Name: production of chemicals by klebsiella pneumoniae using bamboo hydrolysate as feedstock
Uploaded: 2017-06-29T14:00:00.000+00:00
Duration: 7 min 24 s
Description: Watch this Scientific Journal Video about Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock at JoVE.com

这项工作得到了国家自然科学基金（批准号:21576279,20906076）和KRIBB研究计划（KGM2211531）的支持。

竹水合物的制备<ol><li> 在40 mL NaOH溶液中加入5 g竹粉，使其在250 mL烧瓶中达到10％（g / g）终浓度。使用0.05M至0.50M的一系列NaOH溶液，以0.05M的增量。 <ol><li>将混合物在60℃或100℃的水浴中孵育60分钟。在121℃下在高压釜中孵育。 </li></ol></li><li> 孵育后，将混合物在室温下静置4小时。然后，取出上清液。加入100ml淡水并混合。 <ol><li>重复洗涤5-6次，直到上清液的pH达到6.8。使用所得固体进行下一步酶促水解。 </li></ol></li><li>对于水解，使用98％H 2 SO 4调节50mL上述混合物至pH 5.0。然后加入0.5 mL 200张滤纸活性（FPU）/ mL纤维素酶。 注意:纤维素酶与竹子的最终比例每1g竹子要20 FPU。 </li><li>将混合物在振荡器中在50℃下孵育36小时。 注意:水解后，混合物中将残留一些固体。 </li><li>通过以7,690 xg离心5分钟获得透明的水解产物。 </li><li>用配有折射率检测器和光电二极管阵列检测器的高压液相色谱系统对葡萄糖，木糖和其他化学物质进行定量。使用HPX-87H柱（300 mm x 7.8 mm）和流动相为5 mM H 2 SO 4溶液，流速为0.8 mL / min。 </li><li> 对于大量的水解产物，在30L不锈钢罐中混合2kg竹粉和18L 0.25M NaOH。 <ol><li>在121℃下在高压釜中孵育罐60分钟。孵育后，用60升塑料罐中的40升水清洗混合物。重复洗涤5次。 </li><li>用水调整洗涤的竹子总量为20升。使用98％H 2 SO 4将混合物的pH调节至5.0。 </li><li>在配备有机械搅拌器的改进的摇动水浴中将混合物酶水解，以确保混合物分布良好。加入200 mL 200 FPU / mL纤维素酶，并将混合物在50℃孵育36小时。 </li><li>水解后，以4,700 xg离心混合物10分钟。 </li></ol></li></ol> 2.野生型肺炎支原体的 2,3-丁二醇生产<ol><li>使用3升竹水解产物作为介质制备的溶剂。 </li><li>制备含有4g / L玉米浆，2g / L（NH 4 ） 2 SO 4，6g / LK 2 HPO 4,3g / L KH 2 PO 4和0.2g / L MgSO 4的发酵培养基。使用2.5M NaOH将介质的pH调节至中性。使用含有3升高压灭菌发酵培养基的5升生物反应器进行发酵。 </li><li>使用肺炎支原体细胞储存在-80℃的低温冰箱中。 </li><li>对于种子培养物，在37℃下，以200rpm摇动培养含有50mL Luria-Bertani（LB）培养基的250mL烧瓶过夜。使用含有10g / L胰蛋白胨，5g / L酵母提取物和10g / L NaCl的LB培养基。 注意:种子培养的细胞密度应达到OD 2（600nm处的光密度）。 </li><li>接种一瓶50ml的种子培养物在生物反应器中。在pH 6.0和37℃保持培养。使用2升/分钟的空气补充速率和250rpm的搅拌速率，产生微量厌氧条件。 </li><li> 在发酵过程中每2 h收集5 mL样品，并用高压液相色谱法分析，以确定肉汤中的化学物质浓度。 <ol><li>使用MFCS / DA在线监测添加到生物反应器中的碱。 注意:有机酸在发酵过程中产生，a加入NaOH以保持培养物pH稳定。加入的碱的体积代表产生的酸的量。碱添加线高原时，由于碳源耗尽或细胞死亡，该过程完成。 </li></ol></li></ol> 3.由肺炎克雷伯菌的budC突变体产生R-丝瓜素 <ol><li>制备含有4g / L玉米浆，2g / L（NH 4 ） 2 SO 4，3g / L乙酸钠，0.4g / L KCl和0.1g / L MgSO 4的乙偶姻生产的培养基。 </li><li>使用肺炎支原体ΔbudC进行R-丝氨酸生产。重复步骤2.4-2.5。 注意: budC编码2,3-丁二醇脱氢酶。 肺炎克雷伯菌ΔbudC失去2,3-丁二醇脱氢酶活性，产生R-乙酰胆碱代替2,3-丁二醇。 </li><li>在pH 6.0和37℃保持培养。使用4升/分钟的空气补充率和搅拌n速率为450 rpm，有氧条件。 注:补氧量高于2,3-丁二醇生产。 </li><li>按照步骤2.6测定样品。 </li></ol> 4. 肺炎克雷伯菌的budA突变株的2-酮葡萄糖酸生产<ol><li>对于2-酮葡萄糖酸生产，使用与R-组氨酸生产相同的培养基。 </li><li>使用肺炎克雷伯菌ΔbudA生产2-酮葡萄糖酸。重复步骤2.4-2.5。 注意: budA编码α-乙酰乳酸脱羧酶。 肺炎克雷伯菌ΔbudA失去α-乙酰乳酸脱羧酶活性，并以葡萄糖为底物产生2-酮基葡萄糖酸。 注意:2-酮葡萄糖酸合成是酸性条件依赖性过程。开发了2-酮葡萄糖酸生产的两阶段发酵10 ;在第一阶段，将种子培养物接种在生物反应器中。 </li><li&gt;在pH 7.0和37°C保持培养。空气补充速度为4L / min，搅拌速度为500rpm。 注意:在这些条件下，细胞生长非常快（4小时内细胞密度达到OD 7）。然后，发酵进行到第二阶段，在此期间培养物pH降至5.0。不加酸在培养基中产生的有机酸天然导致pH降低。在这些酸性条件下，细胞生长停止，但合成2-酮葡萄糖酸并积累在肉汤中。 </li><li>按照步骤2.6测定样品。使用竹子的水解物作为碳源。 注意:将培养基中的葡萄糖转化为2-酮葡萄糖酸，并将木糖转化为木糖酸。由此得到2-酮基葡萄糖酸和木糖酸的混合物。 </li></ol>

