Peike Gao

Microbial diversity and abundance in the Xinjiang Luliang long‐term water‐flooding petroleum reservoir

Microbial populations associated with microbial enhanced oil recovery (MEOR) and their abundance in the Xinjiang Luliang water‐flooding petroleum reservoir were investigated using 16S rRNA, nitrate reductases, dissimilatory sulfate reductase, and methyl coenzyme‐M reductase‐encoded genes to provide ecological information for the potential application of MEOR. 16S rRNA gene miseq sequencing revealed that this reservoir harbored large amounts of taxa, including 155 bacterial and 7 archeal genera. Among them, Arcobacter, Halomonas, Marinobacterium, Marinobacter, Sphingomonas, Rhodococcus, Pseudomonas, Dietzia, Ochrobactrum, Hyphomonas, Acinetobacter, and Shewanella were dominant, and have the potential to grow using hydrocarbons as carbon sources. Metabolic gene clone libraries indicated that the nitrate‐reducing bacteria (NRB) mainly belonged to Pseudomonas, Azospirillum, Bradyrhizobium, Thauera, Magnetospirillum, Sinorhizobium, Azoarcus, and Rhodobacter; the sulfate‐reducing bacteria (SRB) were Desulfarculus, Desulfomonile, Desulfosarcina, Desulfotignum, Desulfacinum, Desulfatibacillum, Desulfatibacillum, Desulfomicrobium, and Desulfovibrio; while the methanogens were archaea and belonged to Methanomethylovorans, Methanosaeta, Methanococcus, Methanolobus, and Methanobacterium. Real‐time quantitative PCR analysis indicated that the number of bacterial 16S rRNA reached 106 copies/mL, while the metabolic genes of NRB, SRB, and methanogens reached 104 copies/mL. These results show that the Luliang reservoir has abundant microbial populations associated with oil recovery, suggesting that the reservoir has potential for MEOR.

Microbial Abundance and Community Composition Influence Production Performance in a Low-Temperature Petroleum Reservoir

Enhanced oil recovery using indigenous microorganisms has been successfully applied in the petroleum industry, but the role of microorganisms remains poorly understood. Here, we investigated the relationship between microbial population dynamics and oil production performance during a water flooding process coupled with nutrient injection in a low-temperature petroleum reservoir. Samples were collected monthly over a two-year period. The microbial composition of samples was determined using 16S rRNA gene pyrosequencing and real-time quantitative polymerase chain reaction analyses. Our results indicated that the microbial community structure in each production well microhabitat was dramatically altered during flooding with eutrophic water. As well as an increase in the density of microorganisms, biosurfactant producers, such as Pseudomonas, Alcaligenes, Rhodococcus, and Rhizobium, were detected in abundance. Furthermore, the density of these microorganisms was closely related to the incremental oil production. Oil emulsification and changes in the fluid-production profile were also observed. In addition, we found that microbial community structure was strongly correlated with environmental factors, such as water content and total nitrogen. These results suggest that injected nutrients increase the abundance of microorganisms, particularly biosurfactant producers. These bacteria and their metabolic products subsequently emulsify oil and alter fluid-production profiles to enhance oil recovery.

Spatial isolation and environmental factors drive distinct bacterial and archaeal communities in different types of petroleum reservoirs in China.

To investigate the spatial distribution of microbial communities and their drivers in petroleum reservoir environments, we performed pyrosequencing of microbial partial 16S rRNA, derived from 20 geographically separated water-flooding reservoirs, and two reservoirs that had not been flooded, in China. The results indicated that distinct underground microbial communities inhabited the different reservoirs. Compared with the bacteria, archaeal alpha-diversity was not strongly correlated with the environmental variables. The variation of the bacterial and archaeal community compositions was affected synthetically, by the mining patterns, spatial isolation, reservoir temperature, salinity and pH of the formation brine. The environmental factors explained 64.22% and 78.26% of the total variance for the bacterial and archaeal communities, respectively. Despite the diverse community compositions, shared populations (48 bacterial and 18 archaeal genera) were found and were dominant in most of the oilfields. Potential indigenous microorganisms, including Carboxydibrachium, Thermosinus, and Neptunomonas, were only detected in a reservoir that had not been flooded with water. This study indicates that: 1) the environmental variation drives distinct microbial communities in different reservoirs; 2) compared with the archaea, the bacterial communities were highly heterogeneous within and among the reservoirs; and 3) despite the community variation, some microorganisms are dominant in multiple petroleum reservoirs.

