Ann Microbiol (2015) 65:1699–1707 DOI 10.1007/s13213-014-1009-6 ORIGINAL ARTICLE Isolation and characterization of Achromobacter sp. CX2 from symbiotic Cytophagales, a non-cellulolytic bacterium showing synergism with cellulolytic microbes by producing β-glucosidase Xiaoyi Chen & Ying Wang & Fan Yang & Yinbo Qu & Xianzhen Li Received: 27 August 2014 /Accepted: 24 November 2014 /Published online: 10 December 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract A Gram-negative, obligately aerobic, non- degradation by cellulase (Carpita and Gibeaut 1993). There- cellulolytic bacterium was isolated from the cellulolytic asso- fore, efficient degradation is the result of multiple activities ciation of Cytophagales. It exhibits biochemical properties working synergistically to efficiently solubilize crystalline cel- that are consistent with its classification in the genus lulose (Sánchez et al. 2004;Lietal.2009). Most known Achromobacter. Phylogenetic analysis together with the phe- cellulolytic organisms produce multiple cellulases that act syn- notypic characteristics suggest that the isolate could be a novel ergistically on native cellulose (Wilson 2008)aswellaspro- species of the genus Achromobacter and designated as CX2 (= duce some other proteins that enhance cellulose hydrolysis CGMCC 1.12675=CICC 23807). The strain CX2 is the sym- (Wang et al. 2011a, b). Synergistic cooperation of different biotic microbe of Cytophagales and produces β-glucosidase. enzymes is a prerequisite for the efficient degradation of cellu- The results showed that the non-cellulolytic Achromobacter lose (Jalak et al. 2012). Both Trichoderma reesi and Aspergillus sp. CX2 has synergistic activity with cellulolytic microbes by niger were co-cultured to increase the levels of different enzy- producing β-glucosidase. To our knowledge, this is the first matic components (Kumar et al. 2008). However, the ability of report on the synergetic effect of the combination of non- major cellulolytic member of microbial strains identified so far cellulolytic and cellulolytic microbes, which is significant to produced a limiting level of one or other enzymatic component help understand the cellulolytic mechanism of cellulose- (Maki et al. 2009). Thus, search for the potential source of digesting Cytophagales. cellulolytic activity is continuing in the interest of successful bioconversion of lignocellulosic biomass. Keywords Achromobacter . Cellulolytic . β-Glucosidase . In our study of cellulose degradation, it was found that the Cytophagales . Isolation . Synergic cellulolytic Cytophagales were always associated with some non-cellulolytic microbes in their process of cellulose diges- tion. Kato et al. (2008) also confirmed that the cellulose- degrading culture contains four organisms, but only one can Introduction hydrolyze cellulose, and the others are not directly linked to cellulose hydrolysis. To eliminate those non-cellulolytic con- Cellulose consists of only a linear structure of β-1,4-linked taminants, serial dilution and parallel streaking on the cellu- glucose residues, but is very difficult to be degraded, because lose medium was repeatedly performed. However, even after it is highly crystalline and often tightly intertwined together the cellulolytic Cytophagales were grown as a single colony with hemicelluloses and other polymers presenting a barrier to on the cellulose plate, non-cellulolytic strains were still asso- ciated with it, because such an association can develop upon X. Chen : Y. Q u School of Life Science, Shandong University, Jinan 250100, People’s transferring to the glucose agar plate. In order to understand Republic of China the attribution of those associations on cellulose digestion, one such non-cellulolytic microbe was isolated. It was observed : : : * X. Chen Y. Wan g F. Yang X. Li ( ) that the novel isolate could enhance the cellulolytic activity of School of Biological Engineering, Dalian Polytechnic University, Ganjingqu Dalian 116034, People’s Republic of China its partner Cytophagales when co-cultured together, although e-mail: [email protected] it did not show any activity on cellulose in the pure culture. In 1700 Ann Microbiol (2015) 65:1699–1707 this paper, we report on the isolation and some properties of medium used for the taxonomic assay is 0.05 g yeast extract this novel non-cellulolytic association, Achromobacter sp. in 100 mL of the mineral medium. Soaked agar was prepared CX2, and its synergic effect with cellulolytic microbes by by soaking and washing agar gel with distilled water to the produced β-glucosidase. To our knowledge, it is the first remove soluble contaminant after agar was melted in water report on non-cellulolytic microbes working synergistically by heating and cooled down to form agar gel. with cellulolytic strains to hydrolyze cellulose. Morphological properties Materials and methods The unidentified strain was cultured on the glucose medium plate overnight at 37 °C and the cellular morphology was Strains and culture condition observed by light microscopy. The