J Ind Microbiol Biotechnol DOI 10.1007/s10295-017-1909-0

ENVIRONMENTAL MICROBIOLOGY - ORIGINAL PAPER

Potential of wheat bran to promote indigenous microbial enhanced oil recovery Yali Zhan1 · Qinghong Wang1 · Chunmao Chen1,2 · Jung Bong Kim3 · Hongdan Zhang1 · Brandon A. Yoza4 · Qing X. Li2   

Received: 3 May 2016 / Accepted: 29 January 2017 © Society for Industrial Microbiology and Biotechnology 2017

Abstract  Microbial enhanced oil recovery (MEOR) is an emerging oil extraction technology that utilizes microorganisms to facilitate recovery of crude oil in depleted petroleum reservoirs. In the present study, effects of wheat bran utilization were investigated on stimulation of indigenous MEOR. Biostimulation conditions were optimized with the response surface methodology. The co-application of wheat bran with KNO3 and NH4H2PO4 significantly promoted indigenous MEOR (IMEOR) and exhibited sequential aerobic (O-), facultative (An-) and anaerobic (A0-) metabolic stages. The surface tension of fermented broth decreased by approximately 35%, and the crude oil was highly emulsified. Microbial community structure varied largely among and in different IMEOR metabolic stages. Pseudomonas sp., Citrobacter sp., and uncultured Burkholderia sp. dominated the O-, An- and early A0-stages. Bacillus sp., Achromobacter sp., Rhizobiales sp., Alcaligenes Y. Zhan and Q. Wang contributed equally to this article and are joint first authors. Electronic supplementary material  The online version of this article (doi:10.1007/s10295-017-1909-0) contains supplementary material, which is available to authorized users. * Qing X. Li [email protected] 1

State Key Laboratory of Heavy Oil Processing, China University of Petroleum, Beijing 102249, China

2

Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA

3

Department of Agro‑Food Resources, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju 55365, Republic of Korea

4

Hawaii Natural Energy Institute, University of Hawaii at Manoa, Honolulu, HI 96822, USA





sp. and Clostridium sp. dominated the later A0-stage. This study illustrated occurrences of microbial community succession driven by wheat bran stimulation and its industrial potential. Keywords  Wheat bran · Biostimulation · Response surface methodology · Indigenous microbial enhanced oil recovery

Introduction Increased oil demand has facilitated the development of novel oil extraction technologies. Efforts primarily focus upon methods that allow for efficient crude oil access during tertiary phase recovery [3, 17]. Of those methods, microbial enhanced oil recovery (MEOR) has received significant attention due to cost-efficiency and its environmentally friendly merits. MEOR utilizes complex microbial populations and their specific metabolites to facilitate recovery of crude oil in depleted petroleum reservoirs [34]. Numerous trials have shown that MEOR is an effective means to extend oil field life and enhance oil recoveries [11, 31, 47, 51]. MEOR can be classified into exogenous MEOR (EMEOR) and indigenous MEOR (IMEOR) according to the source of the microbes used in the process [2]. Regardless of how extreme the environments are, petroleum reservoirs contain abundant microbial populations. Indigenous microbes are, however, nutrient limited (C, N, P) and stimulating microbial growth requires addition of nutrients [9]. IMEOR techniques directly inject nutrients into the petroleum reservoir and have advantages over EMEOR as it requires less equipment and the indigenous microbes are already adapted to the environment [37]. Propagation of beneficial microbes that contribute to the