An Exogenous Surfactant-Producing Bacillus subtilis Facilitates Indigenous Microbial Enhanced Oil Recovery

This study used an exogenous lipopeptide-producing Bacillus subtilis to strengthen the indigenous microbial enhanced oil recovery (IMEOR) process in a water-flooded reservoir in the laboratory. The microbial processes and driving mechanisms were investigated in terms of the changes in oil properties and the interplay between the exogenous B. subtilis and indigenous microbial populations. The exogenous B. subtilis is a lipopeptide producer, with a short growth cycle and no oil-degrading ability. The B. subtilis facilitates the IMEOR process through improving oil emulsification and accelerating microbial growth with oil as the carbon source. Microbial community studies using quantitative PCR and high-throughput sequencing revealed that the exogenous B. subtilis could live together with reservoir microbial populations, and did not exert an observable inhibitory effect on the indigenous microbial populations during nutrient stimulation. Core-flooding tests showed that the combined exogenous and indigenous microbial flooding increased oil displacement efficiency by 16.71%, compared with 7.59% in the control where only nutrients were added, demonstrating the application potential in enhanced oil recovery in water-flooded reservoirs, in particular, for reservoirs where IMEOR treatment cannot effectively improve oil recovery.

Compositions and Abundances of Sulfate-Reducing and Sulfur-Oxidizing Microorganisms in Water-Flooded Petroleum Reservoirs with Different Temperatures in China

Sulfate-reducing bacteria (SRB) have been studied extensively in the petroleum industry due to their role in corrosion, but very little is known about sulfur-oxidizing bacteria (SOB), which drive the oxidization of sulfur-compounds produced by the activity of SRB in petroleum reservoirs. Here, we surveyed the community structure, diversity and abundance of SRB and SOB simultaneously based on 16S rRNA, dsrB and soxB gene sequencing, and quantitative PCR analyses, respectively in petroleum reservoirs with different physicochemical properties. Similar to SRB, SOB were found widely inhabiting the analyzed reservoirs with high diversity and different structures. The dominant SRB belonged to the classes Deltaproteobacteria and Clostridia, and included the Desulfotignum, Desulfotomaculum, Desulfovibrio, Desulfobulbus, and Desulfomicrobium genera. The most frequently detected potential SOB were Sulfurimonas, Thiobacillus, Thioclava, Thiohalomonas and Dechloromonas, and belonged to Betaproteobacteria, Alphaproteobacteria, and Epsilonproteobacteria. Among them, Desulfovibrio, Desulfomicrobium, Thioclava, and Sulfurimonas were highly abundant in the low-temperature reservoirs, while Desulfotomaculum, Desulfotignum, Thiobacillus, and Dechloromonas were more often present in high-temperature reservoirs. The relative abundances of SRB and SOB varied and were present at higher proportions in the relatively high-temperature reservoirs. Canonical correspondence analysis also revealed that the SRB and SOB communities in reservoirs displayed high niche specificity and were closely related to reservoir temperature, pH of the formation brine, and sulfate concentration. In conclusion, this study extends our knowledge about the distribution of SRB and SOB communities in petroleum reservoirs.

Diverse algicidal bacteria associated with harmful bloom-forming Karenia mikimotoi in estuarine soil and seawater

Algicidal bacteria associated with Karenia mikimotoi have been isolated, yet the distribution of the algicidal bacteria has been rarely studied. Here, we postulated and demonstrated that terrestrial environment harbors diverse algicidal bacteria, which can survive in seawater along water flowing into marine and suppress Karenia mikimotoi. In summary, 9 and 5 bacteria with algicidal activity on Karenia mikimotoi were isolated from seawater and estuarine soil, respectively. Similar with the marine bacteria (Alteromonas sp., Halomonas sp., Marinobacter sp., Paracoccus sp., Rhodobacteraceae, Idiomarina sp.), the soil strains (Pseudoalteromonas sp. and Flavobaterium sp.) showed high mortality in Karenia mikimotoi with the inhibitory rate of 87% and 93.5%, respectively, after two days co-cultivation. Algicidal activity of the two strains was detected in the cell-free filtrate not in bacterial cells. The results suggest that algicidal bacteria associated with Karenia mikimotoi widely exist in terrestrial and marine environments, and have application potential on controlling Karenia mikimotoi.

