耗化工原料多,且最终产品收率低的问题,本文首次将双水相技术运用到餐厨垃圾发酵中提取乳酸,探究了乳酸菌在双水相体系中的分配规律,确定影响分配的主要因素,分析了聚合物浓度、分子量及乳酸菌接种率对双水相体系发酵的影响,并考查了餐厨垃圾多批次连续发酵提取乳酸的效果。主要研究结果如下:
⑴从餐厨垃圾中分离筛选出若干乳酸菌株,经生理生化试验初筛得到一株同型乳杆菌。进一步经API 50 CH系统及16sDNA分子生物学技术鉴定,鉴定为同型Lactobacillus plantarum乳杆菌,并命名为Lactobacillus plantarum BP04。生长曲线与产酸能力表明该菌株生长周期短,经12 h生长可达到稳定期,且具有良好的产酸能力,可达到138.09 g/L。
⑵研究了投加Lactobacillus plantarum BP04快速保存餐厨垃圾的方法。实验结果表明接种Lactobacillus plantarum BP04有利于在保存前期加速乳酸的生成,快速降低发酵液pH值,达到抑制肠道菌等病原菌与腐败菌的滋生。提高接种率,有利于控制Lactobacillus brevis与Leuconostoc lactis等异型乳酸菌的生长,加速发酵类型由异型向同型转化。乳酸是餐厨垃圾保存过程中最主要的有机酸,其变化趋势反应了不同菌属的细菌在保存过程中的相互作用规律。
⑶运用二阶响应曲面法分析α-淀粉酶、蛋白酶、Yeast extract、温度及CaCO3对餐厨垃圾同步糖化发酵的影响规律。响应曲面法回归模型表明一次项中蛋白酶、温度及CaCO3对乳酸产量有显著正影响,而α-淀粉酶与Yeast extract影响不显著;蛋白酶与温度的交互项对乳酸产量有高度显著的负作用。餐厨垃圾同步糖化发酵产乳酸的优化条件为:α-淀粉酶13.86 U/g,蛋白酶2.12 U/g,温度29.31℃,CaCO3 62.67 g/L,对应的乳酸产量为98.51 g/L,垃圾利用率为88.75%。提高乳酸菌接种率有利于加速餐厨垃圾中可溶性碳水化合物的降解,提高α-淀粉酶的水解效率;增加CaCO3投加量有助于加速还原糖降解,并促使水解与发酵平衡向水解阶段转变。
⑷运用Plackett-Burman与响应曲面法组合设计分析Lactobacillus plantarum BP04乳酸菌在双水相体系中的分配行为。实验结果表明Lactobacillus plantarum BP04在PEG/DEX体系中为单相分配,绝大部分分配于下相与两相界面,其分配系数K不受成相聚合物分子量、浓度、电解质及pH值变化的影响。Plackett-Burman试验结果表明聚合物分子量对体积比有高度显著的负影响,而细胞浓度、电解质、静置时间及pH值无显著影响。响应曲面法结果表明聚合物浓度变化对体积比有高度显著影响,其中PEG10000为正线性相关,DEX20000为负的二次函数关系,而PEG10000与DEX20000无相互影响。响应曲面法所得优化条件为:PEG10000 6%、DEX20000 13.85%,对应体积比最小值为0.815。
⑸研究了餐厨垃圾发酵时PEG/DEX双水相体系提取乳酸的情况。实验结果显示PEG/DEX体系具有良好的生物相容性,能有效从餐厨垃圾发酵体系中提取乳酸。相比于常规发酵体系,PEG10000/DEX20000对Lactobacillus plantarum BP04生长影响不大;而乳酸平均生成速率在12～24 h为0.20 g/(L·h)左右,低于常规发酵体系的0.683 g/(L·h)。上下相聚合物浓度的变化对餐厨垃圾发酵影响不明显。PEG/DEX体系在发酵过程中,体积比稳定,不受产物与基质浓度变化的影响。PEG分子量增加对发酵影响不大;而DEX分子量从20000增加到40000,乳酸生成速率相应由0.631 g/(L·h)降至0.518 g/(L·h),乳酸产量也由33 g/L降至22 g/L。提高接种率可缩短乳酸菌的停滞期,并加速乳酸生成,当接种率为1%、4%和8%时,6～48 h内乳酸平均生成速率分别为0.558 g/(L·h)、0.602 g/(L·h)、0.649 g/(L·h)。在PEG10000/DEX20000体系进行的多批次连续发酵结果表明,由于DEX相很好地富集乳酸菌,每批次发酵时乳酸菌能保持高浓度,乳酸菌生长不经历停滞期、对数期,相比于传统发酵极大缩短发酵时间;在不投加缓冲剂的情况下,餐厨垃圾发酵单批次乳酸产率大于0.30 g/g,累积产率大于0.45 g/g。
Kitchen wastes have been the most abundant and problematic organic solid waste in the world since they are the main source of decay, odor and leachate in collection and transportation due to its high volatile solids and moisture content. However, as kitchen wastes contain rich nutrition, including carbohydrate, lipid, protein and other compounds, it could be a potential raw material for recycling utilization. Compared to the landfill, incineration and compost, lactic acid fermentation is a sustainable technology for kitchen wastes. It can effectively achieve kitchen wastes minimization and generate lactic acid for energy recovery. Lactic acid has both hydroxyl and carboxyl groups with one chiral carbon atom, and it is widely used in the food, pharmaceutical and general chemical industries. In addition, lactic acid can be polymerized to form the biodegradable and recyclable polyester polylactic acid, which is considered a potential substitute for plastics manufactured from petroleum. Thus, it is necessary to develop an efficient lactic acid fermentation process to stabilize the kitchen wastes.
