Isolation and identification of protease‐producing bacteria from sludge and sediment soil around Adama, Ethiopia

Proteases are enzymes used in industries such the production and processing of detergents, food, leather, and silk. The aim of this study was to isolate and identify protease‐producing bacteria from a sludge disposal site and from sediments. Soil samples were collected separately from the selected area. Samples weighing 1 g were serially diluted and spread onto skim milk agar. A total of 16 bacteria species were isolated from the study samples. Four bacterial isolates showed high proteolytic activity and were selected for enzymatic study based on their zone of proteolysis. The isolates were identified based on biochemical tests. The results indicated that the isolated bacteria were E. coli (99.69%), Pseudomonas putrefaciens (Shewanella putrefaciens) (91.61%), Bacillus carboniphilus (92.78%), and Lysinibacillus sphaericus (98.4%). The crude protease enzymes produced by these bacterial isolates showed promising results for application in dehairing and destaining as detergent additives. Bacillus carboniphilus showed the best level of activity and was selected as the most potent protease‐producing bacteria for both dehairing and destaining ability. Soils from sludge disposal sites and sediments from around tannerywastes could be good sources fromwhich to isolate alkaline protease‐producing bacteria.


Introduction
Microbes are a preferred source of enzymes than plants and animals due to their short generation time, cultivat ing in small space, and making it easy for genetically ma nipulated to generate new enzymes for various applica tions ranging from domestic to industrial processes. Ex amples of industrial processes include leather processing, food, chemical, environmental pollution abatement, diag nostic kits development for clinical applications (Kocher and Mishra 2010).
There is a huge demand for protease enzymes im ported for use in leather tanning, textile, and brewing in dustries. Alkaline protease has attracted worldwide atten tion to achieve its physiological and biotechnological ap plications due to diversity and the specificity of action. Isolation and characterization of new bacteria strains are the best ways to increase the diversity and yield of such enzymes. Even if the same species of proteaseproducing microbes, especially bacteria, are isolated from different environments, their effectiveness is completely different. High amounts of protein waste from plants and animals are released annually; one such protein waste is keratin re leased from leather tanning industries in the form of hair. Over 90% of keratins are protein, and are highly crystalline and cannot be digested by most enzymes. But some mi croorganisms can hydrolyze keratin (Gupta et al. 2002). Therefore, bioprospecting for new microorganisms that can be used for protease production is a necessary continuous process for industrial development and envi ronmental conservation. Since, most industrial microbes are patented and/or must be purchased for use outside their country of origin, isolation of bacterial strains are good ways to increase the diversity and yield of such enzymes (Gupta et al. 2002). Alkaline proteaseproducing bacteria isolated from different environments may have different potential in their enzyme's effectiveness. Hence, the use of microbial enzymes for industrial processes has offered a viable option to decrease environmental pollution and leads to improved product quality or process efficiency mi crobes (Gupta et al. 2002; Kocher andMishra 2010).
Ethiopia is on rapid expansion of various industries, like textile, leather, detergent, pulp and paper, food and di etary industry. Some of these industries highly depend on chemical utilizations, which leads to environmental pol lution (Gessesse et al. 2011). According to this report, the market for proteases enzymes in the East African re gion (Ethiopia and Tanzania alone) was estimated to US$ 3.8 million per annum. The massive move to industrialize the country created a demand gap for the enzyme, espe cially protease. This needs the isolation of suitable mi croorganisms from within the country for sustainable uti lization in the rapidly growing Ethiopian industrial sector, which could replace several imported chemicals as pro cessing aids and encourage local industries to adopt en zyme technology. Therefore, the objective of this study was to isolate and identify alkaline proteaseproducing bacteria from sludge disposal sites and sediment located in central Ethiopia.

Study area
Adama is a city in central Ethiopia. It is located at 8.54°N 39.27°E at the elevation of 1712 m above sea level (m.a.s.l) and 99 km southeast of Addis Ababa. Modjo Tannery Share Company is located around Adama city.
Modjo is a small town named after the nearby Modjo River. Modjo is located in the East Shewa Zone of the Oromia region. It has a latitude (8°35 ' 12.44 " N) and longitude (39°07 ' 16.00 " E) and with an elevation be tween 1788 and 1825 m.a.s.l.

Sample collection
Five hundred grams (g) of soil samples were collected from the sludge disposal site of Adama Science and Tech nology University campus and sediments from Modjo Tannery Share Company with a sterile spatula from 5 cm depth into sterile plastic bags. The samples were la beled with site name, date of sample collection, and sam ple type and transported to Oromia Public Health Re search Capacity Building and Quality Assurance Labora tory (OPHRCBQAL) at the bacteriology laboratory. The samples were airdried at room temperature for a week and maintained at room temperature for further analysis.