motility was checked in the hanging-drop mount as described previously (Murray Cytophaga sp. LX-7 was isolated previously in our lab (Li and et al. 1994). Flagella were examined with a transmission Gao 1997) and cultured in the mineral medium in the presence electron microscope (TEM) by negative staining. For TEM, of 0.5 % (w/v) cellulose CF11 (Whatman) and 0.2 % (w/v) a droplet of the diluted culture was fixed with 3 % potassium peptone at 30 °C and 150 rpm. The mineral medium consists phosphotungstate (pH 7.0) and the observation was made on a of (per liter) 0.2 g MgSO4·7H2O, 0.75 g KNO3,0.5g model JEM-2000EX TEM. K2HPO4, 0.02 g FeSO4·7H2O,and0.04gCaCl2·2H2O, pH 7.0–7.2. Penicillium decumbens JU-A10 was obtained Physiological and biochemical characteristics by physical and chemical mutagenesis (Sun et al. 2008)and cultured at 30 °C and 160 rpm in the medium containing (per The Hucker staining method was used for Gram staining liter) 30 g wheat bran, 6 g microcrystalline cellulose, 30 g (Murray et al. 1994). Oxidase activity was detected using corncob residue, 2 g (NH4)2SO4,1gurea,3gKH2PO4,0.5g 1 % tetramethyl-p-phenylene-diamine as substrate, and the MgSO4,0.3gCaCl2, 4 g peptone, and 1 g Tween-80. result was read within 30 s. Catalase activity was determined by mixing 3 % hydrogen peroxide with colonies cultured for Isolation of non-cellulolytic strain 24–48 h, and the formation of gas bubbles within 2 min indicated a positive result. Nitrate reduction was tested on five A soil sample was collected from the surrounding enrichment consecutive days of incubation in culture medium containing with lignocellulose at the Bangcuidao Garden of Dalian, and 0.1 % KNO3 and a negative result was confirmed by adding inoculated on the filter paper agar plate by stamping the soil zinc dust. Urea hydrolysis was observed on Christensen urea sample on the filter paper with the flat end of glass rod. The agar slant and the occurrence of a red-violet color was con- inoculated plates were incubated at 37 °C and the transfers sidered as positive. Hydrolysis of starch, casein, esculin and were made as early as possible from the margin of the gelatin, methyl red (MR) reaction, Voges-Proskauer (VP) test, decomposed filter paper to a fresh filter paper agar plate when indole production, hydrogen sulfide production, and citrate cellulolytic bacteria grew. The transfer was performed by usage were carried out as described elsewhere (Smibert and streaking on the filter paper agar plate, and the plates were Krieg 1994). The assimilation of glucose, xylose, maltose, incubated at 37 °C for 5–6 days. This transfer was carried out sucrose, and cellobiose was determined by incubating the over ten times to set up a steady cellulolytic association. A 10- unidentified isolate in the mineral solution supplemented with fold serial dilution of the bacterial suspension with sterile 0.2 % of each carbohydrate at 37 °C for 7 days. Acid and gas saline was spread on the glucose medium plate, in which the production were tested at the same time as described by bacterial suspension was prepared by mixing cellulolytic bac- Smibert and Krieg (1994). Resistance to sodium chloride teria picked out from the thoroughly digested filter paper with was detected by growing on the glucose medium plate sup- the sterile glucose medium. After incubation at 37 °C over- plemented with 1–6 % NaCl at 0.5 % intervals. night, the individual single colonies were picked out from the glucose medium plates and incubated on the fresh glucose Nitrogen and carbon source usage medium plate. To ensure the strain purity, the isolate was streaked on the glucose medium plate and the single colony Nitrogen sources for cell growth were evaluated by comparing was used for the further study. the turbidity of the culture in mineral solution supplemented Filter paper agar plate was prepared by placing a piece of with 0.3 % of glucose and 0.2 % nitrogen sources including filter paper (Whatman No. 1, 8.5 cm in diameter) on a mineral yeast extract, peptone, beef extract, urea, sodium nitrate, am- medium with 2 % (w/v) soaked agar. Glucose medium con- monium citrate, diammonium phosphate, and ammonium sul- tains (per liter) 15 g glucose, 5 g beef extract, 10 g peptone, fate. To test the carbon sources for cell growth the unidentified and 20 g soaked agar in the mineral medium. The basal strain was incubated in the basal medium supplied with 0.3 % Ann Microbiol (2015) 65:1699–1707 1701 carbon sources including glucose, fructose, maltose, soluble MegAlin program in DNAStar package (DNAStar Inc., Mad- starch, cellobiose, sucrose, lactose, carboxymethyl cellulose, ison, USA). mannose, and inulin. Cellulase preparation Temperature and pH growth Cytophaga sp. LX-7 was cultured in a 250-mL flask contain- The temperature range for cell growth was determined by ing 60 mL of mineral medium in the presence of 0.5 % incubating the unidentified isolate in the glucose medium at cellulose CF11 (Whatman) and 0.2 % peptone at 30 °C and different temperatures ranging from 5 to 60 °C.