Determination of target genes for constructing conditional BC-producing strain
FY-07 produces BC when cultivated with various common sources of carbon, such as molasses, glucose (Fig. 1a), glycerol (Fig. 1b), and sucrose [21, 22]. The biosynthesis pathway of BC from glucose in FY-07 has been described in our previous study [21]; briefly, glucose is consecutively catalyzed by glucokinase (GK), phosphoglucomutase (PM), UDP-glucose pyrophosphorylase, and BC synthase complex, resulting in BC biosynthesis [21]. Inactivation of any enzyme involved in this pathway restricts the ability of FY-07 to produce BC. In addition, GK inactivation affects the normal metabolism of FY-07 as it is a key enzyme in the glycolysis pathway. A feasible strategy to construct a conditional BC-producing strain is thus to decrease the substrate (glucose) concentration in the BC biosynthesis pathway by reducing or inactivating the gluconeogenesis pathway in FY-07 [21]. To reduce the gluconeogenesis pathway, transcription levels of related genes involved in the glycolysis pathway, gluconeogenesis pathway, and glycerol metabolism were analyzed by qRT-PCR upon cultivating FY-07 with glucose or glycerol as the sole carbon source. In comparison to when glucose was used as the sole carbon source, when glycerol was used as the sole carbon source, the expression of genes encoding phosphofructokinase, phosphoglycerate kinase, phosphoglycerate mutase and pyruvate dehydrogenase complex was downregulated, however, the expression of genes encoding glycerol kinase, phosphoglycerol dehydrogenase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase, fructose-1, 6-bisphosphatase (FBP), and phosphoglucose isomerase was upregulated (Fig. 2). The numerical results of relative fold change of these genes can be seen in Additional file 1: Figure S1.
According to the aforementioned results, the gene encoding FBP can be regarded as a target gene, for it catalyzing irreversible reactions in the gluconeogenesis pathway. However, completely inactivating this gene is undesirable because its catalytic product is also used to synthesize essential components of cells such as peptidoglycan and lipopolysaccharide [28]. Genomic sequence analysis suggested that the genome of FY-07 includes three genes (AKI40_1324, AKI40_4472, and AKI40_4858) encoding FBP isoenzymes. The results of qRT-PCR indicated that the transcriptional level of AKI40_4472 was higher than that of AKI40_4858 and AKI40_1324 when glycerol or glucose was the sole carbon source (Additional file 1: Figure S2), suggesting that FBP encoded by AKI40_4472 mainly contributes to the conversion of fructose-1, 6-bisphosphate to fructose-6-phosphate in FY-07. Therefore, AKI40_4472 was selected as the target gene to reduce the gluconeogenesis pathway of FY-07 for constructing a genetically engineered strain that could grow on glycerol and not synthesize substantial quantities of BC.
Construction and characterization of the conditional BC-producing strain
A conditional BC-producing strain was constructed by knocking out AKI40_4472. PCR was used to confirm the successful knock out of this target gene (Additional file 1: Figure S3). We designated this strain as FY-0701. Its growth rate was slightly faster than that of FY-07 when glycerol was used as the sole carbon source (Additional file 1: Figure S4). SEM demonstrated that the BC-producing ability of FY-0701 remained unaffected when glucose was used as the sole carbon source (Fig. 1c); only a few BC fibers were observed when glycerol was the sole carbon source (Fig. 1d). These results suggested that FY-0701 could still synthesize sufficient glucose derivatives via the gluconeogenesis pathway, using FBP isoenzymes encoded by AKI40_4858 and AKI40_1324, to ensure cell growth rather than synthesizing large quantities of BC. Therefore, for microbial EOR (MEOR), preparation of injection cells of FY-0701 using glycerol as the sole carbon source can potentially save substantial costs as special equipment or cellulase is not required.
Plugging capacity of FY-0701 in the core flooding experiment
Core flooding experiments have been extensively used to simulate and evaluate microbial selective plugging for MEOR practices [29,30,31]. Three types of cores are commonly used: sand-packed columns, artificial cores, and natural reservoir cores [15]. We chose artificial cores to evaluate the efficiency of selective plugging by FY-07 and FY-0701 because they are more economical than natural reservoir cores and more suitable for observation under a scanning electron microscope than sand-packed columns. To simulate the heterogeneity of reservoirs, we connected an artificial core with low permeability and another one with high permeability in parallel. In order to avoid the production of high volumes of biopolymers during the injection process, injection cells and nutrients are usually separately injected in the production practice in oil fields. Table 3 shows the details of the core flooding experiment.
