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.