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metabolic production of biosurfactants, acids, solvents, biopolymers and gases, etc. is critical to the successful implementation of IMEOR. Furthermore, low environmental impacts and economical applications are important considerations. A variety of nutrients for the promotion of IMEOR has been screened. Carbohydrates (mainly molasses) have been widely used in trials as carbon sources and inorganic nutrients injected as alternative electron acceptors for O2 [16]. With increasing demand for bio-ethanol, market prices for carbohydrates have risen [23]. However, wheat bran, a low cost agricultural byproduct waste, is abundant and rich in proteins, carbohydrates, minerals and vitamins [29]. Wheat bran has been widely used for enzymatic solid-state fermentations and for the production of biosurfactants, alcohols, hydrogen and acids [21, 33, 38]. Wheat bran has, however, not been investigated as a bio-stimulator to promote IMEOR. Previous studies using specific microbes were usually evaluated by the formation of fermented products [7, 14]. Similarly, metabolite production in MEOR processes is often used to evaluate biostimulation [2, 10], yet little is known about the changes in the indigenous microbial community. Describing the relationship between microbial community structures and their functions will contribute to the understanding of biostimulation mechanisms and promotion of IMEOR [42, 43, 50]. The objectives of this study were to investigate the industrial IMEOR potential using wheat bran. Additionally, biostimulation pathways were studied by microbial community analysis.

J Ind Microbiol Biotechnol

(NH4)2HPO4 and Na3PO4. All nitrogen and phosphorus sources were purchased from Beijing Chemical Reagents Co., China. Optimization of biostimulation conditions

Materials and methods

The nutrient components were screened by single factor optimization (SFO) experiments in which one parameter was changed at a time. SFO experiments were implemented in 250 mL Erlenmeyer flasks. Each flask contained 50 g of formation water, 10 g of crude oil and various nutrient contents, and kept a final volume of 100 mL using distilled water. Erlenmeyer flasks were incubated for 6 days at 37 °C and 110 rpm in an air-bath shaker. All materials, except formation water and crude oil, were autoclaved at 115 °C for 30 min. SFO experiments did not depict the interaction effects of all factors [40]. Response surface methodology (RSM) experiments were implemented to quantify biostimulation conditions as previously described [8, 20, 45]. In RSM experiments, the Plackett–Burman design was used to evaluate the significance of six factors, including wheat bran, molasses, formation water, KNO3, NH4H2PO4, and crude oil. The pair-wise co-effects of the selected critical factors at three levels were further probed by the BoxBehnken design. The analysis of variance (ANOVA) and response surface regression analysis were performed with “Design Expert” software v 8.0.4 (Stat-Ease, Minneapolis, MN, USA). The factors with P value <0.05 were considered significant. The operation conditions of RSM experiments were the same as SFO ones except formation water and crude oil content that were variables. SFO and RSM experiments were carried out in triplicate. Gas production was an indicator of biostimulation performance.

Materials

Simulation of IMEOR

Formation water and crude oil were both sampled from the Menggulin block at Huabei Oilfield, which is located in northern China and has over 30 years of water-flooding operations. After the samples were collected in sterilized plastic bottles, the bottles were stored in cooler boxes and immediately transported to the laboratory. Initial temperature, pH and salinity of the formation water were 37 °C, 8.26 and 1301 mg/L, respectively. Kinematic viscosity (50 °C) and density (20 °C) of the crude oil were 50.45 mm2/s and 0.842 g/cm3, respectively. Nutrients used to stimulate indigenous microbes in a MEOR process mainly included carbon, nitrogen and phosphorus sources. Carbon sources were local wheat bran (particle size of 100 mesh), corn starch residues and molasses. Nitrogen sources were KNO3, NaNO3, NH4NO3, (NH4)2SO4, NH4Cl and urea (CO(NH2)2). Phosphorus sources were NH4H2PO4, NaH2PO4, KH2PO4, K2HPO4,

