Name: Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock (Video) | JoVE Journal
Uploaded: 2017-06-29T14:00:00.000+00:00
Duration: 7 min 24 s
Description: Learn microbial fermentation for sustainable chemical production using Klebsiella pneumoniae with bamboo hydrolysate. This method enables efficient conversion of renewable biomass into valuable bioproducts.

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5 minutes

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 supernatant. Add 100 mL of fresh water and mix.
	<ol>
		<li>Repeat this washing 5-6 times, until the pH of the supernatant reaches 6.8. Use the obtained solid for the next step of enzymatic hydrolysis.</li>
	</ol>
	</li>
	<li>For hydrolysis, adjust 50 mL of the above mixture to pH 5.0 using 98% H2SO4. Then, add 0.5 mL of 200 filter paper activity (FPU)/mL cellulase. 
	NOTE: The final ratio of cellulase to bamboo should be 20 FPU per 1 g of bamboo.</li>
	<li>Incubate the mixture at 50 &#176;C for 36 h in a shaker. 
	NOTE: After hydrolysis, some solid will remain in the mixture.</li>
	<li>Obtain a clear hydrolysate by centrifuging at 7,690 x g for 5 min.</li>
	<li>Quantify the glucose, xylose, and other chemicals with a high-pressure liquid chromatograph system equipped with a refractive index detector and a photodiode array detector. Use an HPX-87H column (300 mm x 7.8 mm) and a mobile phase of 5 mM H2SO4 solution at a 0.8 mL/min flow rate12.</li>
	<li>For a large volume of hydrolysate preparation, mix 2 kg of bamboo powder and 18 L of 0.25 M NaOH in a 30-L stainless-steel tank.
	<ol>
		<li>Incubate the tank at 121 &#176;C in an autoclave for 60 min. After incubation, wash the mixture with 40 L of water in a 60 L plastic tank. Repeat this washing 5 times.</li>
		<li>Use water to adjust the total washed bamboo to a volume of 20 L. Adjust the pH of the mixture to 5.0 using 98% H2SO4.</li>
		<li>Enzymatically hydrolyze the mixture in a modified shaking water bath, equipped with a mechanical agitator to ensure that the mixture is well distributed. Add 200 mL of 200 FPU/mL cellulase and incubate the mixture at 50 &#176;C for 36 h.</li>
		<li>After hydrolysis, centrifuge the mixture at 4,700 x g for 10 min.</li>
	</ol>
	</li>
</ol>
2. 2,3-Butanediol Production by Wildtype K. pneumoniae
<ol>
	<li>Use 3 L of bamboo hydrolysate as the solvent for medium preparation.</li>
	<li>Prepare a fermentation medium containing 4 g/L corn steep liquor, 2 g/L (NH4)2SO4, 6 g/L K2HPO4, 3 g/L KH2PO4, and 0.2 g/L MgSO4. Adjust the pH of the medium to neutral using 2.5 M NaOH. Use a 5-L bioreactor containing 3 L of autoclaved fermentation medium for fermentation.</li>
	<li>Use K. pneumoniae cells stored in a -80 &#176;C low-temperature refrigerator.</li>
	<li>For the seed culture, incubate a 250 mL flask containing 50 mL of Luria-Bertani (LB) medium overnight at 37 &#176;C, with shaking at 200 rpm. Use LB medium containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl. 
	NOTE: The cell density of the seed culture should reach OD 2 (optical density at 600 nm).</li>
