生物制氢最新研究进展与发展趋势

氢能具有清洁、高效、可再生的特点,是一种最具发展潜力的化石燃料替代能源.与传统的热化学和电化学制氢技术相比,生物制氢具有低能耗、少污染等优势.近年来,生物制氢技术在发酵菌株筛选、产氢机制、制氢工艺等方面取得了较大进展.暗发酵和光发酵结合制氢技术是一种新技术,具有较高的氢气产量.以厌氧细菌和光合细菌为发酵菌种,以富含碳水化合物的工农业废弃物为原料,进行暗发酵和光发酵结合制氢,具有广阔发展前途前景.本文综述了国内外生物制氢技术研究进展,展望了未来发展趋势.     

高温条件下混菌发酵合成气产乙酸及其群落结构

合成气(主要包括CO、H_2和CO_2)通过生物转化生产高附加值的生物燃料和化学品已引起人们广泛关注,微生物菌群作为生物转化的酶催化剂对合成气发酵产物组成和效率十分关键.通过富集得到高温条件下分别稳定转化CO、甲酸钠和合成气的厌氧菌群,探究CO与甲酸钠转化菌混培物和合成气转化菌发酵合成气生成乙酸的能力,并分析其微生物群落结构.结果显示,CO-甲酸钠转化菌混培物与合成气转化菌在合成气发酵前期主要进行CO的产氢反应生成H_2和CO_2以及同型产乙酸反应生成乙酸,CO利用率为100%,CO反应速率分别为6.93和6.34 mmol L~(-1)d~(-1);随后同型产乙酸菌利用H_2和CO_2继续合成乙酸,两者的乙酸最大累积量分别为9.11 mmol/L和8.01 mmol/L.CO-甲酸钠转化菌混培物主要菌群为Thermoanaerobacterium、Romboutsia、Ruminococcus、Clostridium、Eubacterium、Moorella和Desulfotomaculum属,合成气转化混菌则主要含有Romboutsia、Thermoanaerobacterium、Moorella、Eubacterium、Acetonema和Clostridium属,其中同型产乙酸菌广泛分布于Ruminococcus、Clostridium、Eubacterium、Moorella和Acetonema属.本研究表明复配CO和甲酸钠转化菌可用于合成气高温发酵产乙酸,且转化能力优于合成气转化菌,结果可为合成气混菌发酵提供微生物资源和技术参考.     

微生物絮凝剂絮凝特性的研究

考察了微生物絮凝剂产生菌Bacillus sp.B-2发酵生产絮凝剂的絮凝特性。研究表明,该菌在生长过程中产生絮凝剂并将其分泌到细胞外,每升发酵液可制得絮凝剂粗品1．7g,絮凝活性最高达97％以上,絮凝效能优于目前市售化学絮凝剂。该絮凝剂凝聚絮凝高岭土悬浊液最适宜的pH值在大于7的偏碱性条件,Ca^2 ,Mg^2 ,Mn^2 ,Al^3 等二价、三价金属离子对其絮凝具有促进作用,且随着Ca^2 浓度的增加,促进作用加强。     

从原料到产品:生物基丁二酸研究进展

丁二酸作为一种重要的有机化工原料及中间体,广泛用于生物高分子、食品与医药等行业,市场潜在需求量巨大;同时作为一种优秀的C4平台化合物,被认为是未来12种最具发展前景的生物炼制产品之一.近年来随着石化资源的日益枯竭及环境污染问题的日益严峻,以生物质为原料生产丁二酸等生物基产品的研究备受国内外研究者的关注.本文从产丁二酸菌种的种类及常见菌株产丁二酸的代谢途径、产丁二酸工程菌的改造、丁二酸发酵过程控制与优化、丁二酸的分离提取工艺等4个方面综述近年来国内外生物基丁二酸研究进展,其中以产丁二酸工程菌的改造为重点展开详细阐述.为提高菌株产丁二酸的能力,研究者们常采用代谢工程技术改造菌株,皆取得显著效果.近来也出现了利用ARTP法和基因组重排技术选育高产丁二酸的菌株.此外,高效的丁二酸发酵与其发酵原料,发酵过程中相关控制因素如p H、CO2和H2浓度以及发酵方式密切相关;相比其他的丁二酸分离法,原位分离法回收丁二酸具备优势.最后对产丁二酸菌种的改造进行展望,认为利用适应性进化和最小基因组等技术筛选优良丁二酸生产菌是未来的趋势.     