The present study dealt with lactic acid production using kitchen wastes as substrate. A homofermentative Lactobacillus plantarum strain, isolated from kitchen wastes, was employed as the starter culture in the quick storage of kitchen wastes to suppress both pathogenic and spoilage microorganisms. Response surface methodology was adopted to optimize lactic acid production during simultaneous saccharification and fermentation of kitchen wastes by examining five independent variables (α-amylase/solid ratio, protease/solid ratio, yeast extract concentration, temperature and CaCO3 concentration). The combination of Plackett-Burman design and response surface methodology was employed to screen and optimize various variables for lactic acid bacteria partitioning in aqueous two-phase system under different operating conditions. Moreover, PEG/DEX systems were also used to extract lactic acid from kitchen wastes fermentation.
⑴A strain of lactic acid bacteria was initially isolated from 10 day fermented kitchen wastes on MRS agar. After identified by normally physiological and biochemical tests and API 50 CH galleries, the strain was a homofermentative Lactobacillus plantarum species and was named Lactobacillus plantarum BP04. Experiments from growth curve indicated that this strain could grow quickly and reach stationary phase within 12 h. In addition, its lactate production capacity could be up to 138.09 g/L.
⑵Lactobacillus plantarum BP04 was employed as starter culture in kitchen wastes storage with different inoculant levels at 0, 2 and 10% (v/w) to suppress the outgrowth of pathogenic and spoilage bacteria. Inoculation was effective in accelerating pH drop and reducing the growth period of enterobacteria to 9.7 and 2 days, corresponding to inoculant levels at 0, 2 and 10% (v/w). Increasing inoculum levels were found to inhibit the growth of Lactobacillus brevis and Leuconostoc lactis. HPLC analysis revealed that lactic acid was the predominant organic acid during the treatment of kitchen wastes. Its concentration varied among the fermented processes reflecting variations of microbial activity in the fermented media.
⑶Central composite design using response surface methodology was employed to optimize parameters ofα-amylase, protease, temperature, CaCO3 and yeast extract in simultaneous saccharification and fermentation process. A satisfactory fit of the quadratic model was realized. Lactic acid biosynthesis was significantly affected by interaction of protease×temperature. Protease, temperature and CaCO3 had significantly linear effects on lactic acid production whileα-amylase and yeast extract had insignificant effects. Yeast extract was proved to be unnecessary in eatery food waste fermentation. The optimum condition was found to beα-amylase at 13.86 U/g dried food waste, protease at 2.12 U/g dried food waste, temperature at 29.31℃and CaCO3 at 62.67 g/l with the maximum lactic acid concentration and yield at 98.51 g/l and 88.75%, respectively. Increase of inoculum size would be suitable by accelerating depletion of initial soluble carbohydrate to enhanceα-amylase efficiency in kitchen wastes fermentation. Increasing magnitudes of CaCO3 supplement could improved the reducing sugars depletion rate and led to the balance between the rates of hydrolysis and fermentation favorable to hydrolysis.
⑷The combination of Plackett-Burman design and response surface methodology was used to examine partitioning parameters for Lactobacillus plantarum BP04 in aqueous two-phase system with polyethylene glycol (PEG)/dextran (DEX) as the biphasic system. One-sided partition of cells to DEX phase was observed in all trials. Results from Plackett-Burman design presented that the molecular weight of PEG and DEX had highly significant and negative effects on volume ratio while cell density, electrolytes, settling time and pH were insignificant. PEG10000 and DEX20000 were further optimized by response surface methodological approach. Volume ratio was strongly affected by the variation of polymer concentration whereas the interaction between PEG10000 and DEX20000 was insignificant. The optimum condition was found to be PEG10000 at 6% (w/w) and DEX20000 at 13.85% (w/w) at the experimental range with the minimal volume ratio at 0.815.