	<li>Inoculate one flask of 50 mL of the seed culture in the bioreactor. Maintain the culture at pH 6.0 and 37 &#176;C. Use an air supplementation rate of 2 L/min and an agitation rate of 250 rpm, creating a microaerobic condition.</li>
	<li>Collect 5 mL samples every 2 h during the fermentation and analyze them with high-pressure liquid chromatography to determine the chemical concentrations in the broth4.
	<ol>
		<li>Monitor the alkali added to the bioreactor online using MFCS/DA. 
		NOTE: Organic acids are produced in the fermentation process, and NaOH is added to keep the culture pH stable. The volume of alkali added represents the amount of acid produced. When the alkali-added line plateaus, the process is finished, either because of carbon source exhaustion or cell death.</li>
	</ol>
	</li>
</ol>
3. R-acetoin Production by the budC Mutant of K. pneumoniae
<ol>
	<li>Prepare a medium for acetoin production containing 4 g/L corn steep liquor, 2 g/L (NH4)2SO4, 3 g/L sodium acetate, 0.4 g/L KCl, and 0.1 g/L MgSO4.</li>
	<li>Use K. pneumoniae-&#916;budC for R-acetoin production. Repeat steps 2.4-2.5. 
	NOTE: budC encodes 2,3-butanediol dehydrogenase. K. pneumoniae-&#916;budC loses the 2,3-butanediol dehydrogenase activity and produces R-acetoin instead of 2,3-butanediol.</li>
	<li>Maintain the culture at pH 6.0 and 37 &#176;C. Use an air supplementation rate of 4 L/min and an agitation rate of 450 rpm, an aerobic condition. 
	NOTE: The oxygen supplementation is higher than that of 2,3-butanediol production.</li>
	<li>Assay the samples as in step 2.6.</li>
</ol>
4. 2-Ketogluconic Acid Production by the budA Mutant of K. pneumoniae
<ol>
	<li>For 2-ketogluconic acid production, use the same medium as for R-acetoin production.</li>
	<li>Use K. pneumoniae-&#916;budA for 2-ketogluconic acid production. Repeat steps 2.4-2.5. 
	NOTE: budA encodes &#945;-acetolactate decarboxylase. K. pneumoniae-&#916;budA loses the &#945;-acetolactate decarboxylase activity and produces 2-ketogluconic acid, using glucose as a substrate. 
	NOTE: 2-ketogluconic acid synthesis is an acidic condition-dependent process. A two-stage fermentation has been developed for 2-ketogluconic acid production10; in the first stage, the seed culture is inoculated in the bioreactor.</li>
	<li>Maintain the culture at pH 7.0 and 37 &#176;C. Use an air supplementation rate of 4 L/min and an agitation rate of 500 rpm. 
	NOTE: In these conditions, cells grow very fast (the cell density reaches OD 7 in about 4 h). Then, the fermentation progresses to the second stage, during which the culture pH decreases to 5.0. No acid is added; the organic acids produced in the culture naturally lead to the pH decrease. In these acidic conditions, cell growth stops, but 2-ketogluconic acid is synthesized and accumulates in the broth.</li>
	<li>Assay the samples as in step 2.6. Using the hydrolysate of bamboo as the carbon source. 
	NOTE: The glucose in the medium is converted to 2-ketogluconic acid, and the xylose is converted to xylonic acid. Thus, a mixture of 2-ketogluconic acid and xylonic acid is obtained.</li>
</ol>