营养和环境条件对生物絮凝剂合成的影响

在考察了红平红球菌Rhodococcus erythropolis CCRC10900基本生长代谢特性的基础上,研究了营养条件对生物絮凝剂合成的影响。R.erythropolis CCRC10909合成的絮凝剂约80％以上分泌至胞外培养基中,仅20％左右的絮凝活性存在于菌体细胞上,蔗糖是该菌生长及絮凝剂合成的最佳碳源;玉米浆既能刺激菌体生长,又能显著提高絮凝剂的水平,生物絮凝剂的合成与培养基碳氮比密切相关,碳氮比在20－30之间时,絮凝活性达到最大,碳氮比低于10或高于100后,絮凝活性迅速下降,提出了促进絮凝剂合成的控制策略,发酵前期适当提高玉米浆的浓度,发酵后期按碳氮比为20－30,补加一定量的蔗糖和尿素,实验结果表明,采用这一策略,最终发酵液的絮凝活性和细胞生长量分别增长了8％和33．3％。     

细胞絮凝及硫酸锌对酿酒酵母乙酸胁迫耐性的影响

酿酒酵母细胞絮凝和外源添加锌离子对其环境胁迫耐受性都具有促进作用,为了解细胞絮凝形态对锌促进乙醇发酵的影响,比较硫酸锌添加对絮凝酿酒酵母SP SC01及其絮凝基因失活突变体SPSC01 FLO1Δ在乙酸胁迫条件下乙醇发酵的影响.结果显示,与野生型絮凝酵母相比,添加锌可更明显改善SPSC01 FLO1Δ在乙酸中的生长和发酵,在10 g L~(-1)乙酸存在的情况下,SPSC01在70 h消耗100 g L~(-1)葡萄糖,锌离子添加后可使发酵终点提前10 h,而SPSC01FLO1Δ在锌离子添加后发酵时间为48 h,可将发酵时间显著缩短138 h.这些结果表明,锌在酿酒酵母细胞缺少絮凝保护的条件下更能有效发挥作用,同时甘油、琥珀酸的增加在絮凝基因敲除突变体中更加明显.本文研究结果可为进一步利用絮凝及锌响应调控基因提高酿酒酵母的环境胁迫耐受性,提高燃料乙醇的生产效率奠定基础.     

温度对赤霉素发酵的影响及优化策略

温度是影响赤霉素发酵的重要因素,研究温度与菌丝生长和次级代谢物合成的关系将有助于发酵工艺优化.采用5 L全自动发酵罐,调控罐温在25-35℃范围内进行赤霉素发酵,分时记录菌体浓度、残糖浓度和产物赤霉素浓度变化情况.发酵过程数据经微分处理后,比较分析在不同温度下赤霉素发酵过程中菌比生长速率、菌得率及赤霉素比合成速率的变化特征.结果显示:最适菌丝生长温度是32℃,其比生长速率和得率分别为0.571/h和0.431 g/g;适宜GA3合成的温度是28℃,最大比合成速率为2.161 mg g~(-1) h~(-1).进而提出赤霉素发酵过程变温控制轨迹:发酵0-18 h,控温28℃;18-40 h,温度32℃;40-60 h控制温度为30℃;60 h后,控制温度28℃.采用此温度控制策略进行GA3发酵,赤霉素终浓度达到2 294 mg/L,比恒温28℃发酵浓度提高了11.14%.本研究表明赤霉素发酵过程分阶段变温控制策略能提高产量,具有产业应用前景.     