The biostimulation effects of wheat bran were evaluated in 250 mL Erlenmeyer flasks. Each flask contained 100 mL of initial broth under optimized biostimulation conditions. An aliquot of 0.05 mL of Resazurin solution (0.1 wt%) was added to each flask as a visual anaerobic indicator. Sterilization followed the protocols described during RSM experimentation, as well as the incubation conditions with an extended 28 day incubation period. Simulated IMEOR experiments were carried out in triplicate. The indicators of biostimulation effects were measured. The produced gases were collected in bottles and analyzed on a HP 6890 gas chromatograph (GC) (Agilent, Wilmington, DE, USA) [5]. The OD600 was recorded with a UV-2550 UV–VIS spectrophotometer (Shimadzu, Kyoto, Japan). The pH was measured with a PHSJ-4 m (Leici, Shanghai, China). The volatile fatty acids (VFAs) were determined on GC-2010 plus GC (Shimadzu, Kyoto, Japan) [46]. The emulsification

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J Ind Microbiol Biotechnol

effects, caused by produced biosurfactants, biopolymer, acids and solvents, were determined by measuring the surface tension and emulsification state, which the former was recorded with a JK99B tension meter (Powereach, Shanghai, China) and the latter was observed under a CX21BIMSET5 microscope (Olympus, Tokyo, Japan) at 200× magnification. Analysis of microbial diversity Fermented broth samples (2 mL) were collected directly from Erlenmeyer flasks. After centrifugation at 8,000 rpm for 10 min, the precipitate was extracted with a KG-201 DNA isolation kit for genomic DNA (TianGen, Beijing, China) according to the instructions. The V3 region of 16S rDNA genes was amplified with universal primers 341FGC and 534R [26]. PCR amplification was carried out in a 50 μL reaction mixture containing 2 μL of DNA template, 2 μL of each primer, 19 μL of nuclease-free water and 25 μL of 2× GoTaq Colorless Master Mix (Promega, Madison, WI, USA). The touchdown PCR procedure was as follows: (1) initial denaturation at 94 °C for 7 min; (2) 20 cycles at 94 °C for 1 min, annealing at 65 °C for 1 min (decrease of 0.5° with every cycle) and extension at 72 °C for 1 min; (3) 10 cycles at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min; (4) final extension at 72 °C for 10 min. PCR products were stored at 4 °C and verified by electrophoresis on 1.2% (w/v) agarose gel. DGGE analysis of PCR products was performed on a D-Code universal mutation detection system (Bio-Rad, Hercules, CA, USA) as described by Ma et al. [22]. Electrophoresis was performed at 60 °C, initially at 60 V for

20 min and then at 100 V for 10 h. The gels were stained with Invitrogen SYBR Green I (Life Tech, Gaithersburg, MD, USA) for 30 min and then photographed with a Gel Doc 2000 gel documentation system (Bio-Rad) to obtain DGGE profiles. DGGE profiles, including the presence, intensity and similarities of the bands, were analyzed with Quantity One software (Version 4.4, Bio-Rad). The microbial diversity was calculated by Shannon-wiener’s indexes (H) [13, 18]. Dominant bands were excised from the gels and then re-amplified. The fragments were recovered and cloned with the procedure previously described by Shi et al. [36]. Positive clones were selected and sequenced (Beijing Genomics Institute, China). Typical sequences were analyzed with the NCBI BLAST database to identify the closest relatives. A phylogenetic tree was constructed with MEGA software (Version 4.0).

Results Optimized conditions of biostimulation Wheat bran, KNO3 and NH4H2PO4 were determined to be the preferred combination of carbon, nitrogen and phosphorus sources, respectively (Fig. S1). The biostimulation conditions were preliminarily determined to be 1.0 g/100 mL of wheat bran, 0.05 g/100 mL of KNO3, 0.05 g/100 mL of NH4H2PO4 and 2.0 g/100 mL crude oil under SFO experiments (Fig. S2). Formation water, wheat bran and KNO3 were determined to be the critical factors as evaluated through Plackett–Burman experiments (Table 1). According to Box-Behnken experiments (Table 2) and followed response surface regression analysis, the optimized

Table 1  Experiment results of Plackett-Burman design for optimization of biostimulation conditions Runs

Concentrations (g/100 mL)

Gas production (mL)

