ISOLATION AND CHARACTERISATION OF A NEW ALKALI-HALOTOLERANT BACILLUS AQUIMARIS STRAIN LGMT10, PRODUCING EXTRACELLULAR HYDROLASES, FROM THE EFFLUENTS OF A THERMAL POWER PLANT, IN ALGERIA

Modern biotechnology takes into account cost, performance, and respect for the environment to set up an industrial process. In this sense, the study's goal is to highlight the presence of indigenous microbial microflora in the Terga thermal power plant effluents, which are capable of secreting extracellular hydrolases. Four different extracellular hydrolases classes, which are most of the time used in bioindustry, namely protease, amylase, lipase, and cellulase, were investigated in agar plate assay, to select microorganisms with an interesting enzymatic potential adapted to this type of environment. Consequently, the results have shown that among twelve isolated bacterial strains, three strains were screened based on their enzymatic potential, and were later identified by 16S rRNA gene sequencing, i.e. showing that the strains belong to the genus Pseudomonas aeruginosa, and Bacillus wiedmannii with a similarity percentage of 99.33% and 100%, respectively, with their corresponding type of strains. Among them, the strain LGMT10 that belongs to the Bacillus genus, and is closely related to Bacillus aquimaris, showed a 16S rRNA sequence similarity with the type of strain of 99.23%. This strain presents adequate characteristics to resist the harsh conditions of pH and NaCl. It could grow against wide ranges of NaCl concentrations between 0-12% (w/v), pH 5.5-12, and was able to produce extracellular hydrolases (i.e., protease, amylase, and cellulase) against pH ranges of 6.8-12 and NaCl concentrations between 0-12% (w/v) at 30 °C. This strain's intriguing enzymatic potential, as well as its pH and salinity tolerance, make it a promising candidate for various biotechnological applications in detergent, leather, textile, and food processing industries.


