Characterization of Variovorax Strain C6d Isolated from the Algae-bacteria Consortia

The rhizosphere microorganisms can form beneficial, pathogenic, or neutral relationships. These relationships can promote plant growth and productivity. Among them, a number of Variovorax isolates from the rhizosphere were isolated. Bacteria Variovorax strain C6d (AB552893) was isolated from the non axenic culture of Chlorella spp., C6. The cell was Gram-negative, motile, non-spore-forming, short and rod-shaped (0.5-1.0x1.5-2.0μm). Colonies were in white colour after 7 days on 10-fold diluted Nutrient Broth. The strain was able to tolerate NaCL to 1.0% but not to 4.0% of NaCl. It grew quite well at temperatures ranging from 10°C to 37°C, yet did not show any growth at 4°C and 42°C. The dominant isoprenoid quinone was ubiquinone 8 (Q8). The major fatty acid composition of this strain was summed feature 3, 16:0 and 18:1:w7. The DNA G+C content of strain C6d was 70.4 mol%.


Introduction
The phycosphere is the zone surrounding an algal cell within which microorganisms are influenced by algal products. Not a small number of microalgae are living in soil, and the phycosphere in soil may represent a good microhabitat for soil bacteria. Studies on the associations between green microalgae (phylum Chlorophyta, kingdom Plantae) and bacteria are considered to be of great value, because the form is not only a microorganism-microorganism association but, at the same time, may also be a primitive plantmicroorganism association.
Rhizosphere microbes that can form beneficial, pathogenic, or neutral relationships. Understanding these associations can inform strategies for manipulating the plant's microbiome to promote plant growth and productivity. Among the microorganisms isolated from the rhizosphere are a number of Variovorax isolates (Jennifer, 2019). Variovorax are known for degrading a variety of toxins and organic compounds (Futamata et al., 2005). Variovorax also plays an important role in the natural cycling of biogenic chemicals including degrading a variety of contaminants such as pesticides (Han et al., 2011).
Variovorax are microorganisms of interested due to their diverse metabolic capabilities. Many species of Variovorax have been isolated from contaminated soils and groundwater around the world and of those, some are able to biodegrade of both biogenic compounds and anthropogenic contaminants. For example, Mahan et al. (2017) isolated Variovorax sp. strain JS1663 that was able to degraded N-nitroglycine (NNG). This is the key enzyme involved in its catabolism. The interaction between Variovorax with other microbe and/or plants makes V. paradoxus ideal for studies investigating its potential for different applications.
Variovorax strain C6d was isolated from the culture of Chlorella spp., and C6 was initially isolated in November 2005 from the soil collected in the experimental field of the University of Tokyo, Japan (Otsuka et al., 2008a). The algal culture has been maintained without purification in C liquid medium (Ichimura, 1971) for long term. The cultivation conditions were a 16:8 light-dark cycle with a light intensity of about 10-20 µmol photons m -2 s -1 at 25°C in the light and 20°C in the dark. The algal culture was serial subcultivated once every four weeks. C medium contains no carbon sources for general heterotrophic bacteria (Vu et al., 2010).
Currently, few studies the characterization of Variovorax isolated from algal cultures have been reported. In this study, the taxonomic position of the bacteria Variovorax strain C6d is discussed based on phenotypic, genotypic and chemotaxonomic properties. This study gave us a good understanding of the bacteria associated with Chlorella, and opened a chance for conducting further studies on the effects of the consortia on the soil and environment.

Identification of bacteria
DNA of the isolate was extracted according to Ashida et al. (2010). The almost full length of the 16S ribosomal RNA (rRNA) gene of the isolate was amplified by polymerase chain reaction (PCR) with the forward primer 27F and the reverse primer 1492R that corresponds to the positions 8-27 (AGAGTTTGATCCTGGCTCAG) and 1510-1492 (GGTTACCTTGTTACGACTT) in the Escherichia coli 16S rRNA gene numbering, respectively and a thermal cycler GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). The amplification was carried out with the initial denaturation step at 94°C for 10min, then 27 cycles including denaturation at 94°C for 30 sec, annealing at 55°C for 30sec, and extension at 72°C for 1min. The final extension was done at 72°C for 7min, and then the mixtures were held at 4°C. Amplification of the PCR products of the proper size was confirmed by electrophoresis through a 1.5% agarose gel, followed by staining with ethidium bromide. MicroSpin S-400 HR columns (GE Healthcare Biosciences, Oslo, Norway) were used to purify the amplified products then directly sequenced as described by Saito et al. (2008). The DNA sequence chromatograms were edited and analyzed by using Finch TV ver. 1.4 (Geospiza, Seattle, WA, USA; http://www.geospiza.com/finchtv/) software and bidirectional sequences were aligned by Clustal W ver. 2.0 (Larkin et al., 2007) to obtain the consensus sequence for each region of the 16S rRNA gene of a strain. The type strains of species phylogenetically closely related to the isolated strains were searched for using the SeqMatch program (Cole et al., 2007) based on the similarity of the almost full-length 16S rRNA gene sequences. Clustal W version 2.0 program was used to analyse the phylogenetic relationships of the isolated strains and the closely related species, and identify the strains at the genus level.