Wheat bran

Formation water

KNO3

NH4H2PO4

Molasses

Crude oil

1 2 3 4 5 6 7 8 9 10 11

0.67 (−1) 0.67 1 (+1) 1 0.67 0.67 1 1 0.67 0.67 1

25 (−1) 50 (+1) 25 25 25 50 50 25 25 50 50

0.1 (+1) 0.1 0.05 (−1) 0.05 0.05 0.05 0.1 0.1 0.1 0.05 0.05

0.1 (+1) 0.1 0.1 0.1 0.05 (−1) 0.1 0.1 0.05 0.05 0.05 0.05

0 (−1) 0.33 (+1) 0.33 0.33 0 0 0 0 0.33 0.33 0

4 (+1) 2 (−1) 4 2 2 4 2 2 4 2 4

24 35 47 55 40 84 61 49 36 56 87

12

1

50

0.1

0.05

0.33

4

71

(−1) for low level and (+1) for high level

13

Table 2  Experiment results of Box-Behnken design for bio-stimulation conditions optimization

J Ind Microbiol Biotechnol Runs

Wheat bran (g/100 mL) Formation water (g/100 mL) KNO3 (g/100 mL) Gas production (mL)

1 2 3 4 5 6 7 8 9

1.6 (+1) 1.6 1.6 1.35 (0) 1.35 1.35 1.35 1.35 1.1 (−1)

70 (0) 70 90 (+1) 70 70 50 (−1) 90 50 70

0.06 (+1) 0.04 (−1) 0.05 (0) 0.05 0.05 0.04 0.06 0.06 0.06

98 99 111 156 155 99 112 118 115

10 11 12 13 14

1.6 1.35 1.1 1.35 1.1

50 90 90 70 50

0.05 0.04 0.05 0.05 0.05

98 113 153 158 114

15

1.1

70

0.04

115

(−1) for low level, (0) for medium level and (+1) for high level

biostimulation conditions were eventually identified as 1.28 g wheat bran, 76.0 g formation water, 0.051 g KNO3, 0.05 g NH4H2PO4 and 2.0 g crude oil in each 100 mL fermented broth (Fig. 1).

accumulated to a peak value of 1950 mg/L on the 16th day when the total VFAs reached a peak value of 2375 mg/L (Table 4). Propanoic acid was first recorded on the 12th day and disappeared in the later A0-stage.

Gas production Emulsification The concentration of oxygen in the field-IMEOR process was changed leading to conversion of microorganism community [44]. In the simulated IMEOR process, the color of fermented broth changed from red to pink and then colorless, which indicated the gradual conversion from aerobic (O-) (0–2nd day), facultative anaerobic (An-) (3–7th day), and anaerobic (A0-) metabolic stages (8–28th day). Gas production lasted for 10 days; a total of 176 mL of gases (1760 mL gases per L fermented broth, 1760 mL/L) was collected mainly during the O- and An-stages. The highest gas production rate was 640 mL/L/days at the end of O-stage (Fig. 2a). The components of produced gases varied among stages (Table 3). The CO2 concentration promptly increased from 0.03 to 37.88 v% along with a sharp depletion of O2 in the O- and An-stages. The H2 concentration increased to 10.87 v% in the A0-stage. Acid production The pH values rapidly decreased from the initial 7.47–5.76 during the O- and An-stages and then slowly declined to 5.32 by the 16th day, followed by a slight increase to 5.58 at the end (Fig. 2b). OD600 increased from zero to 2.73 on the 16th day and then declined gradually in the later A0-stage (16th day later). VFAs were not detected during the O-stage, after which, acetic acid was gradually

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The surface tension dropped from 56.0 to 45.3 mN/m during the O- and early An (2–4th day) -stages, but no significant decline in the later An-stage (5–7th day). After that, the surface tension declined rapidly and reached a value of 36.4 mN/m on the 16th day (Fig. 2c). The surface tension decreased by approximately 35%, and crude oil was highly emulsified (Fig. 2d).