INTRODUCTION
The search for new eco-friendly means potentially involved in various industrial processes has directed the scientific community to microbial enzymes (Ardakani et al., 2012). In recent years, research dealing with enzymes of extremophilic microorganisms has found great interest (Shukla, 2019). However, many microbes such as bacteria, actinomycetes, fungi, and yeast extracellularly or intracellularly produce a group of versatile and attractive enzymes with a wide variety of structures and commercial applications. Many microbial enzymes, such as amylases, proteases, pectinases, lipases, xylanases, cellulases, and laccases are extracellularly produced (Fiedurek and Gromada, 2000; Venkateshwaran et al., 1999). The microbial enzymes have gained recognition globally for their widespread uses in various industrial sectors, e.g., food, agriculture, chemicals, medicine, and energy. Enzyme mediated processes are rapidly gaining interest because of reduced processing time, intake of low energy input, cost-effective, non-toxic and ecofriendly characteristics (Li et al., 2012;Choi et al., 2015). In addition, the microbial enzymes have been given more attention due to their active and stable nature compared to enzymes extracted from plants and animals Gopinath et al., 2013). Most microorganisms are unable to grow and produce enzymes under harsh environments that cause toxicity. However, some microorganisms have undergone various adaptations enabling them to grow and produce enzymes under harsh conditions (Sardessai and Bhosle, 2004; Anbu, 2016). Recently, several studies have been initiated to isolate new bacterial and fungal strains from harsh environments such as extreme pH, temperature, salinity, heavy metal, and organic solvent, in order to the produce different enzymes having the properties to yield higher (Anbu, 2016; Gopinath et al., 2005). Halotolerant bacteria form a versatile group adapted to life at the lower range of salinities, with the possibility of rapid adjustment to changes in the external salt concentration (Litchfield, 2002). This property of halotolerant bacteria makes them better candidates for bio-prospecting than their halophilic counterparts. Enzymes of the halotolerant bacteria find a very interesting field of application. However, halotolerant proteases are hydrolytic enzymes, which are mostly used in industries. For example, detergent industries add halotolerant proteases to their laundry detergent formulations in order to hydrolase proteinaceous stains (Anwar and Saleemuddin, 1998). The tannery industry uses halotolerant proteases to assist in de-hairing of animal hides (Abd Samad et al., 2017). Moreover, the identification of novel bacterial cellulases remains a currently explored route to the development of modern sustainable bio-industries for biofuel generation (Novy et al., 2015). Thermo-alkali-stable cellulases isolated from extremophilic Bacillus strains have shown their potential within conditions that are appropriate for bioconversion processes (Souii et al., 2020). Marine microorganisms have been attracting more attention as a resource for new enzymes. The complexity of the marine environment involving high salinity, high pressure, low temperature, and special lighting conditions may contribute to the significant differences between the enzymes from marine microorganisms and homologous enzymes from terrestrial microorganisms (Zhang and Kim, 2010). Besides that, new approaches such as metagenomics need to be performed to identify new groups of bacteria that remain unexplored in the seas and oceans (Sharma et al., 2019). In this context, the current research highlighted the promising potential of a newly isolated marine source bacterium; Bacillus aquimaris strain LGMT10, isolated from the effluents of the Terga thermal power plant, in western Algeria, after 16S rDNA sequencing. This bacterial strain can produce three extracellular enzymes (i.e., amylase, protease, and cellulase). It is a preliminary screening study that uses a qualitative method of producing extracellular hydrolases by indigenous microorganisms isolated from a new underutilized site in Algeria.
Modern biotechnology takes into account cost, performance, and respect for the environment to set up an industrial process. In this sense, the study's goal is to highlight the presence of indigenous microbial microflora in the Terga thermal power plant effluents, which are capable of secreting extracellular hydrolases. Four different extracellular hydrolases classes, which are most of the time used in bioindustry, namely protease, amylase, lipase, and cellulase, were investigated in agar plate assay, to select microorganisms with an interesting enzymatic potential adapted to this type of environment. Consequently, the results have shown that among twelve isolated bacterial strains, three strains were screened based on their enzymatic potential, and were later identified by 16S rRNA gene sequencing, i.e. showing that the strains belong to the genus Pseudomonas aeruginosa, and Bacillus wiedmannii with a similarity percentage of 99.33% and 100%, respectively, with their corresponding type of strains. Among them, the strain LGMT10 that belongs to the Bacillus genus, and is closely related to Bacillus aquimaris, showed a 16S rRNA sequence similarity with the type of strain of 99.23%. This strain presents adequate characteristics to resist the harsh conditions of pH and NaCl. It could grow against wide ranges of NaCl concentrations between 0-12% (w/v), pH 5.5-12, and was able to produce extracellular hydrolases (i.e., protease, amylase, and cellulase) against pH ranges of 6.8-12 and NaCl concentrations between 0-12% (w/v) at 30 °C. This strain's intriguing enzymatic potential, as well as its pH and salinity tolerance, make it a promising candidate for various biotechnological applications in detergent, leather, textile, and food processing industries.

Sampling
The effluents of a well-known thermal power plant near the sea, in the Terga region of Ain Temouchent (GPS Coordinates: 35°27'43.2"N 1°13'40.5"W), were the subject of this study. Seawater is the main source for the operation of the thermal power plant, which consequently generates effluents. The samples were collected at 60 m before the discharge area into the sea (Figure 1), in sterile flasks, and then transferred directly to the laboratory and placed in a cold room (4 °C) for further analysis. The pH of the effluents at the time of collection was recorded to be 7.5-7.8, the temperature 25 °C, and the concentration of rejected chlorine 0.25 ppm.

Enrichment and isolation of microorganisms
One millilitre of the sample was transferred aseptically to 9 ml of nutrient broth (NB). After incubation at 30 °C for 1-2 days, decimal dilutions series were performed according to the method described by Nandhini and Josephine (2013). For isolation, volumes of the bacterial cultures were diluted with a 0.85% (w/v) sodium chloride pre-sterilized. Decimal dilutions of 10 -1 to 10 -7 were made and 1 ml of the dilutions (10 -5 , 10 -6 , and 10 -7 ) was plated onto NB agar plates containing a nutrient agar composed of: Peptone (10g), yeast extract (5g), NaCl (5g), Agar (15g), and distilled water (1litre). The dishes were incubated at 30 °C under aerobic conditions for 2 days. After incubation, colonies of different morphologies were isolated and purified. The pure isolates were stored at (-20 °C) on a NB medium supplemented with glycerol 20% (v/v) for further studies.