⑸PEG/DEX systems were also used to extract lactic acid from kitchen wastes fermentation. Results showed that PEG/DEX was a good biocompatible system which could effectively extract lactic acid from fermented media. PEG10000/DEX20000 biphasic system had little effect on the growth of Lactobacillus plantarum BP04, whereas lactic acid production rate was about 0.20 g /(L·h) between 12 and 48 h, much lower than 0.631 g/(L·h) in conventional fermentation system. The variation of PEG and DEX concentrations influenced cells growth and lactic acid biosynthesis indistinctively. Volume ratio kept stable throughout the processes, despite the changes of raw material and product concentrations. Different from little influence of PEG molecular weight on fermentation, the increase of DEX molecular weight from 20000 to 40000 led to declines of lactate conversion rate from 0.631 g/(L·h) to 0.518 g/(L·h) and lactic acid concentration from 33 g/L To 22 g/L. Increasing magnitudes of inoculum size could shorten the period of lag phase and enhance the production rate. When inoculum sizes were at 1%、4% and 8% (w/w), the corresponding lactic acid conversion rate were 0.558 g/(L·h) and 0.602 g/(L·h) and 0.649 g/(L·h), respectively. A repeated extractive fermentation was carried out in PEG10000/ DEX20000 biphasic system with four top-phase replacements. Results presented that when cell density reached the stationary phase in the first extractive fermentation, the lactate production in the aqueous two-phase system was maintained.
为提高餐厨垃圾中生物质的降解率、最大程度地实现餐厨垃圾减量化与资源化,本论文对餐厨垃圾同步糖化发酵产乳酸和双水相技术提取乳酸进行了研究,着重对影响这两种过程的限制因素及其改善措施进行系统研究:针对餐厨垃圾极易腐化,容易在运输过程中产生恶臭,滋生腐败菌的问题,本文研究了添加同型乳杆菌快速保存餐厨垃圾的方法;针对传统乳酸发酵工艺流程长,操作复杂,乳酸产率低的问题,本文将同步糖化发酵技术运用到餐厨垃圾发酵中,分析α-淀粉酶、蛋白酶、Yeast extract、温度及CaCO3对餐厨垃圾同步糖化发酵的影响规律,并提出提高餐厨垃圾发酵速率的措施;针对传统的乳酸提取工艺流程长,消耗化工原料多,且最终产品收率低的问题,本文首次将双水相技术运用到餐厨垃圾发酵中提取乳酸,探究了乳酸菌在双水相体系中的分配规律,确定影响分配的主要因素,分析了聚合物浓度、分子量及乳酸菌接种率对双水相体系发酵的影响,并考查了餐厨垃圾多批次连续发酵提取乳酸的效果。主要研究结果如下:
⑴从餐厨垃圾中分离筛选出若干乳酸菌株,经生理生化试验初筛得到一株同型乳杆菌。进一步经API 50 CH系统及16sDNA分子生物学技术鉴定,鉴定为同型Lactobacillus plantarum乳杆菌,并命名为Lactobacillus plantarum BP04。生长曲线与产酸能力表明该菌株生长周期短,经12 h生长可达到稳定期,且具有良好的产酸能力,可达到138.09 g/L。
⑵研究了投加Lactobacillus plantarum BP04快速保存餐厨垃圾的方法。实验结果表明接种Lactobacillus plantarum BP04有利于在保存前期加速乳酸的生成,快速降低发酵液pH值,达到抑制肠道菌等病原菌与腐败菌的滋生。提高接种率,有利于控制Lactobacillus brevis与Leuconostoc lactis等异型乳酸菌的生长,加速发酵类型由异型向同型转化。乳酸是餐厨垃圾保存过程中最主要的有机酸,其变化趋势反应了不同菌属的细菌在保存过程中的相互作用规律。
⑶运用二阶响应曲面法分析α-淀粉酶、蛋白酶、Yeast extract、温度及CaCO3对餐厨垃圾同步糖化发酵的影响规律。响应曲面法回归模型表明一次项中蛋白酶、温度及CaCO3对乳酸产量有显著正影响,而α-淀粉酶与Yeast extract影响不显著;蛋白酶与温度的交互项对乳酸产量有高度显著的负作用。餐厨垃圾同步糖化发酵产乳酸的优化条件为:α-淀粉酶13.86 U/g,蛋白酶2.12 U/g,温度29.31℃,CaCO3 62.67 g/L,对应的乳酸产量为98.51 g/L,垃圾利用率为88.75%。提高乳酸菌接种率有利于加速餐厨垃圾中可溶性碳水化合物的降解,提高α-淀粉酶的水解效率;增加CaCO3投加量有助于加速还原糖降解,并促使水解与发酵平衡向水解阶段转变。
