AlkB and CYP153 are important alkane hydroxylases responsible for aerobic alkane degradation in bioremediation of oil-polluted environments and microbial enhanced oil recovery. Since their distribution in nature is not clear, we made the investigation among thus-far sequenced 3,979 microbial genomes and 137 metagenomes from terrestrial, freshwater, and marine environments. Hundreds of diverse alkB and CYP153 genes including many novel ones were found in bacterial genomes, whereas none were found in archaeal genomes. Moreover, these genes were detected with different distributional patterns in the terrestrial, freshwater, and marine metagenomes. Hints for horizontal gene transfer, gene duplication, and gene fusion were found, which together are likely responsible for diversifying the alkB and CYP153 genes adapt to the ubiquitous distribution of different alkanes in nature. In addition, different distributions of these genes between bacterial genomes and metagenomes suggested the potentially important roles of unknown or less common alkane degraders in nature.

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The distribution of microbial communities in the Menggulin (MGL) and Ba19 blocks in the Huabei Oilfield, China, were studied based on 16S rRNA gene analysis. The dominant microbes showed obvious block-specific characteristics, and the two blocks had substantially different bacterial and archaeal communities. In the moderate-temperature MGL block, the bacteria were mainly Epsilonproteobacteria and Alphaproteobacteria , and the archaea were methanogens belonging to Methanolinea , Methanothermobacter , Methanosaeta , and Methanocella . However, in the high-temperature Ba19 block, the predominant bacteria were Gammaproteobacteria , and the predominant archaea were Methanothermobacter and Methanosaeta . In spite of shared taxa in the blocks, differences among wells in the same block were obvious, especially for bacterial communities in the MGL block. Compared to the bacterial communities, the archaeal communities were much more conserved within blocks and were not affected by the variation in the bacterial communities.

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This study examined and compared the microbial community in three typical fermentation starters (called as Daqu, Xiaoqu, and Fuqu in China) used for liquor production by analysing the 16S and 18S rRNA gene clone library. The results show that the microbial diversity in the three types of fermentation starters (JiuQu) differs significantly. The bacterial species in Daqu and Fuqu were mainly thermophilic or thermotolerant. In Daqu, the dominant bacterial species were Thermoactinomyces sanguinis (53.85%) and Pantoea agglomerans (19.23%), followed by uncultured bacteria (15.39%). The lactic acid bacterium Weissella cibaria (50%) and a member of Enterobacteriaceae, Enterobacter ludwigii (10%), were the dominant bacterial species in Xiaoqu. Low abundances of other bacteria, including Deinococcus radiodurans, Corynebacterium variabile and Acinetobacter baumannii, were reported for Xiaoqu. Enterococcus faecium, Clostridium beijerinckii and Bacillus cereus were observed in Fuqu and accounted for 46.67, 23.33 and 16.67% of the total bacteria identified, respectively. Fungal diversity was high in Daqu and consisted exclusively of thermophilic moulds, such as Aspergillus glaucus (62.5%), Thermomyces lanuginosus (12.5%) and Thermoascus crustaceus (12.5%). Only two fungal species were reported for Fuqu and Xiaoqu and both contained the mould Rhizopus oryzae. Saccharomyces cerevisiae and the non-Saccharomyces yeast (Saccharomycopsis fibuligera) were also identified in Fuqu and Xiaoqu, respectively. This finding suggests that microbial community structure in JiuQu starters is the key factor to determine the variety of flavours.

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Two alkane hydroxylase-rubredoxin fusion gene homologs (alkW1 and alkW2) were cloned from a Dietzia strain, designated DQ12-45-1b, which can grow on crude oil and n-alkanes ranging in length from 6 to 40 carbon atoms as sole carbon sources. Both AlkW1 and AlkW2 have an integral-membrane alkane monooxygenase (AlkB) conserved domain and a rubredoxin (Rd) conserved domain which are fused together. Phylogenetic analysis showed that these two AlkB-fused Rd domains formed a novel third cluster with all the Rds from the alkane hydroxylase-rubredoxin fusion gene clusters in Gram-positive bacteria and that this third cluster was distant from the known AlkG1-and AlkG2-type Rds. Expression of the alkW1 gene in DQ12-45-1b was induced when cells were grown on C 8 to C 32 n-alkanes as sole carbon sources, but expression of the alkW2 gene was not detected. Functional heterologous expression in an alkB deletion mutant of Pseudomonas fluorescens KOB2⌬1 suggested the alkW1 could restore the growth of KOB2⌬1 on C 14 and C 16 n-alkanes and induce faster growth on C 18 to C 32 n-alkanes than alkW1⌬Rd, the Rd domain deletion mutant gene of alkW1, which also caused faster growth than KOB2⌬1 itself. In addition, the artificial fusion of AlkB from the Gram-negative P. fluorescens CHA0 and the Rds from both Gram-negative P. fluorescens CHA0 and Gram-positive Dietzia sp. DQ12-45-1b significantly increased the degradation of C 32 alkane compared to that seen with AlkB itself. In conclusion, the alkW1 gene cloned from Dietzia species encoded an alkane hydroxylase which increased growth on and degradation of n-alkanes up to C 32 in length, with its fused rubredoxin domain being necessary to maintain the functions. In addition, the fusion of alkane hydroxylase and rubredoxin genes from both Gram-positive and -negative bacteria can increase the degradation of long-chain n-alkanes (such as C 32 ) in the Gram-negative bacterium.Alkane hydroxylation is the key step in alkane degradation in microorganisms, and alkane hydroxylases play an important role in the microbial degradation of alkanes (34). There are three classes of alkane hydroxylases in microorganisms, depending on the chain length of the alkane substrate. The soluble nonheme di-iron monooxygenases (sMMO) and membrane-bound particulate copper-containing enzymes (pMMO) are the main enzymes that catalyze the oxygenation of alkanes C 1 to C 5 in length (21). The integral-membrane alkane monooxygenase (AlkB)-related alkane hydroxylases (37) and cytochrome P450 enzymes (35) found in fungi and bacteria can oxidize long-chain alkanes with up to 16 carbon atoms. Among the members of the third class of enzymes, which can catalyze the oxidation of alkanes longer than C 18 , only one C 15 to C 36 alkane monooxygenase (LadA) found in Geobacillus thermodenitrificans NG80-2, which is distinct from other known AlkBtype alkane hydroxylases, has been cloned and the activities of purified LadA on alkanes with different chain lengths have been previously identified (8). In the AlkB system, three individual...