Isolation of protease producing bacteria
Isolation of bacteria was carried out with serial dilution techniques and the spread plate method (Sneath 1986). Onegram soil samples (sediment and sludge) were asepti cally subsampled from 500 g samples after thorough mix ing and then transferred to 9 mL) distilled water and se rial diluted up to 106 (i.e., 101-106). Serially diluted aliquot 1 mL was spread on skim milk agar. The plates were incubated at 37°C for 48 h. Moreover, zones of hy drolysis were recorded. The colonies showing the highest clearance zone were selected for further study and repeat edly purified on nutrient agar plates and preserved at room temperature.

Morphological characterization
The bacterial cultures grown on blood agar were character ized based on colony morphology like colony type, shape, margin, and elevation with reference to Bergey's manual of determinative bacteriology 7ed.

Microscopic characterization
Microscopic examination was carried out from slide preparations and Gram staining methods to distinguish the isolated strains as Grampositive or Gramnegative bacte ria. Every single colony from 24 h incubation was puri fied. Old culture on agar plate was taken with loop, put on the center of the clean slide, and made to smear. The smear was fixed on the slide with heat and stained with crystal violet for one minute. Then iodine was added, waited for one minute, and two drops of alcohol were added as a de colorizing agent. Finally, a counterstain, safranin, was added and rinsed with water after one minute. The slide preparation was viewed under a microscope using oil emulsion objective 1000 × magnification power. The result was recorded as Grampositive (purple) and Gramnegative bacteria (pink color) (Murray et al. 2007).

Enzyme tests
Catalase test. Three drops of 3% of H 2 O 2 solution were added to bacteria cultures grown on a nutrient broth medium. The bubble formation was indicated as a posi tive reaction, while no bubble formation was considered a negative reaction for the catalase test (Nikolic et al. 2008).
Oxidase test. Oxidase test was conducted following the method described in (Nikolic et al. 2008). One drop of 1% pamino dimethylaniline oxalate (Gaby and Hadley reagents) was flooded on bacteria culture on oxidase strip paper. The color was changed to deep purpleblue/blue color within 30 s as positive results.
Urease test. The urea hydrolysis test was conducted according to the method described by (Nikolic et al. 2008). A loopful of bacterial culture was inoculated into a test tube containing urea broth and then incubated for 24 h. at 37°C. The development of bright pink color was indica tive of a positive result for urea hydrolysis.

Biochemical test
The biochemical characterization of bacteria cultures that grow on nutrient agar and/or nutrient broth was carried out using both commercial kits (Biomerieux API kits) and manual methods. The preparation of bacteria suspension and setting up of API strips were done based on the proce dure of Global Health Network Laboratory (Microbiology Standard Operating Procedure). The biochemical tests in clude catalase, oxidase, indole production, citrate utiliza tion, carbohydrate fermentation, urease, hydrogen sulfide production, and nitrate reduction test. Colonies of bacte ria culture were taken from blood agar and then transferred into 5 mL of saline solution. The bottom tray of the API kite strips holder was filled with distilled water. The API strips were elevated at an angle of 30°, then 50 μL of bac terial suspension were added on one side of the tube by touching the inside wall of the cupule with the end of the pipette. Each of the tubes on the API strips was filled with the bacteria suspension from left to right. Next, mineral oil was added on the top of the cupule area of tubes contain ing dehydrated substances (ADH, LDC, ODC, H2S, Urea, and glucose) and incubated at 37°C for 24 h.

Crude enzyme extraction
Loop full of protease producing bacterial culture from broth was inoculated into a test tube containing 30 mL of sterilized skim milk broth (pH. 9.0) and incubated at 37°C for 48 h. After incubation period the cultures were cen trifuged at 10,000 rpm for 10 min. The supernatant was separated from residues and used as crude enzyme for en zymatic activity (Aftab et al. 2006).

Washing test
Application of alkaline protease enzyme by isolated bac teria as a detergent additive was studied according to a method described by (Aftab et al. 2006). For these six white cotton clothes 5 × 5 cm were stained with sheep blood separately and then waited until it becomes well dried. The following setups were designed to test the en zymatic activity of supernatant to washing tests. A blood stained cloth dipped in a plate with distilled water (10 mL). Bloodstained cloth dipped in plate with distilled water (10 mL)+ (1 mL) detergent (7 mg/mL). Bloodstained cloth dipped in a plate with distilled water (10 mL) + (2 mL) crude enzyme solution (supernatant of APBIT3, APBIS 4, APBIT6, and APBIT6) in a separate plate. All plates were incubated at 40°C for 20 min. After incubation, cloths were taken out and were rinsed with distilled wa ter without scrubbing and then dried. Where APBIT= al kaline protease producing bacteria from sediment soil and APBIS= alkaline protease producing bacteria from sludge soil.