⑷运用Plackett-Burman与响应曲面法组合设计分析Lactobacillus plantarum BP04乳酸菌在双水相体系中的分配行为。实验结果表明Lactobacillus plantarum BP04在PEG/DEX体系中为单相分配,绝大部分分配于下相与两相界面,其分配系数K不受成相聚合物分子量、浓度、电解质及pH值变化的影响。Plackett-Burman试验结果表明聚合物分子量对体积比有高度显著的负影响,而细胞浓度、电解质、静置时间及pH值无显著影响。响应曲面法结果表明聚合物浓度变化对体积比有高度显著影响,其中PEG10000为正线性相关,DEX20000为负的二次函数关系,而PEG10000与DEX20000无相互影响。响应曲面法所得优化条件为:PEG10000 6%、DEX20000 13.85%,对应体积比最小值为0.815。
⑸研究了餐厨垃圾发酵时PEG/DEX双水相体系提取乳酸的情况。实验结果显示PEG/DEX体系具有良好的生物相容性,能有效从餐厨垃圾发酵体系中提取乳酸。相比于常规发酵体系,PEG10000/DEX20000对Lactobacillus plantarum BP04生长影响不大;而乳酸平均生成速率在12～24 h为0.20 g/(L·h)左右,低于常规发酵体系的0.683 g/(L·h)。上下相聚合物浓度的变化对餐厨垃圾发酵影响不明显。PEG/DEX体系在发酵过程中,体积比稳定,不受产物与基质浓度变化的影响。PEG分子量增加对发酵影响不大;而DEX分子量从20000增加到40000,乳酸生成速率相应由0.631 g/(L·h)降至0.518 g/(L·h),乳酸产量也由33 g/L降至22 g/L。提高接种率可缩短乳酸菌的停滞期,并加速乳酸生成,当接种率为1%、4%和8%时,6～48 h内乳酸平均生成速率分别为0.558 g/(L·h)、0.602 g/(L·h)、0.649 g/(L·h)。在PEG10000/DEX20000体系进行的多批次连续发酵结果表明,由于DEX相很好地富集乳酸菌,每批次发酵时乳酸菌能保持高浓度,乳酸菌生长不经历停滞期、对数期,相比于传统发酵极大缩短发酵时间;在不投加缓冲剂的情况下,餐厨垃圾发酵单批次乳酸产率大于0.30 g/g,累积产率大于0.45 g/g。
Kitchen wastes have been the most abundant and problematic organic solid waste in the world since they are the main source of decay, odor and leachate in collection and transportation due to its high volatile solids and moisture content. However, as kitchen wastes contain rich nutrition, including carbohydrate, lipid, protein and other compounds, it could be a potential raw material for recycling utilization. Compared to the landfill, incineration and compost, lactic acid fermentation is a sustainable technology for kitchen wastes. It can effectively achieve kitchen wastes minimization and generate lactic acid for energy recovery. Lactic acid has both hydroxyl and carboxyl groups with one chiral carbon atom, and it is widely used in the food, pharmaceutical and general chemical industries. In addition, lactic acid can be polymerized to form the biodegradable and recyclable polyester polylactic acid, which is considered a potential substitute for plastics manufactured from petroleum. Thus, it is necessary to develop an efficient lactic acid fermentation process to stabilize the kitchen wastes.
The present study dealt with lactic acid production using kitchen wastes as substrate. A homofermentative Lactobacillus plantarum strain, isolated from kitchen wastes, was employed as the starter culture in the quick storage of kitchen wastes to suppress both pathogenic and spoilage microorganisms. Response surface methodology was adopted to optimize lactic acid production during simultaneous saccharification and fermentation of kitchen wastes by examining five independent variables (α-amylase/solid ratio, protease/solid ratio, yeast extract concentration, temperature and CaCO3 concentration). The combination of Plackett-Burman design and response surface methodology was employed to screen and optimize various variables for lactic acid bacteria partitioning in aqueous two-phase system under different operating conditions. Moreover, PEG/DEX systems were also used to extract lactic acid from kitchen wastes fermentation.
