Team:Berkeley/HumanPractice

On a sunny Friday afternoon on August 9th, 2013, the UC Berkeley iGEM team went to visit the Advanced Biofuels Process Demonstration Unit (ABPDU) located in Emeryville, California. The ABPDU is a 15,000 square-foot state-of-the art facility affiliated with the Lawrence Berkeley National Laboratory, and it is designed to help expedite the commercialization of advanced next-generation biofuels by providing industry-scale test beds for discoveries made in the laboratory.

The ABPDU is works closely with DOE’s Bioenergy Research Centers, including the Joint BioEnergy Institute (JBEI) located just a floor above. One of the missions of these research centers is to see that scientific advances are translated into commercially viable technologies.

And ABPDU is the fully equipped facility to bridge the gap between laboratory and marketplace, allowing researchers to be better informed of the bottlenecks in the translation of their work into impact on the real world problem.

Excited to see how the facility works, the UC Berkeley iGEM team put on their laboratory safety goggles and followed Dr. Baez into the heart of ABPDU.

First, we saw several reactors to perform pretreatment of biomass such as grass, wood, and agricultural residues. Pretreatment of biomass breaks down the “shields” formed by ligin and hemicellulose, thus reducing the degree of polymerization to faciliate rapid and efficient downstream processes.

Next to the reactors for pretreament of biomass, we saw small reactors used for enzymatic saccharification. Saccharification is literally the process of making sugar from starch reserves. As economical undergraduate researchers who sometimes feel bad for using more than five microliters of BsmBI at a time, we were quite impressed with the scale that ABPDU worked on.

Needless to say, the UC Berkeley iGEM team was soon then awestruck to see ABPDU’s bioreactors, which have the capacity to grow bacteria, fungi and yeast up to 300-liters. The bioreactors were equipped with advanced control systems for pH, temperature, dissolved oxygen and other process conditions.

Finally, the team was quite happy to see some familiar equipments for enzyme purification at ABPDU, such as a high-throughput centrifuge, a large column chromatography system for enzyme separation and purification, and protein analysis equipments such as the HPLC and gas mass spectroscopy.

From this educational field trip to ABPDU, we learned how the facility provided material and energy balance data to help develop parameters for expansion from pilot to commercial scale production.

Now, it was our turn to vision the large-scale biosynthetic and dyeing process of indigo…

*The UC Berkeley iGEM team would like to thank Dr. Julio A. Baez for the wonderful and detailed tour of the Advanced Biofuels Process Demonstration Unit (ABPDU).

• "Berkeley Lab Opens Advanced Biofuels Facility « Berkeley Lab News Center." Berkeley Lab News Center RSS. N.p., n.d. Web. 22 Sept. 2013.
• Yamashita, Y., Sasaki, C., & Nakamura, Y. (2010). Effective enzyme saccharification and ethanol production from Japanese cedar using various pretreatment methods. Journal of Bioscience and Bioengineering, 110, 79–86.
• Zheng, Y., Pan, Z., & Zhang, R. (2009). Overview of biomass pretreatment for cellulosic ethanol production. International Journal, 2, 51–68. doi:10.3965/j.issn.1934-6344.2009.03.051-068

From what we learned from Dr. Baez of ABPDU, we began to think about how we could potentially scale-up the biosynthetic and dyeing process of indigo.

We wanted to see how viable our method would be for dyeing fabric at an industrial scale. To do so we had to think about two parts of the dyeing process: both indican production and indican cleavage using the beta-glucosidase (GLU) enzyme. Although we have not yet isolated a glucosyl-transferase (GT) that can produce indican in e. coli, we wanted to run the calculations in the case that we do find one.

In the ‘dyeing with indican and purified beta-glucosidase’ page, we demonstrated that a deep blue hue could be achieved with just 10g/L indican and 3.4g/L of purified Glucosidase. Even with just 1g/L of indican a light blue hue was achieved, which with multiple dunks could be darkened to the desired hue. Although we are unable to produce indican currently, once a working GT is isolated, it is not improbable that indican could be produced in the grams per liter range allowing for direct dunking of fabric.

In our vision of the dyeing process, one fermentor would culture indican producing cells. Inputs would be tryptophan, glucose, oxygen, and heat. After culturing optimal indican levels, we would centrifuge the liquid culture to purify just the indican containing cells and then lyse them to make a smaller vat of indican and other cell material. If necessary this could be further purified to get rid of the cell junk leaving just indican. Clearly this process is somewhat energy intensive. but, if optimized, it could potentially compete with the petrochemical indigo making process.

The enzyme GLU would be cultured in a separate fermentor because making both indican and GLU in the same fermentor would cause the conversion of indican back to indoxyl. Similar inputs of sugar, oxygen and heat would be used, without the need for supplemental tryptophan.

Although tryptophan is a relatively expensive starting material, because it is a precursor to indican on the biological synthesis pathway, having it in the cell is absolutely necessary. However, to lower the cost of indican production, instead of adding tryptophan to the media it could instead be produced naturally by a tryptophan over-expressing strain of e. coli.

For example, tryptophan is one of the precursors to indican in the biological pathway, we thought we could get an estimate for how much indican could be made based on current levels of tryptophan production.