PCR‑DGGE result of microbial community There were 19 major bands in the DGGE profiles (Fig. 3), which have distinct differences in H-index and band number between lanes (Table S1). Microbial community similarities among eight lanes varied from 18.2 to 77.8% (Table S2). Of those bands, 18 bands were isolated from the gel, re-amplified, and sequenced. Except for bands 6, 16 and 17, all of the identified genera had sequence similarities of 98% or greater. The sequencing of band 19 was unsuccessful, which was consistent with the previous report [32]. The generated phylogenetic tree (Fig. 4) shows that the identified sequences could be divided into three general groups, Proteobacteria, Firmicutes and Bacteroidetes. Sequence of band 7 was not classified into the phylogenetic tree, which was presumably not reported.

J Ind Microbiol Biotechnol

Discussion Gas production (mL)

a

X2

1.10

1.23

1.35

1.60

1.48

X1

Gas production (mL)

b

X3

1.10

1.23

1.35

1.48

1.60

X1

Gas production (mL)

c

X3

50.0

60.0

70.0

80.0

90.0

X2

Fig. 1  Relationships between wheat bran (X1) and formation water (X2) at KNO3 concentration of 0.05 g/100 mL (a), between wheat bran (X1) and KNO3 (X3) at formation water concentration of 70.0 g/100 mL (b), and formation water (X2) and KNO3 (X3) with wheat bran concentration of 1.35 g/100 mL (c) on gases production

Corn starch residue and molasses are widely used carbon sources in MEOR [48, 54]. The performance of biostimulation by wheat bran was compared with that of corn starch residue and molasses. The single factor optimization (SFO) experiments indicated that wheat bran showed the highest biogas production than molasses and corn starch. It is noteworthy that the market price of wheat bran (RMB¥ 800– 1000 per ton, https://detail.1688.com/offer/1010098291. html?spm=a261b.2187593.1998088710.64.ZqUtdz, accessed on December 3, 2016) was similar with that of molasses (RMB¥ 780-1000 per ton https:// detail.1688.com/offer/43589259743.html?spm= a2 61b.2187593.1998088710.26.xWl2QY&tracelog=p4p, accessed on December 3, 2016). However, the oil fields were located in northeast and northwest China. The transport and storage of molasses would add up the costs. The local wheat processing enterprises most probably provide adequate supplies of wheat bran. Therefore, wheat bran offered the best biostimulation and economic performance in comparison with the other two carbon sources. KNO3 contributed significantly to gas production. Moreover, as NO3− is the terminal electron acceptor used by nitrate-reducing bacteria, competitive interactions can inhibit sulfate-reducing bacteria (SRB) [10]. K+ can stimulate indigenous microbes [19]. Phosphorus sources are not critical for gas production. Lower concentrations of carbon source caused indigenous microbes aging, while higher ones affected cellular growth due to an increased osmolality of fermented broth. The concentrations of wheat bran, KNO3 and NH4H2PO4 at 1.0 g/100 mL, 0.05 g/100 mL and 0.05 g/100 mL, respectively, guaranteed balanced nutrient conditions. The concentration of crude oil had a negligible effect on gas production, which indicated that wheat bran was applicable to petroleum reservoirs with different oil contents. Formation water, wheat bran and KNO3 with P values of 0.01, 0.043 and 0.046, respectively, were determined to be significant factors by Plackett–Burman experiments. By applying response surface regression analysis to Box-Behnken experiments, the following second-order equation was established:

GP = 156.333 − 11.375X1 + 7.5X2 + 2.125X3 − 20.542X12 − 16.792X22 − 29.042X32 − 6.5X1 X2 − 0.25X1 X3 − 5X2 X3

(1)