Gram staining and morphological characteristics
Gram staining was conducted based on the described methods by Gerhardt et al. (1994).The morphological characteristics of the cells were observed by photos taken by JEOL Datum (Tokyo, Japan) using transmission electron microscopy with negative staining.

Physiological tests
The physiological profile of the strains was determined using API 20NE microtest galleries (bioMérieux ® S.A., Marcy-l'Etoile, France) according to the manufacture's instructions. In addition, Biolog Gen III MicroPlate (Biolog, Inc) that allows testing 71carbon source and 23 chemical sensitivity was used to determine the assimilation and the oxidation patterns of these strains, respectively.
The growth of the bacteria at 4, 10, 15, 20, 26, 30, 37, and 42°C was determined by adding 100µL of the subculture into a glass tube containing pure 10mL of pure 10 -1 NB medium and evaluated after two weeks of the inoculation started. The growth of the bacteria was determined by the measurement of the OD at 600 nm compared to a blanked control of the culture media without bacteria.

Isoprenoid quinones
The isoprenoid quinones were extracted and analyzed according to the method described by Collins & Jones (1981) with some modifications. The bacterial cells were vacuum freeze-dried in Freevac (Tozai Tsusho Co., Ltd, Japan) to remove the water content. About 100mg of dried cells was transferred to a 100-mL glass flask and covered with by aluminium foil. Twenty milliliters (20mL) of extraction solvent (chloroformmethanol 2:1) was added and stirred for 2h at room temperature. The sample solution was filtered by a 5A Advantec filter paper (Advantec, Toyo Roshi Kaisha, Ltd., Japan). The procedure was repeated. The filtrates were combined and evaporated in vacuo using a rotary evaporator (R-3000 Rotavapor; Büchi Labortechnik AG, Germany) at 40rpm at 30°C. The quinones were extracted again by using 5mL of acetone and evaporated in the vacuo. Twohundred microliters (200µL) of 99.5% methyl ethanol was added to dissolve the quinones. The quinone fractions and the reference quinones were separated by thin-layer chromatography (TLC) using silica gel 60F (Merck, Ltd., Japan) and developed in the chromatographic chamber containing 100mL hexane-diethyl ether (85:15). After drying the quinone spots were detected under UV light at 275nm. The quinone spots were scraped off and collected in a tube and 500µL of acetone was added to extract the quinones. The quinone solution was concentrated by a stream of N2 gas and dissolved in 200µL of ethanol. The extracted quinones and the reference quinones were analyzed by a highperformance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) equipped with a 5C18R Cosmosil column (Nacalai Tesc) and using methanol-isopropanol (2:1) as the solvent. The oven temperature was set at room temperature with a pump rate of 1 mL min -1 , and chromatograms were read at 270nm.

Whole cellular fatty acids
Whole cellular fatty acids of the bacteria were extracted and analyzed according to the Sherlock Microbial Identification (MIDI) systems. The cell sample was saponified with 1mL of Sherlock Reagent 1, shook for 10sec and heated at 100°C for 5min, then shook again for 10sec and re-heated for 25min. After a total of 30 min of saponification in the water bath at 100°C, the sample was cooled in a cold tap water to room temperature. The cell suspension was methylated with 2mL of Sherlock Reagent 2, shook for five to ten seconds, heated at 80°C ± 1°C for 10min, and cooled rapidly to room temperature. The fatty acid methyl esters were extracted by adding 1.25mL of Sherlock Reagent 3 and gently mixed for 10min. The lower phase was removed and discarded by Pasteur pipette. The remaining upper phase was washed with 3mL of Sherlock Reagent 4 and mixed for 5min to remove free fatty acids and residual reagents from the organic extract. The suspension was centrifuged at 2,000×g for 5min to separate to two phases completely. About 2/3 of the upper phase extract solution was transferred to a clean GC sample vial using clean Pasteur pipette and analyzed by the 6890 Series GC system equipped with 7860 Series Injector (Agilent). Helium was used as the carrier gas. A 2µL injection volume of the samples was used. The injector temperature was 250°C, the column temperature was increased from 120 to 240°C at a rate of 5°C min -1 and the gas chromatography-mass spectrometry transfer line temperature was 280°C. Fatty acid methyl esters (FAMEs) were identified on the basis of their retention times and their mass spectra.

