Cultivable Microbial Diversity in Domestic Bentonites and Their Hydrolytic Enzyme Production

Abstract

Clay minerals are classified as kaolinite and montmorillonite minerals, as a class of layered silicates formed from chemical weathering of other silicate minerals on the surface of the earth [1]. Clay minerals with varying amounts of iron, magnesium, alkali metals, alkaline earths, and other cations are present in the interlayer space or in the lattice structure. They have well-known adsorbent properties due to their layered form, with a high internal surface area, high cation-exchange capacity, and good swelling properties in the presence of water [2]. In the Korea Food and Drug Administration, six kinds of clay minerals (diatomaceous earth, kaolin, bentonite, acid clay, talc, and perlite) are listed in the Food Additive Code, and they are used in filtration aids in the beverage, brewing, and fermentation industries [3].Bentonite is a kind of montmorillonite, and it contains various elements such as potassium (K + ), sodium (Na + ), calcium (Ca 2+ ), and aluminum (Al + ) [4]. Bentonites have been investigated for the adsorption of heavy metals and molecular species in a variety of environments and industries and have been identified as low-cost adsorbents [5]. In addition to studies on the physicochemicalWe have isolated and identified 72 bacterial strains from four bentonite samples collected at the mining areas located in Gyeongsangbuk-do, Republic of Korea, and measured their hydrolytic enzyme (α-amylase, protease, and cellulase) activities to identify the isolates with industrial-use potential. Most of the isolates belonged to the Bacillaceae, with minor portions being from the Paenibacillaceae, Micrococcaceae, and Bacillales Family XII at the family level. Of the strains isolated, 33 had extracellular α-amylase activity, 30 strains produced cellulase, and 35 strains produced protease. Strain MBLB1268, having the highest αamylase activity, was identified as Bacillus siamensis (0.38 ± 0.06 U/ml). Bacillus tequilensis MBLB1223, isolated from Byi33-b, showed the highest cellulase activity (0.26 ± 0.04 U/ml), whereas Bacillus wiedmannii MBLB1197, isolated from Zdb130-b, exhibited the highest protease activity (54.99 ± 0.78 U/ml). These findings show that diverse bacteria of the Bacillaceae family adhere to and exist in bentonite and are potential sources of industrially useful hydrolytic enzymes.These authors contributed equally to this work.

Isolation and Characterization of Microbial Strains with Hydrolytic Enzyme Profile from Clay Minerals

“…was isolated in the soil [16]. Taken together with previous studies, our results showed a high microbial diversity in bentonite and zeolite samples, dominated by bacterial phyla Firmicutes and Actinobacteria [15,19,20]. Cultivable isolation of diverse microorganisms from clay minerals triggered the investigation of industrial enzyme productions to promote the industrial application of clay minerals-derived functional microorganisms.…”

“…B. amyloliquefaciens DL-3 and B. licheniformis CDB-12 [25,26]. Our previous study also reported that 30 strains isolated from bentonite samples showed cellulase activity, including B. tequilensis MBLB1223 and B. siamensis MBLB1268 [20]. Pectinase responsible for the degradation of pectin polysaccharides are industrially useful enzyme for the clarification, extraction and concentration of tea and juice as well as textile fibre production [27].…”

“…2). Quantifications of α-amylase, cellulase, and pectinase activity were evaluated by the modified method of DNS (3,5-dinitrosalicylic acid) assay [20], while protease activity was tested by simply modified Lowry method [21]. The positive hydrolytic strains were cultivated Nutrient Broth with 0.5% (w/v) starch, Luria-Bertani broth with 1% (w/v) CMC and 0.5% (w/v) pectin, or TSB for 48 h at 37℃.…”

“…To obtain a crude enzyme solution, the cultures were centrifuged (13,000 rpm, 15 min, 4℃), following the collection of supernatants. The reactions for α-amylase, cellulase and protease were performed as described by Seo et al [20]. To measure the quantification of pectinase activity, the enzyme reaction was carried out in 200 μl reaction mixture containing 0.5% (w/v) polygalacturonic acid in 50 mM sodium acetate buffer (pH 5.0), and 100 μl crude enzyme.…”