Team:BNDS China/Human Practices
Proof of Concept
Integrated Human Practices
Aiming to find a way to increase the efficiency of the enzymes including RhlA, RhlB and RhlC, we
design a way of mutating genes in order to increase production rate of rhamnolipid. To get our
optimal enzyme, it involves multiple steps for us to achieve it including gene mutation and
bacterial selection. In order to achieve each step, we have consulted many organizations and
experts in the related industry. Through the talk with Dr. Li Lijun, an expert for improving
enzymes degrading rhamnolipid in Jimei University. We’ve compared the ways to improve
enzymes and asked her about the validity of our experiment. We’ve also got to understand the
difficulties that we may face during our experiment. These connections largely helped us in
conducting improvements for our design and making reflections of our project.
1. Is it possible for you to give us some advice about increasing the activity of the enzyme through
your experience? How do we choose enzymes for hydrolyzing rhamnolipid?
A: It is really difficult to find an enzyme for hydrolyzing rhamnolipid. The structure of rhamnolipid
may be rigid meaning that it may not be hydrolyzed. It also depends on where did you find this
rhamnolipid. As a common example, cell walls can be made out of pectin which at the very end it
will attach a rhamnolipid. I think you should research more about the enzymes on journals. If you
do succeed, I will be glad you helped me to find out a new application for my enzyme.
2. May we ask how to improve the affinity between the chassis and the enzyme?
A: Different enzymes have different characteristics. It is hard to give you a general explanation
about how to improve your enzyme. But it will be better if you can use protein engineering to
modify this enzyme.
3. We found that Dr. Li has used ways including point mutation, molecular stimulation and directed
evolution for enhancing the activity of enzymes. Could you please share with us about your
experience of modification of enzymes?
A: As you did in your experiment, directed evolution is beneficial that you do not focus on a
single point. Instead, you will get a bunch of results. In case of my early experiments, a total
number of ten to the 36th power of enzymes have been created. It is better for you to use
antibiotics to filter out what you want. Moreover, it will be better if you add a part of designing
enzymes. Overall, it is beneficial for you to use a pre-designed enzyme and directed
evolution. This would probably give you the best result.
Dr. Li also comments on our project design, she suggests that to increase the activity of the three
enzymes, the rate-limiting enzyme activity must be increased first. Also, the rational design of the
enzymes encouraged to be combined with the EvolvR system. To start with Point mutations, and
then use it as the starting point to do directed evolution.
Integrated Human Practice
For integrated human practice, we visited and interviewed two professionals
in the field of Biological Oil Recovery: Dr. Hu and Dr. Lin from Microbial Oil Recovery
Research Center of Shandong Shengli Oilfield, Sinopec, and gain
precious suggestions for technical problem and future direction from them.
Current Progress on Biological Oil Recovery
Biological Oil Recovery(BOR) refers to using the biological product, commonly
biosurfactants, to recover oil. Compared to chemical reagents, Biosurfactants
are more resistant to high temperature and salinity; this overall stability enables
biological agents to withstand the extreme conditions of oil fields. Besides, the
production of chemical agents requires energy that comes from the oil, so it’s
less efficient and unfriendly to the environment. More importantly, some
chemicals involved in the process are harmful to people. Thus there’s a couple of
reasons for using BOR. By using it, the cost of fermentation of biological agents
will be way lower, and sometimes food waste, like fermented molasses, can also
be used. These organic substances will eventually be degraded by the
environment when buried into the ground. The whole process should be
pollution-free, green and low-cost. In all, using BOR will bring us lots of technical
advantages: wide range of adaptation, simple process, low cost, good economic
benefits, zero pollution.
the Emulsifying ability
Rhamnolipid is capable of emulsion
recovery: the cysts formed by the
hydrophilic head out and hydrophobic tail
from biosurfactants- a structure similar to
the phospholipid bilayer, can encase oil in
its cavity and carry it out.
Figure.1 during the interview
Industrial Use: Character of Interest
In practices, two properties used to evaluate the efficacy of a biosurfactant. Two important
properties are used to evaluate the efficacy of biosurfactants.
Concerns about Safety issues
In synthesis process, since the bacteria
that are being used now are all
conditional bacteria like Pseudomonas
aeruginosa, which mainly infects
wounds, the danger is considered
small. In addition, the post-processing
of crude oil eliminates the potential
danger of leakage.
the ability or tendency to spread
over a solid surface. Rhamnolipid
can change the wettability of
underground rocks and gravel,
on whose surface oil is stored.
This change can largely reduce
the interfacial tension, and thus
improve the crude oil recovery
The professor also has pointed out a problem and gave us some advice for the
future. In the production of Rhamnolipid at an industrial scale, the factors to be
considered include the composition and proportion of the fermentation formula,
the culture temperature during the fermentation process, the optimized
temperature, the optimized PH value, the optimized inoculation amount of the
whole seed fluid, the optimized oxygen, carbon, nitrogen and phosphorus and the
optimized concentration of inducer. In fermentation, Rhamnolipid can produce a
lot of foam. The foam has to drain out. Otherwise, it will affect the space. But a lot
of the surfactants are actually in this foam. So the whole yield may be
compromised if absorb the foam out. Therefore, how to prevent it from foaming or
how to collect the foam has become the problem.
For the advice for the future, the professor suggested some ways to increase
production. First, we need to increase the synthetic precursor or promotor and
remove the stimulating metabolites to boost output. Also, we need to transfer the
gene for hemoglobin to the microbe, which will improve the entire organism’s
ability to absorb oxygen.
Figure.3 Fermentation techiniques employed by Sinopec
Our Improvement Afterward
After the visit to the research center, we made some
improvements to our project. We adopted the suggestion for
quantitatively testing Rhamnolipid by using HPLC-MS. In addition,
thanks to Dr. Lin and Dr. Hu sharing one strain of P. aeruginosa
that has been used in industry to produce Rhamnolipid with our
Genetic modification is made on this strain of P.A., in order to
increase its production of Rhamnolipid. Please see more details
of our design and results in the following section.
Figure.2 Illustration of the Emulsifying sbility
Remark: All activities in Human Practice are conducted in accordance with the regulation of iGEM,
and all consent forms are collected properly.