Succession of microbial communities and changes of incremental oil in a post-polymer flooded reservoir with nutrient stimulation

Further exploitation of the residual oil underground in post-polymer flooded reservoirs is attractive and challengeable. In this study, indigenous microbial enhanced oil recovery (IMEOR) in a post-polymer flooded reservoir was performed. The succession of microbial communities was revealed by high-throughput sequencing of 16S rRNA genes and changes of incremental oil were analyzed. The results indicated that the abundances of reservoir microorganisms significantly increased, with alpha diversities decreased in the IMEOR process. With the intermittent nutrient injection, microbial communities showed a regular change and were alternately dominated by minority populations: Pseudomonas and Acinetobacter significantly increased when nutrients were injected; Thauera, Azovibrio, Arcobacter, Helicobacter, Desulfitobacterium, and Clostridium increased in the following water-flooding process. Accompanied by the stimulated populations, higher oil production was obtained. However, these populations did not contribute a persistent level of incremental oil in the reservoir. In summary, this study revealed the alternative succession of microbial communities and the changes of incremental oil in a post-polymer flooded reservoir with intermittent nutrient stimulation process.

A novel bioemulsifier from Geobacillus stearothermophilus A-2 and its potential application in microbial enhanced oil recovery

Biosurfactants can improve the mobility of oils in porous media by changing the rock wettability and emulsifying oils, thus increasing the efficiency of crude oil recovery in the petroleum industry. Therefore, surfactant-producers play important roles in the microbial enhanced oil recovery (MEOR) process. In this study, a thermophilic, facultative anaerobic emulsifier-producing strain was isolated. The emulsifier produced and its potential applications in MEOR were investigated in the laboratory. The stain was identified as Geobacillus stearothermophilus, and was designated as A-2, which could use sodium acetate, which is relative abundant in reservoirs, as the carbon source. The produced bioemulsifier is a novel glycoprotein emulsifier, containing 71.4% sugar and 27.8% protein, wherein the monosaccharides were identified as mannose (33.5%), glucose (30.9%), galactose (29.7%), and glucuronic acid (5.9%); and the protein contained 17 types of amino acids. The bioemulsifier successfully emulsified various hydrocarbons at a wide range of salinity, temperature, and pH. Notably, the emulsion layer of diesel remained stable for 12 months at room temperature, with little change at the micron level in the particle size of oil droplets. Core flooding tests indicated that the fermentation broth of strain A-2 increased the oil recovery efficiency by 6.8% under lower oil saturation condition, showing potential applications in oil exploration in high-temperature oil reservoirs.

Antialgal effects of α-linolenic acid on harmful bloom-forming Prorocentrum donghaiense and the antialgal mechanisms

Harmful algal blooms (HABs) induced by Prorocentrum donghaiense occur frequently and cause a serious threat to the marine ecosystem. In this study, antialgal effects of α-linolenic acid (ALA) that is generally extracted from diverse macroalga on P. donghaiense were investigated. Specifically, the growth, cellular morphology and ultrastructure, reactive oxygen species (ROS) content, mitochondrial membrane potential (MMP), cytochrome C (Cyt-C), and caspase-9,3 activity of untreated and treated P. donghaiense were investigated. The results showed that ALA significantly inhibited the growth of P. donghaiense. Under ALA exposure, the cellular morphology and ultrastructure were damaged. ALA also induced ROS overproduction in the algal cells, decreased MMP, induced Cyt-C release, and activated caspase-9,3, which strongly relates to algal apoptosis. In summary, this study revealed the responses of morphology and physiology of P. donghaiense when exposed under ALA, and shows the potential of biotechnology on controlling P. donghaiense.

The relative abundance of alkane-degrading bacteria oscillated similarly to a sinusoidal curve in an artificial ecosystem model from oil-well products: Population succession pattern in a microbial ecosystem

Microbial phylogenetic diversity and species interactions in natural ecosystems have been investigated extensively, but our knowledge about their ecological roles, community dynamics and succession patterns is far from complete. This knowledge is essential to understand the complicated interactions of microorganisms in natural ecosystems. Here, an artificial ecosystem model of microorganisms was constructed from oil-well products and cultivated in a chemostat to investigate the succession pattern of alkane-degrading bacteria, a functional population in oil reservoirs. Their abundance was quantified by an improved qPCR technique. Our results showed that the phylogenetic structure of this artificial ecosystem model is stable during most of the chemostat cultivation process, while the genotype structure of alkane-degrading bacteria containing alkB genes shifted and their relative abundance oscillated similarly to a sinusoidal curve, like the succession pattern of producers in the Lotka-Volterra model. These results suggest that some theoretical frameworks of macroecology may work well in microbial ecosystems and be an efficient tool to understand them.