<ol>
	<li>Zeng, A. P., Biebl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+chemicals+from+biotechnology:+the+case+of+1,+3-propanediol+production+and+the+new+trends.">Bulk chemicals from biotechnology: the case of 1, 3-propanediol production and the new trends.</a> Adv Biochem Eng Biotechnol. 74, 239-259 (2002).</li><li>Wei, D., Wang, M., Jiang, B., Shi, J., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+dihydroxyacetone+kinases+I+and+II+in+the+dha+regulon+of+Klebsiella+pneumoniae.">Role of dihydroxyacetone kinases I and II in the dha regulon of Klebsiella pneumoniae.</a> J Biotechnol. 177, 13-19 (2014).</li><li>Celi&#324;ska, E., Grajek, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotechnological+production+of+2,3-butanediol-current+state+and+prospects.">Biotechnological production of 2,3-butanediol-current state and prospects.</a> Biotechnol Adv. 27 (6), 715-725 (2009).</li><li>Chen, C., Wei, D., Shi, J., Wang, M., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+2,+3-butanediol+stereoisomer+formation+in+Klebsiella+pneumoniae.">Mechanism of 2, 3-butanediol stereoisomer formation in Klebsiella pneumoniae.</a> Appl Microbiol Biotechnol. 98 (10), 4603-4613 (2014).</li><li>Wei, D., Wang, M., Shi, J., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+recombinase+assisted+gene+replacement+in+Klebsiella+pneumoniae.">Red recombinase assisted gene replacement in Klebsiella pneumoniae.</a> J Ind Microbiol Biotechnol. 39 (8), 1219-1226 (2012).</li><li>Wei, D., Sun, J., Shi, J., Liu, P., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategy+to+improve+efficiency+for+gene+replacement+in+Klebsiella+pneumoniae.">New strategy to improve efficiency for gene replacement in Klebsiella pneumoniae.</a> J Ind Microbiol Biotechnol. 40 (5), 523-527 (2013).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+RecBCD+in+Klebsiella+pneumoniae+by+Gam+and+its+effect+on+the+efficiency+of+gene+replacement.">Inhibition of RecBCD in Klebsiella pneumoniae by Gam and its effect on the efficiency of gene replacement.</a> J Basic Microbiol. 56 (2), 120-126 (2016).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R-acetoin+accumulation+and+dissimilation+in+Klebsiella+pneumoniae.">R-acetoin accumulation and dissimilation in Klebsiella pneumoniae.</a> J Ind Microbiol Biotechnol. 42 (8), 1105-1115 (2015).</li><li>Wei, D., Xu, J., Sun, J., Shi, J., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Ketogluconic+acid+production+by+Klebsiella+pneumoniae+CGMCC+1.6366.">2-Ketogluconic acid production by Klebsiella pneumoniae CGMCC 1.6366.</a> J Ind Microbiol Biotechnol. 40 (6), 561-570 (2013).</li><li>Sun, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-stage+fermentation+for+2-ketogluconic+acid+production+by+Klebsiella+pneumoniae.">Two-stage fermentation for 2-ketogluconic acid production by Klebsiella pneumoniae.</a> J Microbiol Biotechnol. 24 (6), 781-787 (2014).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gluconic+acid+production+by+gad+mutant+of+Klebsiella+pneumoniae.">Gluconic acid production by gad mutant of Klebsiella pneumoniae.</a> World J Microbiol Biotechnol. 32 (8), 1-11 (2016).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+xylonic+acid+by+Klebsiella+pneumoniae.">Production of xylonic acid by Klebsiella pneumoniae.</a> Appl Microbiol Biotechnol. 100 (23), 10055-10063 (2016).</li><li>Kumar, P., Barrett, D. M., Delwiche, M. J., Stroeve, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+pretreatment+of+lignocellulosic+biomass+for+efficient+hydrolysis+and+biofuel+production.">Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production.</a> Ind Eng Chem Res. 48 (8), 3713-3729 (2009).</li><li>Brisse, S., Grimont, F., Grimont, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genus+Klebsiella.">The genus Klebsiella.</a> The Prokaryotes. , 159-196 (2006).</li><li>Hao, J., Lin, R., Zheng, Z., Liu, H., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+microorganisms+able+to+produce+1,+3-propanediol+under+aerobic+conditions.">Isolation and characterization of microorganisms able to produce 1, 3-propanediol under aerobic conditions.</a> World J Microbiol Biotechnol. 24 (9), 1731-1740 (2008).</li><li>Hong, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+alkaline+pretreatment+on+corn+stover+for+enhanced+production+of+1.3-propanediol+and+2,+3-butanediol+by+Klebsiella+pneumoniae+AJ4.">Optimization of alkaline pretreatment on corn stover for enhanced production of 1.3-propanediol and 2, 3-butanediol by Klebsiella pneumoniae AJ4.</a> Biomass Bioenerg. 77, 177-185 (2015).</li><li>Pienkos, P. T., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+pretreatment+and+conditioning+processes+on+toxicity+of+lignocellulosic+biomass+hydrolysates.">Role of pretreatment and conditioning processes on toxicity of lignocellulosic biomass hydrolysates.</a> Cellulose. 16 (4), 743-762 (2009).</li></ol>

