Isolation of thermophilic enzyme-producing Parageobacillus bacteria from chipped woody waste

Abstract

The morphological and biochemical properties were investigated for 18 types of thermophilic bacteria isolated from a woody-chip pile at a wood processing plant in Northern Russia. Genetic fingerprinting and 16S rRNA identification were used to divide the investigated microorganisms into groups according to their genetic affiliation. It was found that the isolated bacteria belonged to a minimally studied genus of Parageobacillus and exhibited optimum temperature and pH in the ranges of 57 to 60 °С and 7.0 to 8.5, respectively. The amylase activity was determined for all of the 18 isolated strains. Catalytic properties of the bacteria-produced xylanases were evaluated with respect to their activity towards xylan and xylan-containing carbon substrates. Biotechnological potential of the two most promising bacterial strains (Parageobacillus caldoxylosilyticus and Parageobacillus thermoglucosidasius) and their possible use in xylanase production was evaluated. The results showed that bacteria present in the chipped woody waste is an important source of thermoalkalophilic enzymes.

Full Article

Isolation of Thermophilic Enzyme-producing Parageobacillus Bacteria from Chipped Woody Waste

Olesya Yunitsyna,^a Igor Sinelnikov,^a Oksana Kisil,^a Ksenia Bolotova,^a Andrey Aksenov,^a and Galina Simonsen ^b

The morphological and biochemical properties were investigated for 18 types of thermophilic bacteria isolated from a woody-chip pile at a wood processing plant in Northern Russia. Genetic fingerprinting and 16S rRNA identification were used to divide the investigated microorganisms into groups according to their genetic affiliation. It was found that the isolated bacteria belonged to a minimally studied genus of Parageobacillus and exhibited optimum temperature and pH in the ranges of 57 to 60 °С and 7.0 to 8.5, respectively. The amylase activity was determined for all of the 18 isolated strains. Catalytic properties of the bacteria-produced xylanases were evaluated with respect to their activity towards xylan and xylan-containing carbon substrates. Biotechnological potential of the two most promising bacterial strains (Parageobacillus caldoxylosilyticus and Parageobacillus thermoglucosidasius) and their possible use in xylanase production was evaluated. The results showed that bacteria present in the chipped woody waste is an important source of thermoalkalophilic enzymes.

Keywords: Parageobacillus caldoxylosilyticus; Parageobacillus thermoglucosidasius; Woody-chip pile; Thermophiles; Amylases; Xylanases

Contact information: a: Department of Biology, Ecology and Biotechnology, Northern (Arctic) Federal University, Arkhangelsk, Russia; b: Department of Chemical Engineering, Norwegian University of Science and Technology (NTNU), Trondheim, Norway; *Corresponding author: galinanorge@gmail.com

INTRODUCTION

Modern industrial biotechnology demands the development of high-performance biocatalysts, or enzymes, applicable in a variety of industrial operations (Choi et al. 2015; Jemli et al.2016; Blamey et al. 2017; Madhavan et al. 2017). Enzyme-assisted processes are of great importance in waste minimization and recycling due to the high efficiency of enzymes, their low consumption, and selectivity in removal of unwanted compounds. Cellulases constitute a well-known class of enzymes capable of hydrolyzing 1,4-β-D-glucosidic linkages in cellulose, which is a polymer available globally for bioconversion into numerous products. Cellulases have a long history of applications, including processing of wood biomass, textile and laundry, food, and bioethanol production (Coughlan 1985; Hardiman et al. 2010; Behera et al. 2017). In addition to cellulases, other enzymes capable of hydrolyzing non-cellulosic polysaccharides, such as amylases and xylanases (Hu et al. 2015), are continuously gaining importance in the global market of bioconversion and recycling processes (Pandey et al. 2000; Saxena and Singh Chauhan 2016). Amylases is a group of enzymes capable of hydrolyzing glycosidic bonds in starch, namely, α-amylase, β-amylase, and glucoamylase. The amylases are traditionally used at elevated temperatures and can be derived from a variety of plant and microbial sources (Gopinath et al. 2017). They are commonly utilized in the processing of raw grain materials (Nigam and Singh 1995), removal of starch present in waste paper and paperboard (Saxena and Singh Chauhan 2016), and paper recycling (Raul et al. 2014). The hydrolysis of xylan, a major component of hemicelluloses, can be achieved by xylanases or xylanase mixtures containing endo-β-1,4-xylanases and β-D-xylosidases (Polizeli et al. 2005). Xylanases have been used by the industry since the early 1990’s. They are recognized for their catalytic effects in improving efficiency of the existing pulp bleaching processes (Viikari et al. 1994) and bioconversion of agricultural waste into fuel and chemicals (Nigam and Pandey 2009). One of the main challenges in xylanase production from bacterial sources is the high cost of the xylanase inducer, pure xylan (Alves et al. 2016). Waste materials from wood processing plants, as well as other lignocellulosic sources, can serve as alternative carbon substrates for the production of xylanases. The use of such substrates can reduce the cost of the entire process. At present, researchers are looking for novel bacterial strains capable of assimilating lignocellulosic waste as a cost-effective carbon source for the production of xylanases (Bhalla et al. 2015).

