Team:ITB INDONESIA
RhamCOLIpid
Engineered E. coli producing rhamnolipid for green enhanced oil recovery
Problem
Indonesia’s oil production has been decreasing from 315 million barrels in 2012 into 238 million barrels in 2014.
Data Source
This affects oil price and thus goods price that has been increasing steadily. One contributing aspect is low recovery in conventional oil recovery method that can only recover 20-40% oil from reservoir. This production can be increased into 30-60% by Enhanced Oil Recovery (EOR). Until 2011, implementation of EOR by waterflooding and steamflooding has contributed a 40% increase in Indonesia’s oil production [1]. In the future, chemical injection can become another EOR alternative.
One of EOR methods by chemical injection is surfactant injection. The need of biodegradable and environment-friendly surfactant is important. One of biosurfactant is rhamnolipid, but it’s naturally produced by opportunistic pathogen Pseudomonas aeruginosa. Thus, ITB_Indonesia designs GRAS bacteria, E. coli, to be able to produce rhamnolipid in a controllable and observable manner.
Idea
One of EOR methods by chemical injection is surfactant injection. The need of biodegradable and environment-friendly surfactant is important. One of biosurfactant is rhamnolipid, but it’s naturally produced by opportunistic pathogen Pseudomonas aeruginosa. Thus, ITB_Indonesia designs GRAS bacteria, E. coli, to be able to produce rhamnolipid in a controllable and observable manner.
Using certain autoinduction media, rhamnolipid production is controlled by glucose-lactose composition in media. By utilizing glucose first, E coli can grow without producing rhamnolipid. After glucose exhaustion, lactose can be utilized to induce rhamnolipid production. While producing, a chromoprotein reporter will be turned on and turn E. coli into observable colour. When lactose is depleted and no rhamnolipid is produced, E coli will revert into being colourless, thus making observation much easier for further induction. E. coli is also controlled to not produce rhamnolipid when no lactose is present.
Rhamnolipid will be released into medium,making separation much easier. Only centrifugation is necessary. The medium supernatant containing rhamnolipid can be used for EOR.
This rhamnolipid-producing E. coli is called RHAMCOLIPID.
Method
We utilise The Biosurfactor biobrick (BBa_K653000) designed by Panama 2011 that encodes rhlA and rhlB, two enzymes for rhamnolipid production. We put the biobrick under T7lac promoter with strong RBS, and double terminator.
We designed our Rhamncolipid to express reporter protein when producing rhamnolipid. We tried to find best reporter for our system that has fast turnover and doesn’t have significant fitness cost to the bacteria. We tested two reporters, RFP and aeBlue, with each reporter hasdifferent tags, either not tagged, LAA-tagged, or LVA-tagged. The LAA and LVA tags have the shortest half-life in 37oC (our cultivation temperature) in RFP and GFP, respectively.
We also designed our control device which encodes LacI under different promoter strength, strong, medium, or even without the control.
Result
Reporter Selection
We transformed E. coli BL21(DE3) with different reporter proteins (RFP and aeBlue) with different tags (none, LAA, LVA). We induced the transformants using IPTG and observe the OD600 for bacteria growth and measure each reporter.
The results showed that [to be continued]. Therefore, we selected [to be continued] as our reporter device.
Rhamnolipid Production
We transformed E. coli BL21(DE3) with The Biosurfactor and reporter. We also include control system with different strength in expressing lacI. We grew our transformants in autoinduction media (as described by [to be continued]) and check OD600, rhamnolipid production.
The result showed that expressing LacI under [to be continued] gave the highest OD600 and rhamnolipid concentration.
Rhamnolipid Test
We tested our produced rhamnolipid for its characteristics and surfactant activites. The results showed that [to be continued]
Modelling
Stystem
The effect of reporter protein on growth
The effect of LacI expression on growth and rhamnolipid production
The rhamnolipid usage in oil recovery