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肺炎 克雷伯杆菌属于肠杆菌科科克雷伯 菌属 。 肺炎克雷伯病在土壤，植被和水等自然环境中广泛分布14 。本研究中使用的野生型肺炎克雷伯杆菌菌株从土壤中分离，用​​于1,3-丙二醇生产15 。这种物种的肺炎支原体和突变体在不同条件下产生许多化学物质。 培养pH和空气补充是影响肺炎克雷伯氏菌化学生产的关键因素。野生型肺炎克雷伯菌在pH6下产生2,3-丁二醇作为主要的分解代谢物。然而，当在pH5或更低的pH 9培养时，2-酮基葡萄糖酸变为主要的分解代谢物。增加空气补充剂减少了2,3-丁二醇的合成，而R-乙酰胆碱的合成改善了8 </s了&gt;。使用促进稳定培养物pH和精确空气补充的生物反应器代替烧瓶用于细胞培养。因此，需要升升介质。本研究使用的预处理方法遵循Hong 等人提出的方法16 ，这在实验室很容易执行，特别是在放大级。 较高的预处理温度有利于酶水解过程中的糖生产。在水浴中和121℃下在高压釜中进行≤100℃的处理。这两台设备在实验室中很常见，每批处理几升液体。较高的温度需要高压反应堆。例如，我们的实验室有50 mL和1 L高压反应堆，但这些反应堆的体积限制了其在生物量预处理中的使用。 本研究中显示的竹子水解产物在烧瓶中易于进行。然而，当大量进行时，有时会获得较低水平的葡萄糖和木糖。水解混合物必须分布良好。生物量的沉淀将导致较低的水解水平。 与纯葡萄糖和木糖不同，生物质水解物被许多可能抑制微生物生长的有毒化学物质污染。已经开发了许多方法去除这些抑制剂，但是它们增加了工艺成本，并且倾向于降低糖产量17 。在这项研究中，没有做任何特别的工作去除抑制剂。使用本工作中生成的竹水解产物作为原料，当葡萄糖消耗时，第一阶段葡萄糖与2,3-丁二醇的生产率（0.95g / Lh）和转化率（0.25g / g）较低比纯化葡萄糖作为碳源时，先前报道（分别为1.4g / Lh和0.3g / gm） 4 。 2,3-丁烷的生产率在第二阶段，当木糖消耗时，iol比第一阶段慢。然而，木糖与2,3-丁二醇的转化率达到0.34g / g，高于纯化葡萄糖作为碳源4时的转化率。使用竹水解产物作为原料的R-维生素B的生产率和底物转化率分别为0.92g / Lh和0.29g / g。这两个比例都低于使用纯化葡萄糖作为底物（分别为1.7g / Lh和0.34g / g）。本研究中2-酮基葡萄糖酸的生产率和转化率分别为2.3 g / Lh和0.91 g / g，分别低于纯化葡萄糖作为碳源时（4.2 g / Lh和1g / g） ） 10 。 葡萄糖后，木糖是自然界中第二大丰富的糖。与葡萄糖不同，大多数微生物不容易利用木糖。在这个螺柱y，木糖完全被K. pneumoniae用于化学生产。 2,3-丁二醇， R-乙酰胆碱，2-酮基葡萄糖酸和木糖酸都是有价值的化学物质，其生产工艺从微需氧到高需氧条件不同。发酵结果表明，竹子水解产物中的糖是不同条件下肺炎克雷伯杆菌生长和化学生产的合适碳源。 使用生物质水解产物作为原料是化学生产的有希望的方法。然而，仍然有许多缺点必须克服，例如洗涤预处理的生物质所需的大量水和酶水解所需的漫长时间。

