Microaerobic Fermentation of Bamboo Hydrolysate by Klebsiella pneumoniae

Abstract

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.

Protocol

1. 2,3-Butanediol Production by Wildtype K. pneumoniae

1. Use 3 L of bamboo hydrolysate as the solvent for medium preparation.
2. 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 medium's pH to neutral using 2.5 M NaOH. Use a 5-L bioreactor containing 3 L of autoclaved fermentation medium for fermentation.
3. Use K. pneumoniae cells stored in a -80°C low-temperature refrigerator.
4. For the seed culture, incubate a 250 mL flask containing 50 mL of Luria-Bertani (LB) medium overnight at 37 °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).
5. Inoculate one flask of 50 mL of the seed culture in the bioreactor. Maintain the culture at pH 6.0 and 37 °C. Use an air supplementation rate of 2 L/min and an agitation rate of 250 rpm, creating a microaerobic condition.
6. 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 broth.
7. Monitor the alkali added to the bioreactor online using microbial fuel cell system/data acquisition or 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.

2. R-acetoin Production by the budC Mutant of K. pneumoniae

1. 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.
2. Use K. pneumoniae-ΔbudC for R-acetoin production. Repeat steps 1.4-1.5.
   NOTE: budC encodes 2,3-butanediol dehydrogenase. K. pneumoniae-ΔbudC loses the 2,3-butanediol dehydrogenase activity and produces R-acetoin instead of 2,3-butanediol.
3. Maintain the culture at pH 6.0 and 37 °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.
4. Assay the samples as in step 1.6.

3. 2-Ketogluconic Acid Production by the budA Mutant of K. pneumoniae

1. For 2-ketogluconic acid production, use the same medium as for R-acetoin production.
2. Use K. pneumoniae-ΔbudA for 2-ketogluconic acid production. Repeat steps 1.4-1.5.
   NOTE: budA encodes α-acetolactate decarboxylase. K. pneumoniae-ΔbudA loses the α-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 production; in the first stage, the seed culture is inoculated into the bioreactor.
3. Maintain the culture at pH 7.0 and 37 °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 a pH decrease. In these acidic conditions, cell growth stops, but 2-ketogluconic acid is synthesized and accumulates in the broth.
4. Assay the samples as in step 1.6. 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Autoclave	SANYO	3780
Bioreactor	Sartorius stedim biotech	Bostat Aplus
Agitator	IKA	RW 20
High performance liquid chromatograph system	Shimadzu Corp	20AVP
Bamboo powder	Purchased from Zhejiang Province, China		Mesh number, 50
Cellulase	Youtell Biochemical, Shandong, China		200 PFU/mL