Dehairing activity
Dehairing activity was performed by the method described by (Dudhgara et al. 2015). Sheepskin was cut 3 × 2 cm pieces and washed gently with tap water and then rinsed with distilled water to remove chemicals from the skin, which may hinder enzyme activity during dehairing ac tivity. Four (3 × 2 cm) pieces of skins were inserted into 15 mL) tubes separately. Five milliliters (5 mL) of crude enzyme solution (supernatant of APBIT3, APBIS 4, APBIT6, and APBIT7) were added sequentially and incubated at 40°C for 48 h. After incubation, the skin was taken out and peel with a swab stick and then observed the dehairing ability of the crude enzymes.

Data analysis
The experiment was performed in triplicate and the data were analyzed and interpreted using Microsoft Excel 2013. Using Bergey's Manual of Determinative Bacteri ology 7ed. Furthermore, ABIS version 12 online software was used for the identification of bacterial species.

Screening of protease producing bacteria
A total of 16 bacterial isolates showing zone of proteoly sis were obtained in this study on skim milk agar plates inoculated with the soil samples, as shown in Table 1. Among the sixteen isolates obtained through screening by growing on skim milk agar, four bacterial cultures were selected, which showed zones of hydrolysis ≥ 25 mm. From these, APBIS4 from the soil of sludge disposal site at Adama Science and Technology University (ASTU) campus and APBIT3, APBIT6 and APBIT7 from sedi ment soil collected from Modjo Tannery Share Company (MTSC) showed a promising zone of hydrolysis (≥ 25 mm). These four isolates were subsequently selected for further study.
In this finding, four potential alkaline protease producing bacterial isolates obtained from sludge disposal site and tannery waste showed promising proteolytic ac tivity on skim milk agar. This implies that sludge dis posal sites and soil sediment around tannery waste are good proteaseproducing bacteria sources. The potential of sludge disposal site soils in ASTU Campus and sed iment at Modjo Tannery Share Company waste has not been reported as a source of alkaline proteaseproducing bacteria so far. Hence the need to search for potential protease producing bacteria in unexploited sites such as sediments and sludge disposal sites for commercially ex ploitable species of bacteria.

Cultural characterization bacteria isolated from sediment
Table 2. shows that the selected bacteria isolate (APBIT 3, PBIS4, APBIT6 and APBIT7) showed different mor phological characteristics (colony type, shape, texture, el evation, margin and pigmentation or color) as described in Figure 1.

FIGURE 1
Morphologies of isolated colonies.

FIGURE 2
Washing abilities of crude enzymes extracted from bacteria.

FIGURE 3
Dehairing abilities of crude enzymes extracted from isolated bacteria.

Microscopic characterization
The morphological features of the isolated bacteria were determined by the Gram staining technique. The APBIS 4 and APBIT3 isolates were rodshaped Gramnegative bacteria, but APBIS4 was nonmotile and APBIT3 was motile. APBIT6 and APBIT7 were motile, rodshaped Grampositive bacteria, as stated in Table 3.

Enzyme tests of the isolated bacteria
Isolates (APBIT3, APBIS4, APBIT6 and APBIT7) showed positive results for the catalase test and negative results for the urease test. APBIT3, APBIT6 and APBIT 7 were oxidase positive, but APBIS4 was shown oxi dase negative. APBIT3 and APBIT6 were shown pos itive results on L Arginine dihydrolase, but APBIS4 and APBIT7 shown negative results as shown Table 4.

Biochemical profile of the isolated bacteria
As indicated in the

Application of crude alkaline protease enzyme
The cellfree supernatants as crude enzymes were ex tracted from bacteria culture by inoculating the isolated bacteria into skim milk broth medium and incubated at the appropriate temperature (37°C for 48 h). The cellfree su pernatant was separated from residues using a centrifuge at 10,000 rpm for 10 min, and then the solids were removed. The supernatant was used as a crude enzyme for washing and dehairing activities.

Washing and Dehairing ability of crude enzyme extracted from bacteria
From this finding, all crude enzymes extracted from the isolated bacteria could remove blood. Their effective ness to remove proteinaceous stain (blood) were APBIT 6 (Bacillus carboniphilus) ≥ APBIT3 (Pseudomonas pu trefaciens (Shewanella putrefaciens)) ≥ APBIS4 (Es cherichia coli) > APBIT7 (Lysinibacillus sphaericus); which mean that APBIT3, APBIS4 and APBIT6 had almost similar efficiency for blood stain removal and APBIT7 moderately effective at 40°C for 20 min as shown Figure 2. These results indicated that the enzymes were active for destaining activity and could use as deter gent additives. The overall washing abilities of all crude enzymes were summarized in Table 6.