肺炎克雷伯杆菌是一种在有氧和无氧条件下都能良好生长的细菌。 肺炎支原体是用于生产许多化学物质的重要工业微生物。 1,3-丙二醇是一种有价值的化学物质，主要用作合成聚对苯二甲酸丙二醇酯的单体。聚对苯二甲酸丙二醇酯是一种可生物降解的聚酯，其表现出比1,2-丙二醇，丁二醇或乙二醇1更好的性能。 1,3-丙二醇由肺炎链球菌在氧气限制条件下使用甘油作为底物生产2 。 2,3-丁二醇及其衍生物在塑料，溶剂生产和合成橡胶领域有应用，有潜力被用作生物燃料3 。以葡萄糖为底物，2,3-丁二醇是野生型菌株4的主要代谢物。 2,3-丁二醇是合成的从丙酮酸。首先，两分子丙酮酸缩合得到α-乙酰乳酸;该反应由α-乙酰乳酸合成酶催化。然后通过α-乙酰乳酸脱羧酶将α-乙酰乳酸转变成乙偶姻。当丁二醇脱氢酶催化时，β-维生素可进一步还原成2,3-丁二醇。已经探索了适用于肺炎克雷伯菌的有效基因替代方法，并且已经构建了许多突变体5,6,7 。失去其2,3-丁二醇脱氢酶活性的budC突变体在培养液中积累高水平的乙偶姻。乙酰头痛被用作增加食物味道的添加剂。当编码α-乙酰乳酸脱羧酶的budA被突变时，2-ketogluconic acid积聚在肉汤中。 2-酮基葡萄糖酸用于合成异抗坏血酸（异抗坏血酸），用于食品工业的抗氧化剂9 。 2-酮葡萄糖酸是葡萄糖氧化途径的中间体;在该通路中，位于周质空间中，葡萄糖被氧化成葡萄糖酸，然后进一步氧化成2-酮基葡萄糖酸。葡萄糖酸和在周质中产生的2-酮葡萄糖酸可以转运到细胞质进一步代谢。 2-酮基葡萄糖酸的积累取决于酸性条件，更高的空气补充有利于2-酮葡萄糖酸生产10 。葡萄糖酸脱氢酶，由gad编码， 催化葡萄糖酸转化为2-酮聚糖酸。 肺炎克雷伯氏菌的gad突变体产生高水平的葡萄糖酸而不是2-酮葡萄糖酸，并且该过程也依赖于酸性条件。葡萄糖酸是一种大量有机酸，用作添加剂以增加水泥1的性能1。葡萄糖氧化成葡萄糖酸由葡萄糖脱氢酶催化。木糖也是葡萄糖脱氢酶的合适底物。当木糖用作底物时， 肺炎克雷伯菌产生木糖酸12 。 使用生物质作为原料的化学生产是生物技术的热门话题13 。生物质的主要成分是纤维素，半纤维素和木质素。然而，这些大分子化合物不能被大多数微生物（包括肺炎克雷伯菌 ）直接分解代谢。生物质中的纤维素和半纤维素必须水解成葡萄糖和木糖，然后可以被微生物使用。在木质纤维素中木质素的存在产生防止酶的生物质水解的保护屏障。因此，消除木质素和半纤维素并降低纤维素的结晶度的预处理过程总是在通过麦克风的生物量利用期间进行roorganisms。已经开发了许多预处理方法:酸，碱，氨和蒸汽预处理是常见的。 竹子在热带和亚热带地区丰富，是重要的生物质资源。本文介绍了竹水解产物的制备和竹水解产物的化学生产

