The mechanism of petroleum hydrocarbon degradation by the selected bacteria Brevibacillus brevis and Bacillus cereus is studied based on the metabolite analysis. The result shows that bio-oxidation processes, especially those called unconventional subterminal oxidation, terminal and double-terminal oxidation, are the main pathway by which the bacteria will convert petroleum hydrocarbons into fatty monoacids, hydroxyl fatty acids and dicarboxylic acids. This, for the first time, makes an important correction to the prevailing opinion which says that long-chain hydrocarbon molecules can be degraded into short-chain hydrocarbon molecules instead of acid compounds. The study explains how the bacteria ingest petroleum hydrocarbons as well as the correlation between ingestion modes and the active substances present in crude oil. The pathway to produce bio-surfactants and the correlation between produced bio-surfactants and oil metabolism are also discussed in this paper.From 2002 to 2004, Brevibacillus brevis and Bacillus cereus were applied in the microbial huff and puff as well as the Microbial enhanced waterflood test in Daqing excessively low permeability (1.0md-25md) reservoir. After bacteria injection, 44 among the 60 huff and puff wells shows a significant oil production response - the accumulative oil increment reaches 6012 tons and the input-output ratio amounts to 1: 6. The Microbial enhanced waterflood test carried out in June 2004 also provides a satisfying result. After the injection of bacteria, the pressure in the two water injectors decreased more than 2MPa, which results in an injection capacity increase. In late 2004, the fluid production of the 5 producers (the total number is 8) is increased from 29.5 tons per day (before bacteria injection) to 44.4 tons per day, and the corresponding oil production is increased from 20.8 tons per day (before bacteria injection) to 36.9 tons per day. These make MEOR a very promising oil recovery method in Daqing Oilfield. Introduction With the development of biotic technology and ever-increasing difficulty in EOR.the advantages of MEOR have been further recognized. MEOR has been applied in some countries [1-8] successfully and has enter a new era. MEOR has showed [9] a promising outlook. But the studies of MEOR mechanisms have not gone deeply, the success ratio of application is not high, and this technology cannot play an important role in improving oil recovery to the full extent. Thereby, MEOR mechanisms, nutritions and economic effect should be thoroughly researched. MEOR dominantly takes advantage of the motabolites of microorganisims, including biosurfactants, gases acids and alcolhols. In the process of fermentation, microorganisms could degrade some heavy hydrocarbon and wax, and simultanesouly reduce viscosity of oil. Some research results show the light hydrocarbons would be generated as the heavy hydrocarbons were degraded, improving the quality of oil and increasing oil recovery [14-16]. The machanimisms of hydrocarbon degradation by Brevibacillus brevis and Bacillus cereus have been researched here. Pilot test have been performed with two types of microorganisms in the reservoirs with extra low permeability. 1. The Mechanism of MEOR by Brevibacillus Brevis and Bacillus Cereus Liquid medium, confected with sewage water from No. 1 Oil Plant, was injected into tri-angle bottles for sterilization. Then the inocula (activated Brevibacillus brevis and Baillus cereus) were introduced into bottles of sterilized medium with one bottle blank as contrast. Dehydrated and degassed oil was introduced, and incubated at a rotate speed of 120 rpm at 45.5 day later, fermentation liquid experienced refrigerated centrifugation at a rotate speed of 3000 rpm for 20mins. As the result, oil and water were separated. Oil above the water was sampled and stored in refrigator or performed analysis immediately. 1.1 Changes of saturated hydrocarbon in oil After treatment by Brevibacillus brevis and Bacillus cereus, the distribution curve of saturated hydrocarbon in oil distinctly diff rent with the blank. The content of heavy component decreases and the content of the light component increases relatively. The ratio of ∑nC 21-/∑ nC 22+ increases from 1.35 to 1.73 and 1.87 respectively, and the ratio of Pr/nC 27 and Ph/nC 28 also appears some changes. After treatment by Brevibacillus brevis, the ratio of Pr/nC 27 and Ph/nC 28 increases 19.0%and 17.9% respectively. After treatment by Bacillus cereus, the ratio increases 9.5%and 23.1% respectively. It means that two strains can play the role in the degradaton of heavy satruarated hydrocarbon in oil.