<ol><li>Zeng, A. P., Biebl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bulk+chemicals+from+biotechnology%3A+the+case+of+1%2C+3-propanediol+production+and+the+new+trends%5BTitle%5D)+AND+%22Adv+Biochem+Eng+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Role+of+dihydroxyacetone+kinases+I+and+II+in+the+dha+regulon+of+Klebsiella+pneumoniae%5BTitle%5D)+AND+%22J+Biotechnol%22%5BJournal%5D)">Role of dihydroxyacetone kinases I and II in the dha regulon of Klebsiella pneumoniae.</a> J Biotechnol. 177, 13-19 (2014).</li>
<li>Celińska, E., Grajek, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biotechnological+production+of+2%2C3-butanediol-current+state+and+prospects%5BTitle%5D)+AND+%22Biotechnol+Adv%22%5BJournal%5D)">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/pubmed?term=((Mechanism+of+2%2C+3-butanediol+stereoisomer+formation+in+Klebsiella+pneumoniae%5BTitle%5D)+AND+%22Appl+Microbiol+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Red+recombinase+assisted+gene+replacement+in+Klebsiella+pneumoniae%5BTitle%5D)+AND+%22J+Ind+Microbiol+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((New+strategy+to+improve+efficiency+for+gene+replacement+in+Klebsiella+pneumoniae%5BTitle%5D)+AND+%22J+Ind+Microbiol+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Inhibition+of+RecBCD+in+Klebsiella+pneumoniae+by+Gam+and+its+effect+on+the+efficiency+of+gene+replacement%5BTitle%5D)+AND+%22J+Basic+Microbiol%22%5BJournal%5D)">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/pubmed?term=((R-acetoin+accumulation+and+dissimilation+in+Klebsiella+pneumoniae%5BTitle%5D)+AND+%22J+Ind+Microbiol+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((2-Ketogluconic+acid+production+by+Klebsiella+pneumoniae+CGMCC+1.6366%5BTitle%5D)+AND+%22J+Ind+Microbiol+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Two-stage+fermentation+for+2-ketogluconic+acid+production+by+Klebsiella+pneumoniae%5BTitle%5D)+AND+%22J+Microbiol+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Gluconic+acid+production+by+gad+mutant+of+Klebsiella+pneumoniae%5BTitle%5D)+AND+%22World+J+Microbiol+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Production+of+xylonic+acid+by+Klebsiella+pneumoniae%5BTitle%5D)+AND+%22Appl+Microbiol+Biotechnol%22%5BJournal%5D)">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://pubs.acs.org/doi/abs/10.1021/ie801542g">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. The genus Klebsiella. <a target="_blank" href="http://link.springer.com/referenceworkentry/10.1007%2F0-387-30746-X_8">The Prokaryotes</a>. , Springer. 159-196 (2006).</li>
<li>Hao, J., Lin, R., Zheng, Z., Liu, H., Liu, D. <a target="_blank" href="http://link.springer.com/article/10.1007/s11274-008-9665-y">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.sciencedirect.com/science/article/pii/S0961953415000963">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://link.springer.com/article/10.1007/s10570-009-9309-x">Role of pretreatment and conditioning processes on toxicity of lignocellulosic biomass hydrolysates.</a> Cellulose. 16 (4), 743-762 (2009).</li>
</ol>