<table><tbody><tr><td>autoclave</td><td>SANYO</td><td>3780</td><td></td></tr><tr><td>bioreactor</td><td>Sartorius stedim biotech</td><td>Bostat Aplus</td><td></td></tr><tr><td>agitator</td><td>IKA</td><td>RW 20</td><td></td></tr><tr><td>water bath shaker</td><td>Zhicheng</td><td>ZWY-110X50</td><td></td></tr><tr><td>high performance liquid chromatograph system</td><td>Shimadzu Corp</td><td>20AVP</td><td></td></tr><tr><td>centrifuge</td><td>Hitachi</td><td>CR22G III</td><td></td></tr><tr><td>Bamboo powder</td><td>purchased from Zhejiang Province, China</td><td></td><td>mesh number, 50</td></tr><tr><td>cellulase</td><td>Youtell Biochemical, Shandong, China</td><td></td><td>200 PFU/mL</td></tr></tbody></table>

production of chemicals by klebsiella pneumoniae using bamboo hydrolysate as feedstock

竹是重要的生物质，竹科水解产物被克雷伯杆菌用作化学生产的原料。这里，用NaOH预处理竹粉，洗涤至中性pH。将纤维素酶加入预处理的竹粉中以产生含有30g / L葡萄糖和15g / L木糖的水解产物，并用作碳源以制备用于化学生产的培养基。当在微需氧条件下培养时，野生型肺炎克雷伯菌产生12.7g / L 2,3-丁二醇。在需氧条件下， 肺炎支原体 芽胞芽孢杆菌突变体产生了13.0g / L的R-丝氨酸。在肺炎链球菌的budA突变体的两阶段，pH控制的发酵中补充高空气产生了25.5g / L的2-酮基葡萄糖酸和13.6g / L的木糖酸的混合物。在发酵的第一阶段，将培养物保持在中性pH值;细胞生长后，发酵n进入第二阶段，在此阶段，文化被允许变成酸性。