⑴A strain of lactic acid bacteria was initially isolated from 10 day fermented kitchen wastes on MRS agar. After identified by normally physiological and biochemical tests and API 50 CH galleries, the strain was a homofermentative Lactobacillus plantarum species and was named Lactobacillus plantarum BP04. Experiments from growth curve indicated that this strain could grow quickly and reach stationary phase within 12 h. In addition, its lactate production capacity could be up to 138.09 g/L.
⑵Lactobacillus plantarum BP04 was employed as starter culture in kitchen wastes storage with different inoculant levels at 0, 2 and 10% (v/w) to suppress the outgrowth of pathogenic and spoilage bacteria. Inoculation was effective in accelerating pH drop and reducing the growth period of enterobacteria to 9.7 and 2 days, corresponding to inoculant levels at 0, 2 and 10% (v/w). Increasing inoculum levels were found to inhibit the growth of Lactobacillus brevis and Leuconostoc lactis. HPLC analysis revealed that lactic acid was the predominant organic acid during the treatment of kitchen wastes. Its concentration varied among the fermented processes reflecting variations of microbial activity in the fermented media.
⑶Central composite design using response surface methodology was employed to optimize parameters ofα-amylase, protease, temperature, CaCO3 and yeast extract in simultaneous saccharification and fermentation process. A satisfactory fit of the quadratic model was realized. Lactic acid biosynthesis was significantly affected by interaction of protease×temperature. Protease, temperature and CaCO3 had significantly linear effects on lactic acid production whileα-amylase and yeast extract had insignificant effects. Yeast extract was proved to be unnecessary in eatery food waste fermentation. The optimum condition was found to beα-amylase at 13.86 U/g dried food waste, protease at 2.12 U/g dried food waste, temperature at 29.31℃and CaCO3 at 62.67 g/l with the maximum lactic acid concentration and yield at 98.51 g/l and 88.75%, respectively. Increase of inoculum size would be suitable by accelerating depletion of initial soluble carbohydrate to enhanceα-amylase efficiency in kitchen wastes fermentation. Increasing magnitudes of CaCO3 supplement could improved the reducing sugars depletion rate and led to the balance between the rates of hydrolysis and fermentation favorable to hydrolysis.
⑷The combination of Plackett-Burman design and response surface methodology was used to examine partitioning parameters for Lactobacillus plantarum BP04 in aqueous two-phase system with polyethylene glycol (PEG)/dextran (DEX) as the biphasic system. One-sided partition of cells to DEX phase was observed in all trials. Results from Plackett-Burman design presented that the molecular weight of PEG and DEX had highly significant and negative effects on volume ratio while cell density, electrolytes, settling time and pH were insignificant. PEG10000 and DEX20000 were further optimized by response surface methodological approach. Volume ratio was strongly affected by the variation of polymer concentration whereas the interaction between PEG10000 and DEX20000 was insignificant. The optimum condition was found to be PEG10000 at 6% (w/w) and DEX20000 at 13.85% (w/w) at the experimental range with the minimal volume ratio at 0.815.
⑸PEG/DEX systems were also used to extract lactic acid from kitchen wastes fermentation. Results showed that PEG/DEX was a good biocompatible system which could effectively extract lactic acid from fermented media. PEG10000/DEX20000 biphasic system had little effect on the growth of Lactobacillus plantarum BP04, whereas lactic acid production rate was about 0.20 g /(L·h) between 12 and 48 h, much lower than 0.631 g/(L·h) in conventional fermentation system. The variation of PEG and DEX concentrations influenced cells growth and lactic acid biosynthesis indistinctively. Volume ratio kept stable throughout the processes, despite the changes of raw material and product concentrations. Different from little influence of PEG molecular weight on fermentation, the increase of DEX molecular weight from 20000 to 40000 led to declines of lactate conversion rate from 0.631 g/(L·h) to 0.518 g/(L·h) and lactic acid concentration from 33 g/L To 22 g/L. Increasing magnitudes of inoculum size could shorten the period of lag phase and enhance the production rate. When inoculum sizes were at 1%、4% and 8% (w/w), the corresponding lactic acid conversion rate were 0.558 g/(L·h) and 0.602 g/(L·h) and 0.649 g/(L·h), respectively. A repeated extractive fermentation was carried out in PEG10000/ DEX20000 biphasic system with four top-phase replacements. Results presented that when cell density reached the stationary phase in the first extractive fermentation, the lactate production in the aqueous two-phase system was maintained.
引文
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