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J Ind Microbiol Biotechnol

c

b A0-stage

200

600 500

160

400

120

300

80

200 100

40 0

0 0

1

2

3

4

5

6

7

8

7.5

O-stageAn-stage A0-stage

3.0 2.5

7.0

9 10 total

2.0

6.5

1.5

6.0

1.0

5.5

0.5

5.0

OD600

An-stage

Surface tentison (mN/m)

O-stage

pH

240

Gas producing rate (mL/L/d)

Cumulative gas production (mL)

a

0.0

56

O-stageAn-stage A0-stage

52 48 44 40 36

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Time (d)

Time (d)

Time (d)

d

Fig. 2  Gas production and production rate (a), pH and OD600 changes (b), surface tension changes (c) and crude oil micro-emulsification effect (d) of fermentation broth in simulated IMEOR process

Table 3  Component changes of produced gases in simulated IMEOR processes

Components

Initial concentration v%

O-stage (2nd day) v%

An-stage v%

A0-stage v%

3rd day

6th day

8th day

10th day

O2 CO2 H2

21.00 0.03 0

8.15 28.54 0

0.09 35.77 2.72

0.05 37.88 4.12

0 38.81 6.84

0 38.74 10.87

N2

78.97

63.31

61.42

57.95

54.35

50.39

Table 4  Acid changes of fermentation broth in simulated IMEOR processes VFAs

O-stage (mg/L)

An-stage (mg/L)

A0-stage (mg/L) Early stage

Later stage

2nd day

4th day

8th day

12th day

16th day

20th day

24th day

28th day

Acetic acid Propionic acid

0 0

679 0

919 0

1838 422

1950 425

1261 0

1044 0

1003 0

Total VFAs

0

679

919

2260

2375

1261

1044

1003

where GP was the gas production (mL), X1, X2 and X3 were the concentrations (g/100 mL) of wheat bran, formation water and KNO3, respectively. The ANOVA with a P value of 0.01 and a R2 value of 94.8% indicated Eq. (1) was highly reliable for predicting gas production. X21, X23 and X1 showed significant influence with P values of 0.006, 0.001 and 0.013, respectively. The predicted maximum gas production (159 mL) was obtained when the

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concentrations of formation water, wheat bran and KNO3 were 76.0 g/100 mL, 1.28 g/100 mL and 0.051 g/100 mL, respectively. The experimental gas production of 165 mL under optimized biostimulation conditions from triplicate determinations was consistent with the predicted value. Indigenous microbes were quickly stimulated and produced gases on the 1st day. The concentration changes of CO2 and O2 in the O- and An-stages indicated the rapid

J Ind Microbiol Biotechnol

A

B8

8

19

19

1 15 10 2 11 3 4 12 13 16

1 15 10 2 11 3 4 12 13 16

5 17 6 14 7 18

5 17 6 14

A03

A01

An1

A02

O1

A0

A05

1

7 18

100.0% 80.0% 70.0% 70.0% 55.6% 44.4% 35.3% 16.7%

Fig. 3  DGGE profile of bacterial samples from different stages (a) and schematic diagram of relative band intensities in DGGE profiles (b). Lane number represented the bacterial samples collected in dif-

ferent dates. Lane 1 the zero day; Lane O1 the 2nd day; Lane An1 the 4th day; Lane A01 the 8th day; Lane A02 the 12th day; Lane A03 the 16th day; Lane A04 the 23rd day; Lane A05 the 28th day