The authors have nothing to disclose.

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.
<p class="jove_content"...

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><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

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 °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 °C was selected as the optimal temperature. The glucose and xylose produced in the hydrolysate increa...

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.

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

The overall goal of this experiment is to observe production of the chemicals 2, 3-Butanediol, Acetoin, 2-ketogluconic acid and xylonic acid by Klebsiella pneumoniae using the hydrolysate of bamboo as the feedstock. To begin, using a series of sodium hydroxide solutions in 250 milliliter flasks, add five grams of bamboo powder to 40 milliliters of sodium hydroxide to make a 10%solution. Incubate the solutions in a water bath at 60 degrees Celsius, or 100 degrees Celsius for 60 minutes, or incubate the solutions in an autoclave at 121 degrees Celsius for 60 minutes.Following the incubation, centrifuge the mixtures for five minutes then remove the supernatant, add 100 milliliters of fresh water, and mix. Repeat the washing five to six times until the pH of the supernatant reaches 6.8, then to carry out enzymatic hydrolysis, use 98%sulfuric acid to adjust 50 milliliters of each solution to pH 5.0. Next add 0.5 milliliters of 200 filter paper activity, or FPU, per milliliter of cellulase.The final ratio of cellulase to bamboo should be 20 FPU per one gram of bamboo. Incubate the mixture at 50 degrees Celsius with shaking for 36 hours. Following hydrolysis, some solid will remain in the mixture.To obtain a clear hydrolysate, centrifuge the solutions at 7, 690 times g for five minutes. To prepare a large volume of hydrolysate, follow a similar procedure as just demonstrated, except replace the 250 milliliter flasks with a 30 liter stainless steel tank, a 60 liter plastic tank, and a modified shaking water bath which is equipped with a mechanical agitator. After preparing fermentation medium according to the text protocol, prepare a seed culture by adding K-pneumoniae cells to a plate, and culture overnight, then transfer the culture to a 250 milliliter flask containing 50 milliliters of LB medium.Incubate the culture at 37 degrees Celsius and 200 RPM overnight. Wild-type, the bud C mutant and the bud A mutant are used for 2, 3-Butanediol, Acetoin, and 2-ketogluconic acid production respectively. Inoculate 50 milliliters of the seed culture into the bioreactor.Maintain the culture at 37 degrees Celsius with a pH of 6.0 and 7.0 for Acetoin and 2, 3-Butanediol respectively. For 2-ketogluconic acid production, maintain the culture at pH 7.0 for the first four hours, then switch to a pH of 5.0. For 2, 3-Butanediol production, maintain the culture in a microaerobic environment with air supplementation at two liters per minute and 250 RPM.For Acetoin production, maintain the culture in an aerobic environment with air supplementation at four liters per minute at 450 RPM. For 2-ketogluconic acid production, maintain high aerobic conditions with air supplementation and agitation rates at four liters per minute, and 500 RPM respectively. Finally collect five milliliter samples every two hours during the fermentation, and use high-pressure liquid chromatography to analyze them to determine the chemical concentrations in the broth.Using the protocol demonstrated in this video, higher temperatures and sodium hydroxide concentrations between 0.25 and 0.40 molar favored production of hydrolysate from bamboo. About 20 grams per liter of glucose and 10 grams per liter of xylose were produced during the flask scale enzymatic hydrolysis and 30 grams per liter of glucose and 15 grams per liter of xylose were obtained from the large volume hydrolysate preparation. As shown here, 2, 3-Butanediol was produced by K-pneumoniae in microaerobic conditions.The process was divided into two periods, in the first glucose was used by the cells to produce 7.6 grams per liter of 2, 3-Butanediol. In the second period, the xylose in the broth was used by the cells, and an additional 5.1 grams per liter of 2, 3-Butanediol was produced. Acetoin was produced by the bud C mutant of K-pneumoniae under aerobic conditions.As in 2, 3-Butanediol production by the wild-type strain, glucose and then xylose were used in sequence and K-pneumoniae delta bud C produced 13 grams per liter of Acetoin. 2-ketogluconic acid and xylonic acid were produced by the bud A mutant of K-pneumoniae, and this process required high air supplementation. The glucose in the medium was first converted to gluconic acid, and further converted to 2-ketogluconic acid.At the same time, xylose was converted to xylonic acid. 25 grams per liter of 2-ketogluconic acid and 14 grams per liter of xylonic acid were finally produced.

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

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Source: Wei, D., et al., Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock. J. Vis. Exp. (2017)This video demonstrates the microbial conversion of bamboo-derived sugars into 2,3-butanediol by culturing Klebsiella pneumoniae in a hydrolysate-based medium under microaerobic and mildly acidic conditions.

Microaerobic Fermentation of Bamboo Hydrolysate by Klebsiella pneumoniae

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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.

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

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 Brønsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

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

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.

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

This protocol presents a scalable method for converting food waste into biodegradable polyhydroxyalkanoate (PHA) bioplastics. It utilizes arrested anaerobic digestion for food waste pretreatment, halophilic microbial fermentation for PHA biosynthesis, and a chemical-free downstream process for PHA recovery.

Scalable Step-by-Step Approach of Sustainable Bioplastic Production from Food Waste

Learn microbial fermentation for sustainable chemical production using Klebsiella pneumoniae with bamboo hydrolysate. This method enables efficient conversion of renewable biomass into valuable bioproducts.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

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