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

This work was supported by the National Natural Science Foundation of China (Grant No. 21576279, 20906076) and the KRIBB Research Initiative Program (KGM2211531).

1. Preparation of Bamboo Hydrolysate
<ol>
	<li>Add 5 g of bamboo powder to 40 mL of an NaOH solution to achieve a 10% (g/g) final concentration in a 250 mL flask. Use a series of NaOH solutions ranging from 0.05 M to 0.50 M in increments of 0.05 M.
	<ol>
		<li>Incubate the mixture for 60 min at 60 &#176;C or 100 &#176;C in a water bath. Incubate at 121 &#176;C in an autoclave.</li>
	</ol>
	</li>
	<li>After incubation, leave the mixture to stand for 4 h at room temperature. Then, remove the supe...

<ol>
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K. pneumoniae belongs to the genus Klebsiella in the family Enterobacteriaceae. K. pneumoniae is distributed widely in natural environments such as soil, vegetation, and water14. The wildtype K. pneumoniae strain used in this research was isolated from soil and is used for 1,3-propanediol production15. K. pneumoniae and mutants of this species produce many chemicals under different conditions.
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Klebsiella pneumoniae is a bacterium that grows well under both aerobic and anaerobic conditions. K. pneumoniae is an important industrial microorganism used to produce many chemicals. 1,3-propanediol is a valuable chemical that is mainly used as a monomer to synthesize polytrimethylene terephthalate. Polytrimethylene terephthalate is a biodegradable polyester that exhibits better properties than those of 1,2-propanediol, butanediol, or ethylene glycol1. 1,3-propanediol is produced by K. pneumoniae using glycerol as a substrate in oxygen-limited conditions2. 2,3-butanediol and its derivatives have applications in the field of plastics, solvent production, and synthetic rubber and have the potential to be used as biofuel3. With glucose as the substrate, 2,3-butanediol is the main metabolite of the wildtype strain4. 2,3-butanediol is synthesized from pyruvate. First, two molecules of pyruvate condense to yield &#945;-acetolactate; this reaction is catalyzed by &#945;-acetolactate synthase. &#945;-acetolactate is then converted to acetoin by &#945;-acetolactate decarboxylase. R-acetoin can be further reduced to 2,3-butanediol when catalyzed by butanediol dehydrogenase. An efficient gene replacement method suitable for K. pneumoniae has been explored, and many mutants have been constructed5,6,7. A budC mutant, which lost its 2,3-butanediol dehydrogenase activity, accumulates high levels of acetoin in culture broth. Acetoin is used as an additive to enhance the flavor of food8. When budA, which encodes &#945;-acetolactate decarboxylase, is mutated, 2-ketogluconic acid accumulates in the broth. 2-ketogluconic acid is used for the synthesis of erythorbic acid (isoascorbic acid), an antioxidant used in the food industry9. 2-ketogluconic acid is an intermediate of the glucose oxidation pathway; in this pathway, located in the periplasmic space, glucose is oxidized to gluconic acid and then further oxidized to 2-ketogluconic acid. Gluconic acid and 2-ketogluconic acid produced in the periplasm can be transported to the cytoplasm for further metabolism. The accumulation of 2-ketogluconic acid is dependent upon acidic conditions, and higher air supplementation favors 2-ketogluconic acid production10. Gluconate dehydrogenase, encoded by gad, catalyzes the conversion of gluconic acid to 2-ketolguconic acid. The gad mutant of K. pneumoniae produced high levels of gluconic acid instead of 2-ketogluconic acid, and this process is also dependent upon acidic conditions. Gluconic acid is a bulk organic acid and is used as an additive to increase the properties of cement11. Glucose oxidation to gluconic acid is catalyzed by glucose dehydrogenase. Xylose is also a suitable substrate of glucose dehydrogenase. When xylose is used as a substrate, K. pneumoniae produces xylonic acid12.
Chemical production using biomass as a feedstock is a hot topic in biotechnology13. The main components of biomass are cellulose, hemicellulose, and lignin. However, these macromolecular compounds cannot be directly catabolized by most microorganisms (including K. pneumoniae). Cellulose and hemicelluloses in the biomass must be hydrolyzed to glucose and xylose and can then be used by microorganisms. The presence of lignin in lignocelluloses creates a protective barrier that prevents biomass hydrolyzation by enzymes. Thus, a pretreatment process that removes lignin and hemicelluloses and reduces the crystallinity of cellulose is always performed during biomass utilization by microorganisms. Many pretreatment methods have been developed: acid, alkaline, ammonia, and steam pretreatments are common.
Bamboo is abundant in tropical and subtropical regions and is an important biomass resource. Here, the preparation of bamboo hydrolysate and chemical production using bamboo hydrolysate are presented

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">autoclave</td>
			<td style="width:136px;">SANYO</td>
			<td align="right" style="width:119px;">3780</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">bioreactor</td>
			<td style="width:136px;">Sartorius stedim biotech</td>
			<td style="width:119px;">Bostat Aplus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">agitator</td>
			<td style="width:136px;">IKA</td>
			<td style="width:119px;">RW 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water bath shaker</td>
			<td style="width:136px;">Zhicheng</td>
			<td style="width:119px;">ZWY-110X50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">high performance liquid chromatograph system</td>
			<td style="width:136px;">Shimadzu Corp</td>
			<td style="width:119px;">20AVP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">centrifuge</td>
			<td style="width:136px;">Hitachi</td>
			<td style="width:119px;">CR22G III</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bamboo powder</td>
			<td style="width:136px;">purchased from Zhejiang Province, China</td>
			<td style="width:119px;"></td>
			<td>mesh number, 50</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">cellulase</td>
			<td style="width:136px;">Youtell Biochemical, Shandong, China</td>
			<td style="width:119px;"></td>
			<td>200 PFU/ml</td>
		</tr>
	</tbody>
</table>

production of chemicals by klebsiella pneumoniae using bamboo hydrolysate as feedstock

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

In this protocol, the bamboo was pretreated using alkali. The optimal incubation parameters-a temperature of 121 &#176;C and 0.25 M NaOH-were determined in Figures 1 and 2. The pretreated bamboo was enzymatically hydrolyzed, and the glucose and xylose concentrations obtained in the hydrolysate were measured. Higher temperatures favored sugar production, so 121 &#176;C was selected as the optimal temperature. The glucose and xylose produced in the hydrolys...

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

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

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

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