(Fig1) As seen in Table 1, the ratio of Pr/nC 17 and Ph/nC 18 in oil treated by Brevibacillus brevis and Bacillus cereus almost have no difference with blank. It is concluded that nC 17 and nC 18 in oil are not degraded, and no nC 17 and nC 18 are generated at the same time when the heavy hydrocarbon is degraded, the change law of nC 12∼nC 16 in oil studied here also verify the point. (Table2) Based on above-mentioned discussions, it is concluded that Brevibacillus brevis and Bacillus cereus could only degrade heavy hydrocarbon, not light hydrocarbon, and no light hydrocarbon are generated. The conclusion may be inconsistent with the result showed in Fig 1. Seen from Fig 1, light saturated hydrocarbon increases, comparing with heavy saturated hydrocarbon. The fact is that the heavy hydrocarbon decreases and the light one retains. Thus, some wrong conclusions were drew from these similar figures [17-22]. 1.2Analysis and comparison of polar components in oil samples before and after microbial treatment. To study the microbial treatment on Daqing crude oil, the blank sample was first analysed with GC-MS method. There are 56 kinds of polar materials present in the blank, in which alcohol, aldehyde/ketone and ester compounds respectively amount to 31, 11 and 7. Furthermore, the alcohols and esters are dominant and both account for 97.46 percent of the oxygen-bearing polar materials. The content of another kind of oxygen-bearing compound, fatty acid, however, remains much lower in the blank, it proves that the oil of Daqing field has lower acidity (lower than 0.01mg/g). The kinds of extracellular fatty acids in samples treated by Bacillus cereus and Brevibacillus brevis increase from the original 2 to 17 and 24 respectively, and their relative contents rise from the original 1.05wt% to 60.05wt% and 61.02wt% respectively. They are mainly composed of monoatomic acids and binary acids with carbon number ranging from 2 to 20. The study also indicates that the alkyl acids, especially those with linear, saturated alkyl acids, are dominant in the newly generated acids. A certain amount of naphthenic, alkenyl and aromatic acids are also generated in the degraded samples. 1.3 The decomposition of alkanes Base on the above-mentioned facts, it is deduced that bio-oxidation is the main way in which Brevibacillus brevis and Bacillus cereus degrade crude oil in Daqing oilfield (Fig2). Carbon number of n- alkanes in oil of Daqing oilfield ranges from 2 to 40. The analysis of the oil composition treated by Brevibacillus brevis and Bacillus cereus showes that the blended fatty acids are mainly composed with carbon number ranging from 2 to 20. Both microorganism degrade only the saturated heavy hydrocarbon (carbon number higher than 20), the fact which proves there is probably a so-called unconventional subterminal oxidation in which Brevibacillus brevis and Bacillus cereus convert one long-chain hydrocarbon into two short-chain fatty monoacids or alcohols.(Fig3) 1.4 Bio-surfactants generated in the microbial metabolism Several measurements, such as TLC, ultraviolet absorption, infrared spectrum and elementary analysis, were performed to check bio-surfactants from two fermentation liquids. The measurements showed the bio-furfactants were the mixture of paraffin esters and glycerides, very complicated compounds with a wide range of molecular weight, not sugar esters and phosphorus esters bio-surfactants. The study demonstrates that hydrocarbons at first go though bio-oxidation pr cess to produce fatty acids and fatty alcohols, which, at the presence of catalyst - extracellular esterase, will be converted into a mixture of compounds with a certain molecular weight distribution. They are paraffin esters, paraffin esters with alcoholic group or poly-paraffin esters. 1.5 The modes of hydrocarbon ingestion by Bacillus cereus and Brevibacillus brevis There are generally three modes of hydrocarbon ingestion by the bacteria: mode I :the reciprocity between bacteria and solved alkyl ; mode II : what the bacteria contact and ingest are minute petroleum drops; mode : what the bacteria contact and ingest are the large hydrocarbon drops. In order to validate the hydrocarbon ingestion modes, three parallel experiments have been performed: 1 To apply specified medium, oil was the only carbon source ; 2 Base on former condition, a little tween80 was added into medium; 3 Base on former condition, a little yeast extract was added into medium.Based on experiments, it is deduced that mode is the maim way on the condition 2 and 3; however, on the condition1, there are two hydrocarbon ingestion modes (mode and mode) and the two hydrocarbon ingestion modes could turn from each other depending on the specific period. Due to the low content of fatty acid in Daqing oil, it is difficult for microbial to ingest hydrocarbon by micelle or micro-micelle. At the beginning of microbial incubation, hydrocarbons are ingested mainly in the mode, resulting in a slow increase in bacteria concentration and production of bio-surfactants. Copyright 2005, Society of Petroleum Engineers.