Identification and characterization of Burkholderia multivorans CCA53

Objective A lignin-degrading bacterium, Burkholderia sp. CCA53, was previously isolated from leaf soil. The purpose of this study was to determine phenotypic and biochemical features of Burkholderia sp. CCA53. Results Multilocus sequence typing (MLST) analysis based on fragments of the atpD, gltD, gyrB, lepA, recA and trpB gene sequences was performed to identify Burkholderia sp. CCA53. The MLST analysis revealed that Burkholderia sp. CCA53 was tightly clustered with B. multivorans ATCC BAA-247T. The quinone and cellular fatty acid profiles, carbon source utilization, growth temperature and pH were consistent with the characteristics of B. multivorans species. Burkholderia sp. CCA53 was therefore identified as B. multivorans CCA53.


Introduction
The genus Burkholderia was firstly proposed by Yabuuchi et al. [1], and was classified as Gram-negative and nonspore forming β-proteobacteria. To date, more than 80 Burkholderia species have been reported, and two major clusters and several subgroups have been proposed based on phylogenetic analyses of the 16S rRNA, acd, gyrB, recA and rpoB gene sequences, as well as their genome sequences [2]. Group A contains plant-associated and saprophytic species [2]. For example, nitrogen fixation in legumes is facilitated by B. mimosarum, B. nodosa, B. sabiae, B. tuberum and B. phymatum [3]. Also, growth rates of a few plants are promoted by B. phytofirmans and B. unamae [3]. On the other hand, group B contains opportunistic pathogens that infect animals, humans and plants [2]. B. cenocepacia, B. latens and B. multivorans infect to cystic fibrosis patients, which leads to pneumonic illness [4,5]. B. cenocepacia, B. multivorans and B. vietnamiensis show infectivity to alfalfa and lettuce [6].
Several Burkholderia species are now being utilized in industrial applications as biocatalysts [7,8], for biodegradation [9] and as plant growth-promoting rhizobacteria [3]. For example, B. fungorum DBT1 is capable of assimilating polycyclic aromatic hydrocarbons, which is useful for bioremediation of contaminated soils [10]. B. cepacia GS3C exhibits highly efficient degradation during bioremediation of oil-contaminated soil [11,12], and B. cepacia PCL3 is useful for treating carbofuran-contaminated water [13]. In addition, several antibiotics, including cepaciamide A [14], glidobactin A [15], pyrrolnitrin [16] and xylocandins [17] are produced by Burkholderia species. Several Burkholderia species showed lignin degradation capabilities [18], which are favorable to produce second-generation biofuels from lignocellulosic biomass. Thus, Burkholderia species are versatile bacteria with potential applicability in the biochemical and pharmaceutical industries. We previously isolated Burkholderia sp. CCA53 from leaf soil [19] and determined the draft genome sequence of the strain [20]. In this study, we report the phenotypic and biochemical characterization of Burkholderia sp. CCA53.
Burkholderia sp. CCA53 (strain number: HUT-8135) was cultured in Nutrient Broth (Kyokuto, Tokyo, Japan). The OD 600 was monitored by measuring the difference between the cell and cell-free culture turbidity values using an Eppendorf BioSpectrometer (Eppendorf, Hamburg, Germany). Carbon source utilization was determined using API 20E (bioMérieux, Marcy-l'Etoile, France) and API 50CHE (bioMérieux) according to the manufacturer's instructions. The effects of temperature (10-60 °C) and pH (3.0-10.0) on the growth were studied.
The lipid was extracted from lyophilized cells according to the method of Bligh and Dyer [24], and then loaded onto a Sep-Pak Plus Silica cartridge (Waters, Milford, MA, USA). After washing the cartridge, the quinone was eluted. Quinone quantification was performed using an ACQUITY UPLC system (Waters) with an Eclipse Plus C18 column (Agilent technologies, Santa Clara, CA, USA). The chromatographic conditions were as follows: mobile phase, methanol/isopropanol (3:1 v/v); flow rate, 0.5 mL min −1 ; the column oven temperature, 35 °C. The identification of quinone forms was carried out as previously described [25].
The cellular fatty acid compositions were determined using the Sherlock Microbial Identification System Version 6.0 (MIDI, Newark, DE, USA) and TSBA6 database (MIDI).
When B. multivorans CCA53 was cultured in Nutrient Broth at various temperatures (10-60 °C), the maximum growth rate was achieved at 20 °C (Fig. 2a). The strain was capable of growing at temperatures between 20 and 50 °C, but no growth was seen at 60 °C (Fig. 2a). At 30 °C, the maximum growth rate of B. multivorans CCA53 was at pH 4.0 (Fig. 2b). Moreover, the strain grew effectively at pHs between 4.0 and 9.0, but growth rates were sharply lower at pHs below 3.0 or above 10.0 (Fig. 2b). These characteristics were nearly the same as those of B. multivorans NKI379, which was also isolated from soil samples in the Er-Ren River Basin, Taiwan [33].