利用鱼粉废水生产生物絮凝剂及其性能研究

研究了不同培养时间下鱼粉废水中假单胞菌(Pseudomonas sp.)GX4—1菌的生长状况、培养液pH值和絮凝活性变化，发现该絮凝剂产生于菌生长过程中的，培养基灭菌与否对菌合成絮凝剂的特性无大影响．与其它几种常用絮凝剂相比，该絮凝剂对高岭土悬液的絮凝活性较高(絮凝率达95％以上)．发酵液经中速离心获得的上清液作为絮凝剂样品不仅具有良好的热稳定性，且在低温储存170d内活性稳定，对土壤悬液和大肠杆菌悬液表现出良好的絮凝能力。     

微生物絮凝剂生产菌T1的鉴定及其对生活污水絮凝特性

从活性污泥中分离筛选絮凝剂产生菌,得到一株对高岭土悬浊液具有较高絮凝活性的微生物絮凝菌T1.根据菌株的生理生化鉴定以及16S r DNA基因序列分析,确定该菌株为芽孢杆菌(Bacillus sp),该菌的絮凝活性物质分布在发酵液上清液中.通过正交实验确定最佳生活污水絮凝条件为絮凝剂投加量5%,污水p H值7.0,助凝剂Ca Cl2投加量为2%,在此条件下,T1发酵上清液对生活污水的絮凝率为83.85%.     

利用杂草培养料栽培双孢蘑菇的可行性

充分利用当地杂草资源生产双孢蘑菇对于降低生产成本和环境保护具有重要意义.在密云库区杂草多样性调查和配方工艺调整的基础上,对3个批次库区杂草的双孢蘑菇(Agaricus bisporus)生产进行全程监测,包括堆肥期和出菇期的培养料理化性质、物质变化、相关降解酶活性和培养料发酵过程中细菌菌群变化,统计产量等.结果显示,库区杂草种类繁多,株高茎粗,茎杆坚硬紧实,打捆后的杂草饱和吸水量为76.78%.堆肥期培养料的含水量、含碳量和碳氮比逐渐下降,出菇期变化不大;培养料的含氮量在培养料一次发酵结束(PI)均有下降的现象,二次发酵结束(PII)含氮量升高.培养料二次发酵过程中纤维素和半纤维素的利用率均介于40%-60%之间;木质素利用率介于20%-30%.在双孢蘑菇菌丝生长和出菇过程中木质素的利用率为16%-21%.培养料纤维素和半纤维素的含量变化与相关降解酶变化情况相一致.堆肥期样品Illumina测序得到432 595条有效序列,序列平均长度为441 bp;分类分析表明,堆肥时期优势菌群为拟杆菌门(Bacteroidetes)的普氏菌属(Prevotella),厚壁菌门(Firmicutes)的芽孢杆菌属(Bacillus),栖热菌门(Deinococcus-Thermus)的Thermus属、Truepera属、Caldicoprobacter属(一种木聚糖降解菌),放线菌门(Actinobacteria)的热多孢菌属(Thermopolyspora),变形菌门(Proteobacteria)的假黄单胞菌属(Pseudoxanthomonas). 3个批次产量在17.1-19.7 kg/m2范围.本研究表明库区杂草取代麦草进行双孢蘑菇工厂化生产是可行的,结果可为其合理利用和工艺改进提供理论依据.     

