Ethanol production system from livestock increments by transgenic thermophile

用聚合酶链式反应从杆菌中分离出纤维素酶基因后，利用转座子将该片段转入乙醇型嗜热厌氧杆菌。然而，没有获得任何有效的重组体。为了进行同源重组，我们克隆了氢酶基因。分离到一个可能的氢酶基因片段，但同源重组尚未成功。由于转基因嗜热菌的产生很困难，因此也尝试了从环境中分离出有用的嗜热菌。我们成功地分离到了高产乙醇厌氧菌003和004。它们表现出广泛的底物特异性，包括木聚糖，但不包括纤维素。但在实际应用中，还会出现除氧、厌氧发酵产生恶臭等问题。为了解决这些问题，人们认为与有效的好氧嗜热菌共培养将是活跃的。分离到好氧细菌A1和B3，提高了厌氧菌产乙醇的能力。然而，在任何情况下共培养都只能产生3-4 mM的乙醇，这可能是因为木质纤维素降解产生的抑制剂可能会抑制它们的活性。因此，在去除培养基中的大量植物纤维碎片后，与需氧菌A1菌株和厌氧菌003菌株共培养，可产生24 mM的乙醇。综上所述，成功地分离到了将畜禽增量转化为乙醇的有效微生物，为利用畜禽增量构建乙醇生产系统提供了可能。为了进一步提高其产量，纤维素降解菌和乙醇产生菌的分离是必不可少的。这场审判正在进行中。

After isolation of cellulase gene by PCR from Clostridium stercorarium, this fragment was transfected into Thermoanaerobacter ethanolicus by using transposon. However, any effective recombinant Was not obtained. For homologous recombination, hydrogenase gene was isolated. A fragment of putative hydrogenase gene was isolated, but the homologous recombination was not succeeded yet. As the creation of genetically modified thermophile was difficult, isolation of useful thermophile was also tried from environment. We succeeded in the isolation of highly ethanol producing anaerobic bacteria, strain 003 and 004. They showed broad substrate specificities including xylan, but not cellulose. For their practical use, another problems must be occurred such as removing oxygen from the fermentor, generating bad smell because of anaerobic fermentation. To resolve these problems, it is thought that co-culture with an effective aerobic thermophile would be active. Aerobic bacteria, strain A1 and B3 were isolated, which enhanced ethanol production by the anaerobes. However, only 3-4 mM ethanol was produced in any case of co-culture, probably because inhibitors derived from lignocellulose degradation may suppress their activities. So, after removing large debris of plant fiber in the medium, co-culture with aerobe A1 strain and anaerobe 003 strain led to 24 mM ethanol production. In conclusion, the isolation of effective microorganisms converting livestock increments to ethanol was succeeded, which making it possible to construct the ethanol production system by utilizing live stock increments. To enhance its production further more, it is thought that the isolation of cellulose degrading and ethanol producing bacteria is essential. This trial is in progress now.