Ethanol production system from livestock increments by transgenic thermophile

用PCR方法从粪状梭菌中分离出纤维素酶基因后，利用转座子将该片段转染到乙醇热厌氧菌中。然而，没有获得有效的重组。对氢化酶基因进行同源重组。已分离出推测为氢化酶基因的片段，但同源重组尚未成功。由于基因改造嗜热菌的制备困难，人们也尝试从环境中分离出有用的嗜热菌。我们成功地分离出了产乙醇量高的厌氧细菌菌株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。为了实际应用，必须出现另一个问题，例如从发酵罐中除去氧气，由于厌氧发酵而产生难闻的气味。为了解决这些问题，认为与一个有效的好氧嗜热菌共培养将是积极的。分离出好氧菌A1和B3菌株，提高了厌氧菌的乙醇产量。然而，在任何情况下，共培养仅产生3-4毫米乙醇，可能是因为来自木质纤维素降解的抑制剂可能抑制其活性。因此，在去除培养基中的大量植物纤维碎片后，与需氧菌株A1和厌氧菌株003共培养，乙醇产量为24 mM。综上所述，成功分离出了将家畜饲料转化为乙醇的有效微生物，为构建家畜饲料乙醇生产体系提供了可能。为了进一步提高其产量，纤维素降解菌和乙醇生产菌的分离是必不可少的。这个试验正在进行中。

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.