During the injection process, injection pressures of FY-07 were slightly higher than those of FY-0701, suggesting that residual BC fibers on FY-07 cells impeded their migration in artificial cores. After 3 days of incubation at 30 °C, although plugging ratios of both FY-07 and FY-0701 in all artificial cores were > 80%, subsequent water flooding pressures of artificial cores with high permeability had no obvious difference whereas the pressure of the core B plugged by FY-0701 was 66.67% higher than that of core D plugged by FY-07 (Fig. 3). These results suggested that the migration ability of FY-0701 in oil-bearing formations with low permeability was higher than that of FY-07. The reduction in the permeability of artificial cores represented by the increase in the plugging ratio demonstrated that both FY-07 and FY-0701 could be used to redirect the flooding water into low permeability zones where the oil was left.
BC production and location of the plugging position of FY-0701 in artificial cores
Microbial plugging has great potential in EOR, but the plugging position may largely influence the sweep volume of flooding water, consequently affecting oil recovery [18, 32]. The results of a mathematical model showed that high permeability zones are plugged when biopolymers are produced in situ in oil reservoirs [33]. Flooding water is then redirected into low permeability zones, thereby enlarging sweeping volume [16, 34]. The deeper the plugging position, the larger is the volume between the plugging position and entrance, and the higher is the sweep volume [25, 26]. This implies that the sweep volume of flooding water in deep profile control was higher in comparison to non-deep profile control. Accordingly, oil displacement efficiency should be improved in deep profile control than in non-deep profile control [35].
In practice, it is difficult to measure variations in sweep volume and profile control both in oil reservoirs and artificial cores. Therefore, cell concentration and BC production in the front-, middle-, and rear-ends of artificial cores were quantified to determine the deep profile control ability of FY-0701 and FY-07. The obtained results indicated that the cell concentration of FY-0701 gradually increased from the front- to rear-ends of the cores, while the highest cell concentration of FY-07 was observed at the middle-end of the core (Fig. 4).
To visually compare the deep profile control abilities of FY-07 and FY-0701, BC production in artificial cores was observed by SEM (Fig. 5). In accordance with variations in cell concentration, the quantity of BC in artificial cores plugged by FY-0701 (Fig. 5, cores A and B) also showed a gradual increment. These results indicated that FY-0701 is more suitable for deep profile control than FY-07.
EOR of the genetically engineered strain in oil displacement experiment
The ultimate goal of deep profile control is to increase the sweep volume of injected water and EOR in low permeability zones. Therefore, the oil displacement abilities of FY-07 and FY-0701 were compared in the core flooding experiment using two sand-packed columns with similar permeability and PV. During the subsequent water flooding process, EOR increased to 12.09% in the sand-packed column plugged by FY-0701, while that of FY-07 plugged sand-packed column only increased to 8.23% (Fig. 6). In addition, during the cell injection process, the injection pressure of FY-07 rapidly increased to its highest, becoming approximately 3.16-fold that of FY-0701. In comparison with the water flooding process, the injection pressure of subsequent water flooding only increased by twofold. These results indicated that the residual BC fibers on FY-07 cells can considerably increase the difficulty and cost of MEOR techniques, as they can contribute toward the retention of bacterial cells at the region near injection wells and increase injection pressure. Unlike FY-07, pressure when using FY-0701 did not show an obvious change during the cell injection process, but it increased by approximately 3.29-fold in the subsequent water flooding process (Fig. 6a, b), which conforms to the general trait of profile control [10, 16, 26]. The schematic diagram of FY-07 and FY-0701 in oil displacement processes are shown in Fig. 6c, d.
Although premature plugging in the core flooding experiment could increase ORE during the injection process (Fig. 6a), this is difficult to achieve in a long-term water-flooded reservoir due to less quantity of residual oil near the injection wells [7]. Moreover, with the increase of injection time, the drastic increase of injection pressure will be more serious, which will affect the subsequent injection. In addition, biopolymer production during the injection process can inevitably cause plugging in supply pipes and walls of the well bore [16, 27]. Deep profile control may thus be more effective for long-term water flooding reservoirs. Our results indicated that during the subsequent water flooding process, EOR increased to 12.09% in the sand-packed column plugged by FY-0701, which was 3.86% higher than when the column was plugged by FY-07 (Fig. 6). This implies that FY-0701 is more suitable for deep profile control than FY-07.
Considering the size of sand-packed columns, it is challenging to completely simulate the injection process in an oil field. Bacterial cells could sufficiently be injected in our core flooding experiment, but in actual production practice, proliferation of bacterial cells is essential during the injection process to reduce the mining cost. FY-0701 is therefore a good candidate for deep profile control in oil reservoirs. Glycerol-based nutrient solutions can supply nutrients to ensure FY-0701 growth and transport the cells to high permeability zones in oil-bearing formations. Also, glucose-based nutrient solutions can activate in situ BC-producing ability of FY-0701 to increase sweep volume and enhance oil recovery by reducing the permeability of such formations.