Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Summary

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.

Abstract

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.

Introduction

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 h....

Protocol

1. Preparation of Bamboo Hydrolysate

1. 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.
   1. Incubate the mixture for 60 min at 60 °C or 100 °C in a water bath. Incubate at 121 °C in an autoclave.
2. 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.
   1. Repeat this washing 5-6 times, until the pH of the supernatant reaches 6.8. Use the o....

Results

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.......

Discussion

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.

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
water bath shaker	Zhicheng	ZWY-110X50
high performance liquid chromatograph system	Shimadzu Corp	20AVP
centrifuge	Hitachi	CR22G III
Bamboo powder	purchased from Zhejiang Province, China		mesh number, 50
cellulase	Youtell Biochemical, Shandong, China		200 PFU/mL