growth of aerobic bacteria shortly after biostimulation. The concentration alteration of H2 in the A0-stage signified the activation of anaerobic metabolisms and inhibition of aerobic bacteria. Gas production is helpful to repressurize the pressure-depleted petroleum reservoirs and reduce crude oil viscosity by partial dissolution of the gas. CO2 can increase carbonate rock porosity and permeability with its sub-acidity, increasing the exudation rate of remaining crude oil [53]. Methane production in MEOR process was observed in the previous research [4]. However, it was not observed in the present study, which might be due to the absence of methanogens in the formation water. Gas production is an important effect for promoting IMEOR which mainly occurred during the O-stage. To produce more gases, the O-stage can be extended intentionally in field trials by co-injecting air into a petroleum reservoir. VFAs were not detected during the O-stage while pH decreased to 6.5, which might be attributed to neutralization of the produced VFAs by initial alkaline substances. The peak value of VFAs on the 16th day indicated substantial propagation and metabolism of indigenous microbes in the An- and early A0-stages. Gradual reduction of acetic acid and disappearance of propanoic acid were associated with a slight increase in pH throughout the later A0-stage. The metabolites produced here were reused by anaerobic microbes as substrates. Acid production is beneficial to IMEOR having similar mechanisms to CO2 [34]. However, an excessive amount of acids may be toxic to some species of indigenous microbes. Acid production, therefore, should be controlled during field trials. The present results demonstrated that the indigenous microbial community in

petroleum reservoirs itself can regulate their living conditions through co-metabolism. Aerobic microbes that were stimulated in the O- and early An-stages due to the presence of O2 resulted in decreased surface tension because most biosurfactantproducing microorganisms are aerobic [25]. Aerobic microbes were inhibited during the gradual depletion of O2, while anaerobic microbes were acclimated in the later An-stage, remaining surface tension stable. Stimulated anaerobic microbes produced emulsifiers during the early A0-stage. After that, surface tension remained stable due to anaerobic bacteria inhibition caused by nutrient depletion. The decrease in surface tension of the fermentation broth is the result of emulsification. Among the metabolites, biosurfactants have the strongest emulsifying effects, while biopolymers, acids and solvents also contribute to the emulsification of crude oil [52]. Emulsifier production plays a critical role in IMEOR with their combined functions of emulsifying crude oil, reducing oil–water interfacial tension, lowering the viscosity and promoting the fluidity of crude oil in petroleum reservoirs [15, 28, 41]. A continuous supply of O2 and nutrients to a petroleum reservoir will contribute to the efficient emulsification. Changes in microbial diversity occurred in a simulated IMEOR process. A number of light bands were presented in lane 1 (H-index at 1.66), which indicated that the initial formation water had an established microbial community having low in cell counts due to low nutrient content. Addition of nutrients resulted in the stimulation of Bacillus sp., Pseudomonas sp., Burkholderia sp. (band 6) and Citrobacter sp. communities, dominating the O-stage (Lane O1) having an H-index reaching 1.97. The added nutrients and

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J Ind Microbiol Biotechnol

Fig. 4  Phylogenetic tree of 16S rDNA sequences from DGGE profiles. The bootstrap values less than 50% are not shown in the topology

O2 were rapidly utilized by indigenous microbes to proliferate and grow, accelerating gas production, as well as VFAs and emulsifier generation. Indigenous microbes competed

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mutually in the An-stage (lane An1), increasing microbial diversity (H-index at 2.25), but due to reduced oxygen and changes in available carbon gas production decreased

J Ind Microbiol Biotechnol

during this stage. Microbial diversity declined on lane A01 (H-index at 2.11), and Bacillus sp. was no longer represented. The anaerobic bacterium, Paenibacillus sp. emerged at the beginning of A0-stage. Thereafter, microbial diversity significantly increased again (H-index at 2.35) on lane A03. Anaerobic bacteria such as uncultured Bacteroidetes, Clostridium xylanolyticum and Citrobacter sp. became active. Facultative bacteria such as Pseudomonas sp., Burkholderia sp. and Rhizobiales during aerobic growth persisted in the anaerobic environment. This explains why pH and surface tension reached their minimum levels as well as OD600 and acid production reached its maximum level during the 16th day. Microbial diversity displayed a remarkably decline on lanes A04 and A05. Only Bacillus thuringiensis, Rhizobiales bacteria, Achromobacter sp., Clostridium sp., Alcaligenes sp. and band 19 remained, which can be principally attributed to the complete depletion of available nutrients. In addition, SRB had not been detected, which suggested its growth inhibition by KNO3 [41]. Pseudomonas sp., Citrobacter sp., and uncultured Burkholderia sp. were the dominant bacteria during the O-, An- and early A0- stages with the relative abundance ranges of 20–24, 6–14, and 10–19%, respectively (Fig.  5). Pseudomonas sp. can decompose crude oil and produce biosurfactants [1, 30, 49]. Citrobacter sp. can use carbohydrates to produce acids and gases under anaerobic conditions [49]. Burkholderia sp. can produce biosurfactants by degrading petroleum hydrocarbons [35]. Bacillus sp., which has an excellent capability of