Study of enzymatic activities of isolates
This paper is interested in the study of four hydrolase classes that have a wide range of biotechnology applications:

Protease activity
In order to select the proteolytic microorganisms, a milk agar medium composed of: Yeast extract (3g), agar (15g), and distilled water (1 litre), was used. This mixture was adjusted to pH 7.8 and autoclaved at 121 °C for 15 min. Then, 100 ml of skimmed milk (manufactured by Soummam, Algeria) was added sterilely after cooling the mixture. Microorganisms that hydrolyze milk casein show lightening halos around colonies (Ardakani et al., 2012).

Cellulase activity
This medium was composed of two media as described by Koraichi et al. (2015). A minimum medium M9 and a carboxymethylcellulose (CMC) medium. The M9 medium consists of Na2HPO4 (6g), KH2PO4 (3g), NH4Cl (1g), NaCl (0.5g), and distilled water (1 litre). The medium was autoclaved for 15 min at 121 °C. Then, 1 ml of a 0.1 molar solution of CaCl2 and 1 ml of a 1 molar solution of MgSO4 are added. The CMC medium was composed of CMC (10g), yeast extract (5 g), glycerol (50%, v/v) (2 ml), agar (20 g) and the M9 buffer (quantity per litre). This final medium was adjusted to pH 7.8, autoclaved for 15 min at 121 °C, and then poured into Petri dishes. After incubation for 2 days at 30 °C, the cellulase activity was demonstrated by adding a solution of lugol for 15 min followed by three rinses with a molar solution of NaCl. Microorganisms that have cellulase activity show yellowish rings around the colonies.

Characterization of bacteria
The isolated strains were identified based on phenotypic and biochemical characteristics such as sugars fermentation (i.e., glucose, lactose, saccharose, and mannitol), citrate utilization test, mobility test and others, using the Bergey's Manual of Determinative Bacteriology as a guide, and on molecular characteristics by the sequencing of 16S rRNA gene after extraction and Polymerase Chain Reaction (PCR) amplification, using the boiling method. This later consisted of bringing a pure colony to the boil for 10 min, and centrifuged at 15,000 rpm for 5 min at 4 °C (Dutka-Malen et al., 1995). The amplification of the 16S rRNA gene was carried out in a TC3000 Thermocycler using the following PCR program: Predenaturation 95 °C for 15 min, denaturation 94 °C for 1 min, hybridization 60 °C for 1 min, and elongation 72 °C for 1.

Growth curves
Two growth curves of bacterial culture of the strain LGMT10 were plotted as a function of time at different pH (pH 5.5, 6.5, 8, 10, and 12) and concentrations of NaCl (0, 4, 8, 12, and 16 %, w/v) to study respectively the alkali tolerance and halotolerance in NB medium composed of (g/l): casein peptone (10g), yeast extract (5g), NaCl (at the studied concentrations) and distilled water (1 litre). The bacterial cultures were taken aseptically every 12 h and the optical density (OD) was measured at a wavelength of 600 nm (Shivanand and Jayaraman, 2009). Results are expressed as the mean of two replicates tests ± standard deviation. Principal component analysis (PCA) was used to highlight the strain LGMT10's optimal pH and NaCl growth parameters.

Effect of pH and concentration of NaCl on the enzymatic activities
In order to investigate the influence of pH and NaCl concentrations on the production of extracellular enzymes (protease, amylase, and cellulase) by the strain LGMT10, different enzymatic assays were carried out in agar plate assay, with different pH (pH 6.8, 8.5, 10, and 12), and different concentrations of NaCl (0, 4, 8, and 12%, w/v) at a constant temperature of 30 °C. After incubation for 48 h, the secretion of enzymes was manifested by the formation of halos around the colonies. Another experiment on enzymatic activities was conducted by combining the optimal pH and NaCl growth parameters.

Statistical analyses
Principal component analysis (PCA) was used to compare the effects of different NaCl concentrations and pH ranges for the LGMT10 strain growth, using the XLSTAT® software (trial version). The LGMT10 strain's growth at different NaCl concentrations and pH ranges was performed at two replicates tests. Means and standard deviations were calculated using GraphPad Prism 9 (Trial version).

