Team:NKU China/Description

0. Abstact
In the process of oil recovering by water injection method, it will form large water channels in the bottom. Water mainly flows through these channels, which stops brushing the oil hidden in the minor channel branches in soil, so that the remaining oil will be trapped into the branches. Our project is aiming to engineer Enterobacter sp. FY-07 (which is separated from a oilfield produced water and able to produce cellulose naturally), so as to controllably produce rhamnolipid and cellulose. Rhamnolipid is a biosurfactant with the ability to emulsify oil, so the oil can be easily washed out. Cellulose can clog large water channels, helping water enter into the gaps of soil and wash out the oil within them. Our project is aimed at improving oil recovery rate in the oil harvesting process using this kind of bacteria.
1. Motivation
Petroleum is the blood of the world industry and the pillar energy for the rapid development of the world economy. Oil, however, is a kind of non-renewable and limited energy resource. In recent years, Chinese demand for oil resources continues rising, some areas even appeared the situation where there is insufficient supply of oil resources. In order to meet the normal supply of domestic oil, crude oil has a high degree of dependence on foreign countries. In this condition, the oil recovery factor needs improving, while the fact is that in China the reservoir is sponge-like and heterogeneous whose pores vary in size and the crude oil is contained in the pores, which considerably increase the difficulty of oil recovery. Some oil fields are even abandoned after little exploitation compared with the whole oil content. Thus it is necessary to propose an efficient way to enhance the oil production.

Before this, let us have a glance at conventional oil recovery measures.Primary oil recovery means the crude oil moves to the ground automatically by the earth’s crust energy. And then there is secondary oil recovery, in which crude oil is driven by efforts provided by other things such as waterpower. One of the most commonly used methods is to drill water into the oil reservoir through the injection well to push the oil to the easiest and shortest path. Then with automatically erupting or oil extractor, crude oil finally reaches the ground. But there is a problem in the long run of using method. There is a tendency that liquids flow along the pathway with least resistance. Water mainly flows through existing and dilating channels, which stops brushing the oil hidden in the soil, so the remaining oil will be hard to exploit. So on the base of this, there is tertiary oil recovery, which is enhanced water injection technique by means of physical, chemical and biological ways for higher oil recovery efficiency.
In tertiary oil recovery, there are two common techniques. First, block. After injecting the bacterial culture, bacteria preferentially flow through high osmosis zones and some finally settled down in the pores, where bacteria begin to grow. Then at an appropriate time we add inducer to lead the expression of bacterial metabolites, which contribute to form embolism. With the existence of embolism in water path, when we inject water once again, it flows to the low osmotic zone, which makes it rather easy to wash off crude oil in newly formed water passage, consequently enhance the oil production. Besides, emulsification is a process to help the oil be released from the pores, in which emulsifiers emulsify incompatible oil and water to form relatively stable emulsions. Due to the presence of surfactants, non-polar hydrophobic droplets become charged colloidal particles, which increase surface area and surface energy. Because of the polarity and surface energy, the charged oil droplets adsorb the reverse or polar water molecules in the water to form colloidal double layer, which prevents the collision between oil droplets, and makes the oil droplets stably exist in water for a long time. Obviously, this is beneficial for the oil in the pores to be washed out easily.
2. Our Choice
We choose MEOR—Microbial Enhanced Oil Recovery. So what is MEOR? It is one tertiary oil recovery method that uses the interaction between microbes and their metabolites and crude oil reservoir to enhance the oil recovery efficiency. As is known to us, MEOR is an effective oil recovery technique with simple construction and low cost. It is expected to become one of the main techniques for stabilizing oil and water and improving oil recovery in the later period of oilfield development.
We even want to find a kind of bacteria that is suitable for transformation so that it can combine the functions of blocking and emulsification.
3. Introduction To FY-07
So Enterobacter sp. FY-07 is exactly the one we are aiming to engineer.
What characteristics make FY-07 suitable for us?
First, it is facultative anaerobic, which makes it possible that it can grow under anaerobic condition. The reservoir is in anoxic state, but the absolute anaerobic state cannot be maintained during the reservoir treatment, so the best strain is facultative anaerobe. Besides, another advantage of facultative anaerobe is that it can be cultured in aerobic condition to shorten culture time because aerobic metabolism is faster than anaerobic metabolism. Second, it is easy for genetic manipulation. Similar to E.coli, it has fast growth and clear genetic background (sequenced). Third, one of its most outstanding features is that it is separated from oilfield and able to produce cellulose naturally under anaerobic condition. It is important because we have already confirmed that cellulose is able to block water passages through physical simulation and profile control experiments at our instructor's Lab. Interestingly, its cellulose yields in stone crevice are higher than that in shake flasks. This is more beneficial for cellulose production in the oil reservoir that is sponge-like, therefore takes effect in blocking.
4. Our Design
To combine the functions of blocking and emulsification, our project is aiming to engineer Enterobacter sp. FY-07 so as to controllably produce cellulose and rhamnolipid.
Rhamnolipid, a kind of biosurfactant with the ability to emulsify oil, has three important characteristics. Surface activity. It can promote capillarity by reducing the water surface tension. Interfacial activity. It can break the water-oil interface and mix them by reducing interfacial tension. Emulsify property. It can stabilize the water-oil mixture, promote emulsification.

So how to achieve our goal?

RhlAB is the essential gene in rhamnolipid biosynthetic pathway. And bcs is the essential gene in cellulose synthetic pathway. We can see that the Glucose-1-phosphate is the common intermediate metabolite. So it will be great if at first the cellulose is produced to block the existing channels, then with the injection of water, some small channels begin to form, so that the water can enter into the gaps of soil. And then the inducer is added to induce rhamnolipid to help the oil be washed out.
To make it controllable, we introduce fimS. In E. coli, the expression of the fimbriae component, FimA, is controlled in a binary fashion through the inversion of a 314 bp segment of DNA (fimS) that contains the FimA promoter. The inversion of fimS is performed by the DNA recombinase FimE, which binds to two inverted repeat sequences (Inverted Repeat Left and Right, IRL and IRR, respectively) that flank the fimS element. FimE has different binding affinities for IRL and IRR depending on the orientation of fimS, as a result, FimE is able to efficiently cause recombination only when the promoter faces IRR. Therefore, switch inversion is permanent and heritable.
In our design, rhl-BcsA switch will expresses nothing in nature condition.
●When nothing is added, there is no product because of the presence of the lactose operon.
●After we use IPTG to induce, it will constitutively express BcsA when fimS is oriented toward IRR, corresponding to the OFF state, and FY-07 will start to produce cellulose.
●Following when arabinose is added, fimE is produced due to arabinose operon. Unidirectional inversion (activation) of the switch by FimE, fimS is reoriented toward IRL and causes the constitutive expression of rhlABC, which corresponds to the ON state, and FY-07 will start to produce rhamnolipid.
In this condition FY-07 can controllably produce rhamnolipid and cellulose if we control the time and amount of the IPTG and arabinose.



Nankai University
No.94 Weijin Road, Nankai District
Tianjin, P.R.China 300071