在这个方案中，竹子是用碱预处理的。在图1和图 2中确定了最佳孵育参数 - 温度为121℃和0.25M NaOH。将预处理的竹子酶水解，测定水解产物中得到的葡萄糖和木糖浓度。更高的温度有利于糖的生产，所以选择121°C作为最佳温度。在水解产物中产生的葡萄糖和木糖在NaOH浓度范围内在0.05-0.25Ma范围内增加。NaOH浓度的进一步增加对糖的产生没有积极的影响。因此，选择0.25M的NaOH作为最佳浓度。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55828/55828fig1.jpg" /> 图1:预处理温度对葡萄糖和木糖的影响竹子水解物的浓度。红柱:葡萄糖;蓝色柱:木糖。更高的温度有利于糖的生产，并且选择121℃用于大量水解产物。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55828/55828fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55828/55828fig2.jpg" /> 图2:预处理过程中NaOH浓度对竹子水解产物葡萄糖和木糖浓度的影响。红线:葡萄糖;蓝线:木糖。在水解产物中产生的葡萄糖和木糖在0.05-0.50M的范围内随着NaOH浓度的增加而增加，对于大体积的水解产物，选择0.25M。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55828/55828fig2large.jpg" target="_blank">请点击他</a>重新查看这个数字的较大版本。 在烧瓶规模酶水解过程中，在水解产物中产生约20g / L葡萄糖和10g / L木糖;从大容量水解产物得到30g / L葡萄糖和15g / L木糖。这是因为在开放式水浴摇床中进行了大批量的制备，并且在该过程中蒸发了一些水，导致了水解产物的浓缩。竹子水解液中的葡萄糖和木糖被肺炎克雷伯杆菌用作化学生产的碳源。水解产物中的其他化合物为:纤维二糖（1.4g / L），阿拉伯糖（8.9g / L），乙酸（1.9g / L）和甲酸（0.2g / L）。 2,3-丁二醇由肺炎支原体在微需氧条件下生产（ 图3 ）。该过程分为两个时期。在f首先，细胞使用葡萄糖产生7.6g / L 2,3-丁二醇，而肉汤中的木糖水平保持不变。葡萄糖在8小时内耗尽，这时候转移到下一个时期。在第二时期，细胞中使用肉汤中的木糖，并产生另外的5.1g / L 2,3-丁二醇。第二期2,3-丁二醇的生产速度较慢。在该方法结束时，已经生产了总共12.7g / L的2,3-丁二醇。该方法的副产物是乳酸，乙酸和乙醇。在第一阶段（当葡萄糖用作碳源时）主要合成乳酸和乙醇，并连续合成乙酸。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/55828/55828fig3.jpg" /> 图3:使用竹水解产物作为原料的2,3-丁二醇生产。红线:葡萄糖;蓝线:木糖;嘛红线:2,3-丁二醇;橙线:乳酸;黑线:乙酸;绿线:乙醇。水解产物中的葡萄糖和木糖均用于2,3-丁二醇合成，但首先使用葡萄糖，其次是木糖。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55828/55828fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> R-乙偶姻由有氧条件下的肺炎克雷伯氏菌的budC突变体产生（ 图4 ）。如在野生型菌株的2,3-丁二醇生产中，由肺炎支原体ΔbudC依次使用葡萄糖和木糖。木糖在16 h时耗尽，消耗速度快于野生型2,3-丁二醇生产。该方法结束时， R-乙酰胆碱的产量为13g / L，副产物为2,3-丁二醇，乙酸和eTHANOL。 <img alt="图4" class="xfigimg" src="/files/ftp_upload/55828/55828fig4.jpg" /> 图4:使用竹水解产物作为原料的L-丝氨乙酰胺生产。红线:葡萄糖;蓝线:木糖;紫线:乙偶姻品红线:2,3-丁二醇;黑线:乙酸;绿线:乙醇。水解产物中的葡萄糖和木糖均用于R-丝氨酸合成，其使用顺序为。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55828/55828fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 2-酮葡萄糖酸和木糖酸由肺炎克雷伯氏菌的budA突变体产生（ 图5 ）。这个过程需要高空气补充。培养基中的葡萄糖首先转化为葡萄糖积酸在肉汤中的酸性酸。葡萄糖酸在培养8 h时达到最高水平15 g / L，此后浓度降低。在培养6小时之前不产生2-酮葡萄糖酸。然后以高速率合成，并且在该方法结束时产生25g / L 2-酮基葡糖酸。将培养基中的木糖转化为木糖酸;该反应开始比葡萄糖转化为葡萄糖酸。在该过程中产生一些乙酸作为副产物。 <img alt="图5" class="xfigimg" src="/files/ftp_upload/55828/55828fig5.jpg" /> 图5:使用竹水解产物作为原料的2-酮基葡萄糖酸和木糖酸生产。红线:葡萄糖;橙线:葡萄糖酸;品红线:2-酮基葡萄糖酸;蓝线:木糖;黑线:乙酸;绿线:乙醇。水解产物中的葡萄糖转化为葡萄糖酸并进一步转化为2-酮基葡萄糖酸。将水解产物中的木糖转化为木糖酸。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55828/55828fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock at JoVE.com