Vacuum promotes metabolic shifts and increases biogenic hydrogen production in dark fermentation systems

The successful operation of any type of hydrogen-producing bioreactor depends on the performance of the microorganisms present in the system. Both substrate and partial gas pressures are crucial factors affecting dark fermentation metabolic pathways. The main objective of this study was to evaluate the impact of both factors on hydrogen production using anaerobic granular sludge as inoculum and, secondly, to study the metabolic shifts of an anaerobic community subjected to low partial gas pressures. With this goal in mind, seven different wastewater (four synthetic media, two industrial wastewater, and one domestic effluent) and the effect of applying vacuum on the systems were analyzed. The application of vacuum promoted an increase in the diversity of hydrogenproducing bacteria, such as Clostridium, and promoted the dominance of acetoclastic- over hydrogenotrophic methanogens. The application of different media promoted a wide variety of metabolic pathways. Nevertheless, reduction of the hydrogen partial pressure by application of vacuum lead to further oxidation of reaction intermediates irrespective of the medium used, which resulted in higher hydrogen and methane production, and improved the COD removal. Interestingly, vacuum greatly promoted biogenic hydrogen production from a real wastewater, which opens possibilities for future application of dark fermentation systems to enhance biohydrogen yields.     

Evaluating the impact of sulfamethoxazole on hydrogen production during dark anaerobic sludge fermentation

● SMX promotes hydrogen production from dark anaerobic sludge fermentation. ● SMX significantly enhances the hydrolysis and acidification processes. ● SMX suppresses the methanogenesis process in order to reduce hydrogen consumption. ● SMX enhances the relative abundance of hydrogen-VFAs producers. ● SMX brings possible environmental risks due to the enrichment of ARGs. The impact of antibiotics on the environmental protection and sludge treatment fields has been widely studied. The recovery of hydrogen from waste activated sludge (WAS) has become an issue of great interest. Nevertheless, few studies have focused on the impact of antibiotics present in WAS on hydrogen production during dark anaerobic fermentation. To explore the mechanisms, sulfamethoxazole (SMX) was chosen as a representative antibiotic to evaluate how SMX influenced hydrogen production during dark anaerobic fermentation of WAS. The results demonstrated SMX promoted hydrogen production. With increasing additions of SMX from 0 to 500 mg/kg TSS, the cumulative hydrogen production elevated from 8.07 ± 0.37 to 11.89 ± 0.19 mL/g VSS. A modified Gompertz model further verified that both the maximum potential of hydrogen production (P_m) and the maximum rate of hydrogen production (R_m) were promoted. SMX did not affected sludge solubilization, but promoted hydrolysis and acidification processes to produce more hydrogen. Moreover, the methanogenesis process was inhibited so that hydrogen consumption was reduced. Microbial community analysis further demonstrated that the introduction of SMX improved the abundance of hydrolysis bacteria and hydrogen-volatile fatty acids (VFAs) producers. SMX synergistically influenced hydrolysis, acidification and acetogenesis to facilitate the hydrogen production.     

Effect of different gas releasing methods on anaerobic fermentative hydrogen production in batch cultures

Decreasing hydrogen partial pressure can not only increase the activity of the hydrogen enzyme but also decrease the products inhibition, so it is an appropriate method to enhance the fermentative hydrogen production from anaerobic mixed culture. The effect of biogas release method on anaerobic fermentative hydrogen production in batch culture system was compared, i.e., Owen method with intermediately release, continuous releasing method, and continuous releasing+ CO₂ absorbing. The experimental results showed that, at 35°C, initial pH 7.0 and glucose concentration of 10 g·L^-1, the hydrogen production was only 28 mL when releasing gas by Owen method, while it increased two times when releasing the biogas continuously. The cumulative hydrogen production could reach 155 mL when carbon dioxide in the gas stream was continuously absorbed by 1 mol·L^-1 NaOH. The results showed that acetate was dominated, accounting for 43% in the dissolved fermentation products in Owen method, whereas the butyrate predominated and reached 47%–53% of the total liquid end products when releasing gas continuously. It is concluded that the homoacetogenesis could be suppressed when absorbing CO₂ in the gas phase in fermentative hydrogen production system.     

