Scientific Procedures

Growing Citrus Waste Bacterial Cellulose

Introduction: Bacterial cellulose can be grown in a variety of media such as black tea and citrus juice. In this experiment, we explore how bacterial cellulose grows in citrus juice from lemons, oranges, and tangerines. We expected the bacterial cellulose to grow successfully in our citrus juice mix based on the results of the article Gluconacetobacter sp. gel_SEA623-2, bacterial cellulose producing bacterium isolated from citrus fruit juice by S.S. Kim, et al.

Method: The tea alternative was prepared by combining citrus juice, ethanol, acetic acid, and sugar. Citrus juice was obtained from local organge, lemon, and tangerine waste. This mixture was added to an Erlenmeyer flask. 30 mL of ethanol, 30 mL of acetic acid, and 300g of sugar were added. This was stirred with a stir bar until the sugar dissolved. DI water was added until 3000mL volume was reached. Kombucha tea was added to reach a pH of 3.5 (the ideal pH for the SCOBY) - about 1000mL of kombucha tea was added, but this was not measured precisely as the amount was based on the pH of the liquid, taken with pH paper. The mixture was stirred with a stir stick once more until evenly mixed. This liquid was poured into three plastic tubs (volume split approximately into thirds), and a piece of pellicle (unwashed) was added to each tub. This was left to grow for 1 week at room temperature without disturbance.

Results: A citrus media created from lemons, tangerines, and oranges was successfully prepared for the pellicle to grow in. After one week of growing in the media, the pellicle’s surface area grew enough to span the area of the container in which it was growing in. It appears that the pellicle has a faster growth rate in this citrus media than in its normal tea media. However, the citrus pellicle is more bumpy than the kombucha tea pellicle. The extra bumps on the pellicle may be due to its interactions with the pulp. It also has a more inconsistent thickness, although this may lessen given more time as it grows more. Additionally, because of the lack of tea, the pellicle was not stained a brown color, and looked like pure bacterial cellulose; thinner sections of the pellicle were very translucent. It would be wise to filter the pulp out of the citrus media and redo this experiment in order to understand the pulp’s effect on the growth of the pellicle. Further trials would need to be done, but citrus juice seems to be a more advantageous growth media in comparison to black tea in terms of growth time and pellicle color.

Growing Xanthobacter autotrophicus

Introduction: Xanthobacter autotrophicus is a bacteria that is capable of converting dichloroethane into glycolate. The bacteria can be grown with DI water and nutrient agar containing beef extract, peptone, and agar. In this procedure, we prepare the growth media to propagate our bacteria strain, Xanthobacter Autotrophicus. We prepare two types of media: agar/petri dishes and liquid media and the agar/petri dishes. The intial colonies are grown on a petri dish then added to liquid media in preparation for integrating the bacteria with the pellicle.

Method: To prepare the petri dishes, 23g of nutrient agar were created from 3g beef extract, 5g peptone, 15g agar. The nutrients was mixed with 1000ml of hot water to create the media. The media was autoclaved at 121 °C. The media was cooled in a water bath, and a flame was set up before the agar was poured into 12 petri dishes and 2 500ml media bottles. The petri dishes were stored in a refrigerator, and the media bottles were autoclaved and then stored in the refrigerator.

Integrating Xanthobacter autotrophicus into Bacterial Cellulose

Introduction: We hypothesize that if we can successfully integrate Xanthobacter autotrophicus into bacterial cellulose, then this fabric hybrid will be able to successfully degrade DCE that exists in the environment. In this experiment, we prepare our NaOH-washed pellicle by neutralizing its pH and autoclaving it. We then add the bacteria in liquid media to the pellicle by two different methods: vacuum suction and soaking. The success of each method is assessed by running a PCR.

Method: The bacterial cellulose (pellicle) obtained had been pre-soaked in NaOH at a concentration of 2 N for 20 minutes at 95 °C. After the pellicle was obtained, it was soaked in water before being soaked in an acidic bath of 5M hydrochloric acid to neutralize the base. After the acid bath, it was then rinsed in deionized water and soaked for 24 hours in deionized water (repeated 3 times). On the last rinse/soak, NaOH was added dropwise into the water bath to reach a pH of around 7 (measured using pH paper). The pellicle was left to sit for 24 hours before it was removed from the bath, lightly rinsed, and left to dry in a room temperature environment. While drying, the pellicle was massaged once per day. After drying, the pellicle was cut into 1"x1" squares and autoclaved to ensure purity of the bacterial colonies.

The work area and tools were sterilized with alcohol. A flame was set up. Xanthobacter autotrophicus was added to the pre-made growth media (in a petri dish) using an inoculation loop to spread the bacteria strain. The petri dish was placed in an incubator to grow at 30 °C for 4 days.

Batches of 75 mL of liquid media containing Xanthobacter autotrophicus were combined to make 150 mL of liquid media with bacteria. This mixture was used to incorporate the bacteria into the cellulose.

To incorporate bacteria onto the bacterial cellulose, two methods were used: soaking after vacuuming and soaking. Each method was tested with 3 samples and 1 control (8 total samples). Each petri dish contains one piece of autoclaved bacterial cellulose (1” x 1” each). 5 mL of liquid media containing bacteria was vacuumed through to each sample of bacterial cellulose, then 5 mL of new liquid media containing bacteria was added to each petri dish for samples that were vacuumed then soaked, 3 in total. 5 mL of liquid media containing bacteria was added to each sample petri dish for samples that were soaked, 3 in total. 5 mL of liquid media without bacteria was added to each control petri dish, 2 in total.

The petri dishes were then sealed with parafilm and stored in an incubator (30 °C) for 4 days. DNA samples were then extracted from each of the 8 samples. 1 control DNA extraction was taken from the original liquid media with bacteria added. PCR analysis was run on both the samples and the control, and DNA amplification was checked using Gel electrophoresis.

Results: [procedure in progress]