0% 10%

24%

20%

23%

producing biosurfactants and polymers [35], dominated the O- (relative abundance from 26 to 39%) and later A0stages (relative abundance from 19 to 43%). These four genera were widely reported as functioning bacteria in the MEOR process [24, 27]. Achromobacter sp., Rhizobiales bacteria, Alcaligenes sp. and Clostridium sp. also were dominant bacteria in the later A0-stage. Rhizobiales bacteria can use a variety of substrates to produce acids [6]. Clostridium sp. can produce hydrogen, acids and solvents in anaerobic conditions [6, 39]. Achromobacter sp. and Rhizobiales bacteria which are usually classified as aerobic bacteria survived and dominated in the A0-stage, which suggests the importance of the environment in bacterial community establishment. The role of Achromobacter sp. in a MEOR process has not been previously reported. Alcaligenes sp. should be controlled in MEOR because it can consume acids and produce alkalis, which would result in decreased emulsification [12]. This also explains the reduction of VFAs and slight increase in pH in the later A0-stage. The population structure and relative abundance of microbial communities, especially the interactions between different microbes, have a significant influence on promoting IMEOR. Wheat bran, with the addition of other nutrients, could enhance the indigenous microbial diversity as well as strengthen the functions of gas, acid and emulsifier production. The results indicated that wheat bran can effectively improve IMEOR.

0% 6%

6% 10%

8%

39%

26%

19%

2nd day

10%

15%

11%

13% 0%

0% 4%

24%

11% 0%

0%

22%

14% 0% 5%

19%

6%

9%

16th day

10%

0%

5%

18%

6% 0%

43%

12%

21% 0% 5%

23rd day

28th day

20%

11% 8% 9% 0% 6%

13% 18%

12th day

8th day

10%

18%

8% 0%

6% 0%

4th day

20%

15%

21%

28%

15%

Pseudomonas sp. Citrobacter sp. Burkholderia sp. Bacillus sp. Achromobacter sp. Paenibacillus sp. Clostridium sp. Rhizobiales bacterium Alcaligenes sp. Uncultured bacterium Others

Fig. 5  Relative abundance changes of different microbes in a simulated IMEOR process

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Conclusions The biostimulation performance of wheat bran was investigated for the potential to promote IMEOR. Wheat bran exhibited great promotion effects to produce gases, acids and emulsifiers with the assistance of other nutrients. The re-pressurization and decrease in pH value and surface tension in petroleum reservoirs are important attributes of IMEOR. Microbial diversity showed remarkable successions and beneficial functioning bacteria were stimulated during the experimental IMEOR process. Pseudomonas sp., Citrobacter sp., and uncultured Burkholderia sp. dominated the O-, An- and early A0-stages. Bacillus sp., Achromobacter sp., Rhizobiales bacteria, Alcaligenes sp. and Clostridium sp. dominated the later A0-stage. IMEOR effects were promoted by the interactions between various indigenous microbes. The results indicated an applicable potential of wheat bran for improving IMEOR owing to its effectiveness and low cost. Acknowledgements This study was supported in part by the National Natural Science Foundation of China (No. 21306229), the Korean RDA (No. PJ011884) and the Science Foundation of China University of Petroleum, Beijing (2462014YJRC001).

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