Fermentative hydrogen production from beet sugar factory wastewater treatment in a continuous stirred tank reactor using anaerobic mixed consortia

A low pH, ethanol-type fermentation process was evaluated for wastewater treatment and bio-hydrogen production from acidic beet sugar factory wastewater in a continuous stirred tank reactor (CSTR) with an effective volume of 9.6 L by anaerobic mixed cultures in this present study. After inoculating with aerobic activated sludge and operating at organic loading rate (OLR) of 12 kgCOD?m^-3·d^-1, HRT of 8h, and temperature of 35°C for 28 days, the CSTR achieved stable ethanol-type fermentation. When OLR was further increased to 18 kgCOD?m^-3·d^-1 on the 53rd day, ethanol-type fermentation dominant microflora was enhanced. The liquid fermentation products, including volatile fatty acids (VFAs) and ethanol, stabilized at 1493 mg·L^-1 in the bioreactor. Effluent pH, oxidation-reduction potential (ORP), and alkalinity ranged at 4.1–4.5, -250–(-290) mV, and 230–260 mgCaCO₃?L^-1. The specific hydrogen production rate of anaerobic activated sludge was 0.1 L?gMLVSS^-1·d^-1 and the COD removal efficiency was 45%. The experimental results showed that the CSTR system had good operation stability and microbial activity, which led to high substrate conversion rate and hydrogen production ability.     

Continuous biohydrogen production from diluted molasses in an anaerobic contact reactor

An anaerobic contact reactor (ACR) system comprising a continuous flow stirred tank reactor (CSTR) with settler to decouple the hydraulic retention time (HRT) from solids retention time (SRT) was developed for fermentative hydrogen production from diluted molasses by mixed microbial cultures. The ACR was operated at various volumetric loading rates (VLRs) of 20–44 kgCOD·m^-3·d^-1 with constant HRT of 6 h under mesophilic conditions of 35°C. The SRT was maintained at about 46–50 h in the system. At the initial VLR of 20 kgCOD·m^-3·d^-1, the hydrogen production rate dropped from 22.6 to 1.58 L·d^-1 as the hydrogen was consumed by the hydrogentrophic methanogen. After increasing the VLR to 28 kgCOD·m^-3·d^-1 and discharging the sludge for 6 consecutive times, the hydrogentrophic methanogens were eliminated, and the hydrogen content reached 36.4%. As the VLR was increased to 44 kgCOD·m^-3·d^-1, the hydrogen production rate and hydrogen yield increased to 42.1 L·d^-1 and 1.40 mol H₂·molglucose-consumed^-1, respectively. The results showed that a stable ethanol-type fermentation that favored hydrogen production in the reactor was thus established with the sludge loading rate (SLR) of 2.0–2.5 kgCOD·kgMLVSS^-1·d^-1. It was found that the ethanol increased more than other liquid fermentation products, and the ethanol/acetic acid (mol/mol) ratio increased from 1.27 to 2.45 when the VLR increased from 28 to 44 kgCOD·m^-3·d^-1, whereas the hydrogen composition decreased from 40.4% to 36.4%. The results suggested that the anaerobic contact reactor was a promising bioprocess for fermentative hydrogen production.     

Enhanced hydrogen production in microbial electrolysis through strategies of carbon recovery from alkaline/thermal treated sludge