Discussion
From the viewpoints of economics, ecology and environmental protection, it would be advantageous to produce biofuels from lignocellulosic biomass, which are known as second-generation biofuels [34]. When second-generation biofuels are produced from lignocellulosic biomass, consecutive pretreatment, enzymatic hydrolysis and microbial fermentation steps are required. During the pretreatment step, lignocellulosic biomass is decomposed through heating, which releases cellulose, hemicellulose Phylogenetic tree reconstructed from analysis of the sequences of six housekeeping genes (atpD, gltD, gyrB, lepA, recA and trpB) and showing the relationship of CCA53 with related species. Pandoraea apista DSM 16536 T was used as an outgroup. The tree was reconstructed using the neighbor-joining method with Kimura's two-parameter model [28]. Closed circles show the nodes supported by 80% bootstrap probabilities with 1000 replicates. The bar indicates the 1% nucleotide substitution rate and lignin. At the enzymatic hydrolysis step, cellulose and hemicellulose are converted into saccharified solution, which includes fermentable sugars, aldehyde inhibitors and lignin. In the fermentation step, the fermentable sugars are used as carbon sources by engineered Escherichia coli, Saccharomyces cerevisiae or other microorganisms [35,36]. Although aldehyde inhibitors inhibit microbial growth and interfere with subsequent fermentation, these compounds can be chemically or enzymatically detoxified [34,37]. However lignin is not effectively utilized by the aforementioned microorganisms, causing the yield to be low [35,36]. Microbial degradation of lignin has been primarily studied in brown-and white-rot fungi. Using the Fenton reaction, brown-rot fungi produce free hydroxyl radicals from hydrogen peroxide, after which the free hydroxyl radicals are used in the lignin degradation [38]. Moreover, white-rot fungi are capable of producing several extracellular ligninolytic enzymes, including laccase, lignin peroxidase, manganese peroxidase and versatile peroxidase, which are also useful for lignin degradation [39]. On the other hand, these fungi show slower growth rates and require for long incubation times, which elevates the production costs and draws lower productivities. A few bacterial species belonging to the genera Arthrobacter, Burkholderia, Comamonas, Pseudomonas, Sphingobium, Streptomyces and Rhodococcus show faster growth rates and lignin degradation capabilities, but their capabilities are lower than those of fungi [18]. We therefore screened for lignin-degrading bacteria with rapid growth rates and high capabilities for lignin degradation, and a candidate bacterium was isolated from leaf soil [19]. Based on its 16S rRNA gene sequence homology, the bacterium was identified as Burkholderia sp. CCA53 [19]. This strain was capable of utilizing lignin as a sole carbon source, and it was anticipated that Burkholderia sp. CCA53 would have industrial potential for second-generation biofuel production [19]. In the present study, therefore, we characterized the phenotypic and biochemical features of Burkholderia sp. CCA53. Several Burkholderia species, including B. cepacia KK01 [40] and Burkholderia sp. LIG30 [41] also have a capacity to degrade lignin. In Burkholderia sp. LIG30, the mechanism of its lignin degradation is suggested by its expression of two genes predicted to encode multicopper oxidase and 22 genes encoding putative catalases or peroxidases [41]. Within the draft genome sequence of B. multivorans CCA53, one gene predicted to encode multi-copper oxidase and 21 genes encoding putative catalases or peroxidases were also confirmed [20]. This suggests the mechanism for lignin degradation used by B. multivorans CCA53 may be similar to that used by Burkholderia sp. LIG30.
When saccharified solutions are prepared from sugarcane, cassava and their wastes, d-glucose and l-xylose are the main saccharides [42,43], though small amounts of d-lactose and d-maltose are also present [42,43]. Several Burkholderia species cannot assimilate d-lactose or d-maltose [29], but B. multivorans CCA53 was able to use all of these disaccharides as carbon sources, which means that B. multivorans CCA53 could be a useful strain for production of second-generation biofuels [35,36]. Moreover, we think that B. multivorans CCA53 may have other advantages for industrial application beyond utilization of lignin. The first is that B. multivorans CCA53 showed efficient growth at acidic pH (Fig. 2b). Several lignocellulosic biomass-degrading enzymes showed maximum activities at acidic pH [44][45][46], which means that the saccharified solution pH is also acidic. By contrast, the growth of industrial bacteria such as E. coli is inefficient at acidic pH. Consequently, pH control is required at the fermentation step with engineered E. coli, whereas B. multivorans CCA53 would not require pH control. Second, the optimal growth pH for B. multivorans CCA53 would be expected to prevent contamination by microorganisms in larger scale fermentations. Third, B. multivorans CCA53 showed strong growth at 20-40 °C (Fig. 2a), which is similar to the mesophilic conditions required for E. coli and S. cerevisiae. This means that the existing systems for biofuel fermentation will be applicable for use with B. multivorans CCA53.

Limitations
In this paper, we reported the phylogenetic, phenotypic and biochemical characterization of Burkholderia sp. CCA53. To identify the phylogeny of Burkholderia sp. CCA53, we performed MLST analysis. In addition, results of phenotypic and biochemical analyses were consistent with the characteristics of B. multivorans species. Burkholderia sp. CCA53 was therefore identified as B. multivorans CCA53. These results may give little interest for microbiologists.

Authors' contributions
HA designed this study, performed experiments, participated in the interpretation of the results and drafted the manuscript. ZK and MZMY participated in the design and coordination of this study and helped to revise the manuscript. NN and TH conceived and designed this study, coordinated the experiments, interpreted the results and revised the manuscript for important intellectual content. All authors read and approved the final manuscript.