Team:Berkeley/HumanPractice/Introduction

Visit to the ABPDU

How are bio-manufacturing processes scaled up?

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 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 undergraduate researchers used to laboratory scale experiments where five microliters of enzyme is a lot, 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).


References

Vision for Large-scale Biosynthesis and Dyeing of Indigo

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.

Above is a schematic diagram that we envision the large scale biosynthetic and dyeing process of indigo. On the left, we have a fermentor that will cultivate E. coli that is engineered to produce indican via enzymes FMO and GT. These E. coli will be supplied with compressed and filtered oxygen and premixed nutrients, including tryptophan, which is the starting material for biosynthetically-produced indigo. The agitator will be used to distribute oxygen throughout the fermentor. The temperature control jacket around the fermentor will circulate water around to keep the temperature of the reactor at 37 degrees Celsius.

After E. coli has been fully grown after around 18 hours, the culture will be transferred to a holding tank so that the fermentor can be cleaned and prepared for the next cycle of growth. The volume of the culture will be reduced to about 25 percent by centrifugation, at which point the cells will be homogenized then collected in a vat. The vat will thus contain a heterogenous solution of E. coli, which includes indican.

On the right, we have another fermentor that will cultivate E. coli that is engineered to produce secreted B-glucosidase. Similarly to the first fermentor, the bacteria will be supplied with water and premixed nutrients, but does not necessarily need tryptophan. After around 18-hours of growth, secreted B-glucosidase can go through a simple purification procedure and into a vat.

Utilizing this scheme, one could dye jean by dipping cotton first into a vat of indican, then into a vat of B-glucosidase. For darker shades of blue, one could simply repeat the dipping procedure several times.

Based on the indigo titer we obtained (~200 mg/L), it is not yet feasible to meet the demand of 40,000 tons of indigo per year or compete with the price of chemically synthesized indigo (~ $5.00/kg of Indigo) at the moment. However, biosynthesis and dyeing of indigo may be more promising by using a strain of E. coli that is engineered to overproduce the amino acid tryptophan. Not only will this reduce the cost-limiting reagent (i.e. tryptophan) in our current scheme of our biosynthetic pathway, it can also greatly increase the indigo titer based on our experimental data that indigo titer is strongly dependent on that amino acid. Thankfully, this next step may not be too far away as researchers have already started to engineer strains of E. coli that can overproduce L-tryptophan.

Finally, our proposed biosynthesis and dyeing of indigo has advantages over the current industrial process of producing and dyeing indigo in at least two respects: (1) First, our pathway is not dependent on petroleum based chemicals and the hazardous agents required to work with them, all of which are necessary for the current chemical synthesis of indigo, and (2) second, our pathway simplifies the current dyeing process of indigo by avoiding making indigo first then reducing it into a soluble form, instead we produce stable, soluble indican that can be uncaged into indigo.


References

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