• High hydrogen yield is recovered from thermal-alkaline pretreated sludge. • Separating SFL by centrifugation is better than filtration for hydrogen recovery. • The cascaded bioconversion of complex substrates in MECs are studied. • Energy and electron efficiency related to substrate conversion are evaluated. The aim of this study was to investigate the biohydrogen production from thermal (T), alkaline (A) or thermal-alkaline (TA) pretreated sludge fermentation liquid (SFL) in a microbial electrolysis cells (MECs) without buffer addition. Highest hydrogen yield of 36.87±4.36 mgH₂/gVSS (0.026 m³/kg COD) was achieved in TA pretreated SFL separated by centrifugation, which was 5.12, 2.35 and 43.25 times higher than that of individual alkaline, thermal pretreatment and raw sludge, respectively. Separating SFL from sludge by centrifugation eliminated the negative effects of particulate matters, was more conducive for hydrogen production than filtration. The accumulated short chain fatty acid (SCFAs) after pretreatments were the main substrates for MEC hydrogen production. The maximum utilization ratio of acetic acid, propionic acid and n-butyric acid was 93.69%, 90.72% and 91.85%, respectively. These results revealed that pretreated WAS was highly efficient to stimulate the accumulation of SCFAs. And the characteristics and cascade bioconversion of complex substrates were the main factor that determined the energy efficiency and hydrogen conversion rate of MECs.     

产氢新种Ethanoligenens harbinese B49的氮素营养与代谢特征

从氮源角度研究了一株产氢新种Ethanoligenens harbineseB49发酵葡萄糖和糖蜜的产氢特性及其营养需求,以及酵母粉对产氢菌E.harbinese B49生长和产氢的特殊效应.试验结果表明,在以葡萄糖为底物条件下,以酵母粉替代蛋白胨作为唯一氮源可以大大提高E.harbineseB49的产氢能力,单位体积产氢量从1700mL L-1培养基提高到2400mL L-1培养基,产氢能力提高40.35%.该条件下去除维生素液,E.harbinese B49的产氢能力不受影响.从E.harbinese B49产氢动力学分析可以看出,对数生长期处于12~22h期间.从对数期开始迅速产氢,并且一直持续到稳定前期,产氢速率达到16.8mL/h,生物气中最大氢含量为41%.驯化后的E.harbinese B49利用糖蜜为底物产氢,糖蜜COD为13g L-1时,单位体积最大产氢能力为1576mL L-1.当培养基中额外加入0.5g L-1酵母粉时,可以大大促进E.harbinese B49发酵糖蜜产氢的能力,单位体积氢产量达到1960mL L-1,提高了24.4%,比产氢率达到150.8mL(H2)/g(COD).以牛肉膏、蛋白胨、尿素为氮源时,E.harbineseB49产氢量提高较小.图4表2参16     

Clostridium papyrosolvens厌氧发酵产氢研究

采用分批培养研究了从高浓度厌氧产氢活性污泥中筛选的优势菌种Clostridium papyrosolvens的发酵产氢能力.结果表明:该菌有较强的高糖耐受性和耐酸性,当葡萄糖浓度为30 g/L、pH阶段性控制在4.5时,发酵44 h葡萄糖消耗率为83.7%,总产气量达到3 081.3 mL/L,最高产气率为187.5 mL L-1 h-1,氢气含量为67.5%,比产氢率达1.06 mol(H2)/mol(葡萄糖).研究中选用了廉价的发酵产氢培养基,以玉米浆为氮源,以还原铁粉作氧化还原电位控制剂,省去了牛肉膏、蛋白胨等昂贵氮源以及L-半胱氨酸、维生素、无机离子等高成本组分,显著降低了纯菌发酵的培养基成本,获得了较好的产氢效果.图5表2参23     

几株光合细菌的气体代谢及对产甲烷细菌甲烷生成的影响

三株分别属于光合细菌三个主要类群（紫无硫细菌、紫硫细菌和绿硫细菌）的菌种和两种产甲烷细菌进行了混合培养试验，结果表明，自养生长的光合细菌减少了产甲烷菌有ＣＨ４生成，而异养生长时紫无硫细菌Ｒｈｏｄｏｐｓｅｕｌｏｍｏｕｓ ｐａｌｕｓｔｒｉｓ能利用乙酸放出ＣＯ２供产甲烷菌利用。在光照厌氧条件下，三株光合细菌的生长培养物和静止细胞都能利用有机酸产氢，有时还伴随着ＣＯ２产出。同时试验了不同ｐＨ值、温度和光照