Isolation, screening, and characterization of bacteria
The isolation of microorganisms from the effluents, after enrichment in NB medium, showed a bacterial diversity with interesting enzymatic potential as shown in Tab 1. Among twelve bacterial isolates, three isolates designated LGMT10, LGMT12, and LGMT8 were screened based on their interesting enzymatic potential (Tab 1). The strain LGMT10 was selected for its enzymatic potential amylase, cellulase, and protease, by showing broad halos around the colonies (Figure 2). It is short rod, Gram-positive, catalase-positive, oxidasenegative, which forms pale orange-yellow colonies on the surface of a nutrient agar medium after 24 h of incubation at 30 °C (Figure 3). It grows against wide ranges of NaCl concentrations between 0-12% (w/v) and pH 5.5-12 at 30 °C. Based on phenotypic and biochemical analyzes, and according to Bergey's Manual of Determinative Bacteriology; the strain was tentatively classified as Bacillus sp. (Tab 2). Phylogenetic analysis based on 16S rRNA gene sequencing showed 99.23% homology with the type of strain Bacillus aquimaris strain DSM 16205 T (GenBank Accesion no.: AF483625) (Figure 4). For the strains LGMT12 and LGMT8, the percentage of similarity of the 16S rRNA gene with their corresponding type of strains is 99.93% and 100%, respectively, for Pseudomonas aeruginosa strain DSM 50071 T (GenBank Accesion no.: HE978271) and Bacillus wiedmannii strain DSM 102050 T (GenBank Accesion no.: KU198626). ) ( Figure  4). Rods LGMT9 Rods - Rods -+ ++ -++ + Legend: (-)no halos, (++)medium diameter halos, (+++)large diameter halos

Growth curve
Monitoring the growth of the strain LGMT10 over time has shown its capacity to grow on different concentrations of NaCl (0-12%, w/v), with optimal growth observed in NB medium at a concentration of 4% (w/v) NaCl, i.e., where the best rate growth was noted. This allowed the strain to be classified as moderately halophilic bacterium, indicating that it originated from the sea. (Shivanand and Jayaraman 2009) (Figure 5a). On the other hand, monitoring the growth against various pH ranges has shown that the LGMT10 strain can resist a pH range of 5.5-12, with an optimum growth pH observed at pH 8, i.e., where the best growth rate was recorded. As a result, the strain could be classified as an alkali-tolerant bacterium (Figure 5b). The effect of NaCl and pH on the LGMT10 strain's growth was demonstrated using principal component analysis. Accordingly, the best conditions for bacterial growth have been identified (i.e., 4% NaCl and pH 8) ( Figure 6)