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

竹粉用NaOH预处理，酶水解。竹纤维素水解产物被用作2,3-丁二醇，β-乙酰胆碱，2-酮基葡萄糖酸和肺炎克雷伯菌产生的木糖酸的原料。

化学品生产<em&gt;肺炎克雷伯菌</em使用竹水合物作为原料

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was ...

production-chemicals-klebsiella-pneumoniae-using-bamboo-hydrolysate

Nanyang Technological University

Research

JoVE Journal

Bioengineering

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

8.9K 次观看.  Chinese Academy of Sciences. 竹粉用NaOH预处理，酶水解。竹纤维素水解产物被用作2,3-丁二醇，β-乙酰胆碱，2-酮基葡萄糖酸和肺炎克雷伯菌产生的木糖酸的原料。 

视频: Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

科研

环境科学

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

利用乙烯释放化合物，2-氯乙基膦酸，作为一种工具来研究细菌乙烯反应

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

生物化学

Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal carbonization (HTC) are promising technologies for bioenergy production from biomass in terms of biogas and HTC biochar, respectively. In this study, the combination of AD and HTC is proposed to increase overall bioenergy production. Wheat straw was anaerobically digested in a novel upflow anaerobic solid state reactor (UASS) in both mesophilic (37 &#176;C) and thermophilic (55 &#176;C) conditions. Wet digested from thermophilic AD was hydrothermally carbonized at 230 &#176;C for 6 hr for HTC biochar production. At thermophilic temperature, the UASS system yields an average of 165 LCH4/kgVS&#160;(VS: volatile solids) and 121 L CH4/kgVS&#160;at mesophilic AD over the continuous operation of 200 days. Meanwhile, 43.4 g of HTC biochar with 29.6 MJ/kgdry_biochar was obtained from HTC of 1 kg digestate (dry basis) from mesophilic AD. The combination of AD and HTC, in this particular set of experiment yield 13.2 MJ of energy per 1 kg of dry wheat straw, which is at least 20% higher than HTC alone and 60.2% higher than AD only.

A novel Upflow Anaerobic Solid State (UASS) reactor was used for biogas production from fibrous feedstock. Digestate from UASS reactor was hydrothermally carbonized into HTC biochar in a pressurized batch reactor. The integration of the two bioenergy concepts was applied in this study to increase overall bioenergy production.