One of the important features of microbial enzymes is that they exhibit substrate selectivity and stability under abnormal conditions, mainly high temperatures and pH extremes. Based on their stability, certain enzymes can be categorized as thermophilic, acidophilic, or alkalophilic. These properties make them valuable contributors to multiple industrial processes. One example is the application of thermoalkalophilic enzymes in pulp and paper production and paper recycling, where both the temperatures and pH often rise above 50 °C and 8, respectively. The thermoacidophilic enzymes can contribute to enhancing mass transfer and reducing substrate viscosity during hydrolysis of raw plant materials in industrial processes (Cai et al. 2011).

Microbial enzymes can be produced by different microorganisms, both bacterial and fungal species. Although fungal species are significant producers of thermostable enzymes, the obtained enzymes often lack stability at high temperatures and pH and do not qualify for use in certain biotechnological applications (Kumar et al. 2013). Therefore, the search for new bacteria that are capable of synthesizing stable enzymes is highly relevant. The enzyme-producing bacteria can be isolated from a variety of waste sources, one of which is the chipped woody waste produced in abundance by wood processing plants. The composition, and therefore bacterial presence in such wastes, will vary depending on the season, storage, mixing conditions, type of wood waste, and many other factors. This variety gives endless possibilities for discovering and extracting new bacterial species with unique properties that have not been previously reported.

In this work, several types of bacteria have been isolated from samples of a chipped woody waste pile located at a wood processing plant in Northern Russia. The bacteria are identified and characterized with respect to their amylolytic and xylanolytic properties. Two bacterial strains, P. caldoxylosilyticus and P. thermoglucosidasius, were chosen for detailed study due to their promising ability to produce xylanolytic enzymes. A selection of carbon substrates was used to evaluate the bacterial performance in terms of xylanase activity and xylanase production in industrial conditions.

EXPERIMENTAL

Materials

Study site, sampling, and bacteria isolation

Samples of wood chips were collected from a woody chip pile at a timber mill named Solombalales located in the city of Arkhangelsk in Northern Russia (64°35’19″N, 40°33’20″E) (Fig. 1A). The sampling was carried out at the following depths: pile surface (bark), 2 to 10 cm, 10 to 30 cm, and 80 to 100 cm (Fig. 1B). The pile was semi-anaerobic at each sampling point due to periodic mixing. The samples were placed into sterile plastic containers (500 mL) and transported to the laboratory at ambient temperature.

Once the samples arrived, they were sealed separately into 100-mL airtight bottles containing 0.1% peptone solution and kept at 65 °C for five days stirring at 150 rpm. The primary screening of the bacteria was carried out in a minimal medium (MM) containing potassium hydrogen phosphate trihydrate (К₂НРO₄·3H₂O; 5.24 g/L), magnesium sulfate heptahydrate (MgSO₄·7H₂O; 1 g/L), potassium chloride (KCl; 0.2 g/L), iron(II) sulfate (FeSO₄; 11.4 mg/L), xylan (1%), peptone (0.5%), yeast extract (0.1%), agar (1.5%), and trace element solution (1 mL). The system was incubated for 24 h at 60 °C (Valladares Juárez et al. 2009). Cultures showing different colony morphology were separated and further purified by streaking on the same medium at least three more times. The purified bacteria cultures were stored in glycerol stock (15%) at -80 °C.