DISCUSSION
In developing countries, microbial enzyme research is still in its infancy. However, the exploitation of certain discharge sites for the isolation of indigenous microorganisms should be carried out to better assess their microbial diversity and decipher their capacity for producing various enzymes classes. In this context, the present study highlighted the isolation and characterization of three bacteria designated LGMT10, LGMT12, and LGMT8 belonging respectively to the genus Bacillus aquimaris, Pseudomonas aeruginosa, and Bacillus sp. isolated from the effluents of the thermal power plant of Terga, Algeria. Since the first discovery of the novel strain Bacillus aquimaris sp. nov. by Yoon et al. (2003), there are few reports regarding its enzymatic potential. However, the present study 0% NaCL 4% NaCL 8% NaCL 12% NaCL Variables actives Observations actives highlighted the promising potential of new alkali-halotolerant Bacillus aquimaris strain LGMT10 producing three extracellular hydrolases (i.e., protease, amylase, and cellulase) able to resist different ranges of pH and NaCl concentrations. In the same context, the majority of studies in the literature have shown the halotolerant nature of Bacillus aquimaris and its ability to grow in NaCl concentration between 0-12% (w/v) (Shivanand and Jayaraman, 2009) in agreement with this study. Furthermore, strain LGMT10 has shown excellent properties to resist pH and NaCl for growth and production of the three studied enzymes compared to its counterparts among Bacillus spp and other bacterial genera.
Several bacterial genera that are capable of producing a variety of extracellular enzymes appear to be overly well described in the literature. However, a variety of bacteria has been reported for cellulase production. Trivedi et al. (2011) described the production of an alkalihalotolerant cellulase by Bacillus flexus strain NT. These authors showed that the enzyme, which has a molecular mass of 97 kDa, was stable in the pH range of 9-12 and at a range of NaCl concentration up to 15% (w/v). On the other hand, bacteria such as Klebsiella sp. produce cellulase active at 10 °C and pH 4.5 (Bhat et al., 2013). Cellulase produced by Marinobacter sp. strain MSI032 was alkalotolerant, active at pH 9 (Shanmughapriya et al., 2010).
In contrast to alkaline cellulases, only a few salt-tolerant or halophilic cellulases have been reported ( There are numerous available studies in the literature on alkaline protease production by Bacillus species. However, Bacillus aquimaris strain VITP4 has been reported to be active in the pH range of 7-10, with an optimum at pH 8 Jayaraman 2009, 2011). Currently, there are at least 29 Bacillus species and 17 fungal producers that have been reported to produce alkaline proteases (Veloorvalappil et al., 2013). All the studies published on halotolerant microorganisms have shown that the enzymes from microorganisms which can grow over a concentration range of NaCl between 0-15% (w/v) are of great interest for their industrial use, because of their inherent ability to be active and stable both in the presence as well as in the absence of salt (Chittoor et al.,  2016). Moreover, some extracellular halophile proteases have maximum activity at near-neutral pH that has been reported (Vidyasagar et al., 2006;Norberg and Hofsten, 1969). The Bacillus aquimaris strain VITP4 described by Shivanand and Jayaraman (2011) retained significant activity up to a concentration of 2M NaCl, in agreement with this study, although it exhibited the highest activity in the absence of NaCl. Even in the presence of 4 M NaCl, the protease retained about 40% of its activity, indicating the halotolerant behavior of the enzyme. It has been reported that the addition of NaCl up to a concentration of 5% (w/v) had no effect on the proteolytic function of Bacillus cereus. However, enzymatic activity decreased progressively upon further salt addition, and a 60% reduction in activity was reported when the NaCl concentration was increased to 10% (Joshi et al.,  2007). There are reports of salt-tolerant protease produced by mesophilic or thermophilic Bacillus spp (Joo and Chang, 2005; Bhushan et al., 1999).
In addition, the study of Anupama and Jayaraman (2011) on the production of extracellular amylase by the halotolerant strain Bacillus aquimaris strain VITP4 from the saltern of Kumta coast showed optimal activity at a pH range of 7.5 -9.5 at 40 °C. Likewise, the partially purified a-amylase of Bacillus aquimaris strain MKSC 6.2 displayed optimum activity at pH 6.5 and 50 °C (Puspasari et al.,  2011). It has been reported that amylases produced by certain halophilic microorganisms have optimal activity at high salinities and could, therefore, be used in many severe industrial processes where the concentrated saline solutions used would otherwise inhibit many enzymatic conversions ( , and Bacillus dipsosauri (Deutch, 2002). Based on the literature, the main strains that are described in the production of extracellular enzymes among Bacillus spp are presented in Tab 4. Protease Amylase Cellulase pH 6.8-12; NaCl: 0-12% pH 8.5-12; NaCl: 0-12% pH 6.8-12; NaCl: 4-12%

CONCLUSION
The microbial diversity of the Terga thermal power plant's effluents was investigated in this study, which revealed an intriguing enzymatic potential as well as the identification of a novel alkali-halotolerant bacterium, Bacillus aquimaris strain LGMT10 capable to produce three extracellular hydrolases (i.e., amylase, cellulase, and protease) in the pH range of 6.8-12, and at NaCl concentrations between 0-12% (w/v) at 30 °C. These findings bode well for better understanding the capabilities of novel microorganisms from this new site, in Algeria. In addition, the measurement of the enzymatic activities of purified enzymes and their biochemical characterization will be performed using more accurate techniques, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymograhy, in order to provide the data necessary for their catalyzing capacities, for a possible affiliation of each enzyme towards the corresponding industry.