G + C content of the genomic DNA
The G+C content of the genomic DNA was determined by HPLC according to the method described by Mesbah et al. (1989). Briefly, 25µL of DNA solution containing from 2 to 25µg of DNA in a 1.5 microtube was incubated at 60ºC for 1h, then heated at 100ºC for 2min, and rapidly cooled in an ice bath and flash centrifuged. An equimolar mixture of nucleotides for analysis of DNA base composition (Yamasa Shoyu, Choshi, Japan) was used as the quantitative standard. The standard solution, made up of 10µL of the above nucleotides and 15µL of sterile distilled water was prepared under the same condition. Then, 25µL acetate buffer (40mM, pH 5.3) containing 2mM ZnSO4 , 20µL sterile distilled water, and 5µL P1 nuclease solution (20 U mL -1 ) were added to the DNA solution and incubated at 37ºC for 2h. Then 2.5µL of glycine buffer (0.1 M, pH 10.4) and 2µL of 1/10 alkaline phosphate were added to the DNA solution and incubated overnight at 37ºC. After the incubation period, 10µL of the sample solution was injected into the HPLC equipped with a 5C18R Cosmosil column (Nacalai Tesc), and 0.2M ammonium phosphateacetonitrile (40:1) as solvent. The column temperature was maintained at 40ºC with 1.0mL per min speed and read at 270nm. The G+C content was analyzed by Chromatopac using method 42 to obtain the factors C, G, T, and A.

Identification of bacteria
Strain C6d-NBRC 108627 T was isolated from the non-axenic culture of Chlorella sp and formed colonies with a smooth surface, translucent white color, moist texture, raised elevation, and circular margin on tenfold Nutrient Broth agar. This strain was identified as a Variovorax bacterium based on the neighbourjoining tree (Figure 1). The nearly full length of the 16S rRNA gene sequence of strain C6d, positions 28 to 1491 (Escherichia coli 16S rRNA gene numbering) was compared with those of the type strains of Variovorax bacteria. Sequence similarities of the 16S rRNA gene between strain C6d and the type strains of V. bronicumulans, V. soli, V. paradoxus, V. ginsengisoli, and V. dokdonensis were 96.9%, 96.7%, 96.6%, 96.6%, and 88.9%, respectively (Figure 1).

Morphological and phenotypic characteristics
The cell of the strain C6d was Gramnegative, motile using flagella and occurred as single cells, non-spore-forming, short and rodshaped (Figure 2). The cellular size was about 0.5-1.0×1.5-2.0µm. According to Satola et al. (2013), cells of Variovorax are Gram-negative, motile, and oval or rod-shaped, motile with flagella and live in pairs or single cells, straight to slightly curved rods and the size are amounts to 0.3-0.6×0.7-3.0μm, yellow colonies, slimy, and shiny. The morphological characteristics of strain C6d were nearly the same of other members of the genus Variovorax.

Chemotaxonomic analysis
The dominant isoprenoid quinone of strain C6d was ubiquinone 8 (Q8) as the same as the other type strains of the genus. The major fatty acid composition of this strain was summed feature 3, 16:0, and 18:1:w7, comprising more than 80% in total ( Table 1). Summed feature 7 and summed feature 4 were detected in all members of this genus but not detected in strain C6d. These two fatty acids were probably Vietnam Journal of Agricultural Sciences    identified as 18:1w7c and summed feature 3 in the present study. The differences in minor fatty acid composition between strain C6d and the type strains of Variovorax can also be attributed to the differences in culture conditions such as media used for cultivating the strain for fatty acid analysis. In this study, 1/10 NB was used but in the other studies, R2A was used as the medium to grow the strains. The DNA G+C content of strain C6d was 70.4mol%, which was within the range of 66.0 to 71.2mol% of the genus Variovorax (Im et al. 2010;Tuan et al., 2018).

Conclusion
The results of this study showed that Variovorax not only occurs in contaminated soils and groundwater, but also associates with algae. The strain C6d -NBRC 108627 T isolated from the nonaxenic algal culture had all of the characteristics of the members of Variovorax. These results provide to a chance for continued research on the potential of using C6d in the field Vietnam Journal of Agricultural Sciences of reclamation of polluted soil and the environment.