评定一体化厌氧消化和水热碳化对生物能源生产

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

一个工具包，使水环境中的烃类转化

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

生物工程

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

化学

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, lower vapor pressure, non-flammability, higher heat capacity, and tunable solubility and acidity. Here, we demonstrate a method for the synthesis of C5 sugars (xylose and arabinose) from the pentosan present in jute biomass in a one-pot process by utilizing a catalytic amount of Br&#248;nsted acidic 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogen sulfate IL. The acidic IL is synthesized in the lab and characterized using NMR spectroscopic techniques for understanding its purity. The various properties of BAIL are measured such as acid strength, thermal and hydrothermal stability, which showed that the catalyst is stable at a higher temperature (250 &#176;C) and possesses very high acid strength (Ho 1.57). The acidic IL converts over 90% of pentosan into sugars and furfural. Hence, the presenting method in this study can also be employed for the evaluation of pentosan concentration in other kinds of lignocellulosic biomass.

We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Br&#248;nsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

利用酸性离子液体戊聚糖分析黄麻生物量及其转化为糖单体的新方法

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

Enhanced Oil Recovery using a Combination of Biosurfactants

Biosurfactants are surface-active compounds capable of reducing the surface tension between two phases of different polarities. Biosurfactants have been emerging as promising alternatives to chemical surfactants due to less toxicity, high biodegradability, environmental compatibility and tolerance to extreme environmental conditions. Here, we illustrate the methods used for screening of microbes capable of producing biosurfactants. The biosurfactant producing microbes were identified using drop collapse, oil spreading, and emulsion index assays. Biosurfactant production was validated by determining the reduction in surface tension of the media due to growth of the microbial members. We also describe the methods involved in characterization and identification of biosurfactants. Thin layer chromatography of the extracted biosurfactant followed by differential staining of the plates was performed to determine the nature of the biosurfactant. LCMS, 1H NMR, and FT-IR were used to chemically identify the biosurfactant. We further illustrate the methods to evaluate the application of the combination of produced biosurfactants for enhancing residual oil recovery in a simulated sand pack column.

We illustrate the methods involved in screening and identification of the biosurfactant producing microbes. Methods for chromatographic characterization and chemical identification of the biosurfactants, determining the industrial applicability of the biosurfactant&#160;in enhancing residual oil recovery are also presented.

使用生物表面活性剂组合提高石油采收率

Biosurfactants are surface-active compounds capable of reducing the surface tension between two phases of different polarities. Biosurfactants have been ...

用于从猪废沼气中获取生物甲烷的微藻培养

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of polluting gases such as carbon dioxide and hydrogen sulfide to increase the content of methane. In this study, we used a microalgal cultivation technology to treat and purify biogas produced from organic waste from the swine industry to obtain ready-to-use biomethane. For cultivation and purification, two 22.2 m3 open-pond photobioreactors coupled with an absorption-desorption column system were set up in San Juan de los Lagos, Mexico. Several recirculation liquid/biogas ratios (L/G) were tested to obtain the highest removal efficiencies; other parameters, such as pH, dissolved oxygen (DO), temperature, and biomass growth, were measured. The most efficient L/Gs were 1.6 and 2.5, resulting in a treated biogas effluent with a composition of 6.8%vol and 6.6%vol in CO2, respectively, and removal efficiencies for H2S up to 98.9%, as well as maintaining O2 contamination values of less than 2%vol. We found that pH greatly determines CO2 removal, more so than L/G, during cultivation because of its participation in the photosynthetic process of microalgae and its ability to vary pH when solubilized due to its acidic nature. DO, and temperature oscillated as expected from the light-dark natural cycles of photosynthesis and the time of day, respectively. Biomass growth varied with CO2 and nutrient feeding as well as reactor harvesting; however, the trend remained primed for growth.

作者聚焦:扩展微藻生物技术以提高生物甲烷产量 

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

在半工业化高速率藻类池塘中使用微藻细菌系统进行沼气净化

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of ...

化学品生产

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方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

利用乙烯释放化合物，2-氯乙基膦酸，作为一种工具来研究细菌乙烯反应

评定一体化厌氧消化和水热碳化对生物能源生产

一个工具包，使水环境中的烃类转化

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

利用酸性离子液体戊聚糖分析黄麻生物量及其转化为糖单体的新方法

使用生物表面活性剂组合提高石油采收率

在半工业化高速率藻类池塘中使用微藻细菌系统进行沼气净化

在半工业化高速率藻类池塘中使用微藻细菌系统进行沼气净化

在半工业化高速率藻类池塘中使用微藻细菌系统进行沼气净化