Dehairing ability of crude enzyme extracted from bacteria
All crude enzymes obtained from four isolated bacteria could remove hair. The effectiveness of the crude enzyme extracts obtained from APBIT3 (P. putrefaciens (S. putre faciens)) and APBIT6 (B. carboniphilus) to remove hairs from skin were almost similar efficiencies. However, the quality (softness) of skin treated with crude enzyme ob tained from APBIT6 (B. carboniphilus) was most effec tive. Figure 3 showed that the crude enzyme obtained from E. coli was moderately effective and crude enzyme extract from L. sphaericus was less effective at 40°C within 48 h. The overall dehairing ability of all crude enzymes was summarized in Table 7.

Discussion
In this study, potentially alkaline proteaseproducing bacteria were isolated from the sludge disposal site at Adama Science and Technology University (ASTU) cam pus and sediment soil in Modjo Tannery Share Company (MTSC). From a total of 16 bacterial isolates obtained from sludge disposal site and tannery waste, four isolates showed promising proteolytic activity on skim milk agar. Proteaseproducing bacteria isolated from the study area were identified and their dehairing and destaining abil ities were assessed. All the isolated bacteria could hy drolysis casein protein on skim milk agar, which indi cates that all bacteria could produce the enzyme protease that degraded casein protein. This impels that sludge dis posal sites and soil sediments around tannery waste are   (Tallur et al. 2016) is a Grampositive, mesophilic, rodshaped bacterium commonly found in soil. It is sporeforming bacteria, tolerant to high temperatures, chemicals, and ul traviolet light, and can remain viable for long periods in soil. L. sphaericus' ability of the pure bacterial cultures to produce extracellular protease enzyme was carried out by growing the cultures on skim milk agar. L. sphaericus is a wellknown bacterium used in the biological control of mosquitoes. It is of particular interest to the WHO due to the larvicidal effect of some strains against two mosquito's genera (Culex and Anopheles) (Wirth et al. 2014). Addi tionally, L. sphaericus had the ability to tolerate and re duces heavy metals; hence is commercially important in leather and detergent industries (Tallur et al. 2016). This bacteria species was effective in detergent and can be used as a detergent additive.
P. putrefaciens (S. putrefaciens) (Bayoumi et al. 2012) is Gram negative, facultative anaerobic, non lactose fer menter, on blood agar plate, the colony is typically convex and large, with a brown pigment, and cause "greening" of the agar around the colonies. Shewanella species had the ability to reduce mercury, iron, uranium, and plutonium metabolically. These heavy metals, especially mercury, were released from leather industries and are toxic to the organisms living in water bodies (Bayoumi et al. 2012). These bacteria were isolated from the sediment soil from the leather industry and had the ability to remove hair from the hide and had excellent ability to remove proteinaceous (blood) stain from white cloth.
E. coli, according to (Fu et al. 2003) is Gram nega tive, rod, and mesophilic, facultative anaerobic and lac tose fermenting bacteria. E. coli produces alkaline pro tease enzyme into alkaline culture media (Skim milk agar). This protease enzyme helps to hydrolysis protein (casein) which is found in the culture media (Skim milk agar) as their carbon and nitrogen source (Fu et al. 2003). they reported that the protease enzyme released by E. coli is commercially important, mostly in laundry detergent to re move pretentious stains. Similarly, from the current find ings, E. coli had great activity in blood stain removal. In dehairing activity, E. coli had shown remarkable activity and the softness of skin was promising. These results were encouraging as the isolated bacterial cultures had novel ap plications and also gave an emerging view of the bacterial diversity in the study area since, there was no/ not much information available on bacterial diversity from sediment released from Modjo Tannery Share Company and sludge disposal site at ASTU.

Conclusions
It can be concluded that the soils of sludge disposal sites and sediments around tannery wastes could be good sources for isolating alkaline proteaseproducing bacte ria. The alkaline proteases obtained from bacterial species adapted in the sludge disposal sites and tannery sediments have the potential application in detergent and diaries in dustries. From four bacteria isolates having high prote olytic activity, two isolates were Bacillus species called B. carboniphilus and L. sphaericus. The remaining isolates were Enterobacteriaceae sp. (E. coli) and Pseudomonas sp. (P. putrefaciens (S. putrefaciens). Alkaline protease produced from Bacillus sp. APBIT6 (B. carboniphilus) in the present study had shown high potential in removing hair from animal skin with improved quality and removing proteinacious stain (blood). Finally, alkaline protease pro duced from Bacillus sp. APBIT6 (B. carboniphilus) was selected as the most potent bacteria species due to potential industrial application for both washing ability in detergent and softness and dehairing purpose in leather processing.