1. Introduction
The use of enzymes in ancient fermentation processes has been known since antiquity. Their existence was associated with the history of ancient Greece where enzymes from microorganisms were used in baking, brewing, alcohol production, cheese making, etc and with better knowledge and advances in analytical techniques have demonstrated that can perform a variety of functions such as selective modification of protein and lysis of fibroin clot etc. [9]. According to the market research report on world enzymes published in 2014, the world market for enzymes is expected to record compound annual growth rate (CAGR) of approximately 7.8% during the forecast period of 2015–2020 and reach USD 6.30 Billion in terms of value [54]. This is driven by continued robust growth in pharmaceutical enzyme demand, fine chemical production, bioethanol production and detergent industries [35]. Proteases are important industrial enzymes accounting for 60% of total global enzyme sales and represent one of the three largest groups of industrial enzymes [88].
Proteases are enzymes of ubiquitous nature which catalyse hydrolytic reactions resulting in breakdown of protein molecules into peptides and amino acids[126]. As compared to plants and animals, microorganisms represent an attractive source of proteases as they can be cultured in large quantities in a relatively short time by established fermentation methods, and they produce an abundant, regular supply of the desired product. Also, microbial proteins have a longer shelf life and can be stored under less than ideal conditions for weeks without significant loss of activity. In general, microbial proteases are extracellular in nature and are directly secreted into the fermentation broth by the producer, thus simplifying downstream processing of the enzyme as compared to proteases obtained from plants and animals [70].
In early days, proteases were classified according to their source (animal, plant or microbial), catalytic action (endo or exopeptidases), the molecular size, charge or substrate specificity, however a more rational system was recommended by Enzyme Commission (EC) which provides six major classes for all the enzymes. According to the Enzyme Commission (EC) classification, proteases belong to class three (hydrolases), and sub-group four (which hydrolyse peptide bonds) [126]. Four mechanistic classes are recognized by the Enzyme Commission and within these classes, six families of proteases are recognized till date: serine proteases (EC 3.4.21), serine carboxy proteases (EC 3.4.16), cysteine proteases (EC 3.4.22), aspartic proteases (EC 3.4.23), metallo proteases I (EC 3.4.24) and metallo carboxy proteases (EC 3.4.17) [137]. Alkaline proteases (EC.3.4.21-24, 99) are defined as those proteases, which are active in a neutral to alkaline pH range. They either have a serine centre or are of metallo-type and they are the largely studied group of enzymes because of their wide use in detergent, food, pharmaceutical and leather industries. [47]. The performance of protease is influenced by several factors, such as pH of industrial process, ionic strength, temperature and mechanical handling. Newer enzymes with novel properties that can further enhance the industrial process are always in demand.
Owing to their huge demand in industries, researchers are continuously exploring different aspects of proteases [64], [57], [34], [61]. Therefore, keeping this in view, the review will focus on isolation of alkaline protease producing microorganisms along with production and properties of microbial alkaline proteases. Strategies for exploitation of protease resources and improvement of enzymes to obtain more robust proteases have also been discussed.
2. Isolation of alkaline protease producing microorganisms
Alkaline proteases are indispensable enzymes hence ubiquitous in nature. They are isolated from various sources by surface plating on an alkaline medium and subsequent screening for the desired characteristics. Table 1 shows some protease producing microorganisms isolated from different sources. Natural habitats such as dumping site at Langol, Manipur, India [66]; Egyptian soda lake [53]; Alkaline lonar lake Maharastra, [81]; hot springs, Jordan [10] have been explored for isolation of protease producing microorganisms. Soil samples from leather factories [100], [20], detergent industry [86], wood factory [106], milk processing plant [23] and industrial waste such as tannery waste [37], [11], [123], food processing industrial effluent [104] have also been used. Protease producers have been isolated from soil samples collected from Kurukshetra university campus [70] and sugarcane molasses [141], Thai fish sauce [140], thua nao [26] vegetable waste [55], animal dung [110], [12], [108], degraded and fresh meat [46] and buffalo hide [143]. Other sources used for isolation of alkaline protease producers include mangrove sediment sample [133], compost containing dead animal’s remnants [85], vermicompost pit soil sample [144], sewage sludge sample [22], meat waste contaminated soil [60] and soil from poultry waste site [39]. Alkaline protease has been isolated from fresh fruiting body of the edible mushroom Pleurotus citrinopileatus [24]. Similarly, protease production by mushroom Laccocephalum mylittae has been reported [145].Thirty-nine, protease producing, Streptomycetes were isolated from sewage of tanneries and soil around it, agricultural soil, agricultural fields, water sources from a fish farm, sediments with neutral and alkaline pH and Qaroun lake [102].
Table 1. Different sources of isolation of alkaline protease producing microorganisms.
| Organism | Source of isolation | Reference |
|---|---|---|
| Bacteria | ||
| Bacillus sp. JB 99 | Sugarcane molasses | Johnvesly et al. [58] |
| Pseudomonas aeruginosaMN7 | Tannery wastewater | Ellouz et al. [37] |
| Listeria monocytogenes | Degraded meat of cow | Shumi et al. [115] |
| Geobacillus caldoproteolyticus, sp. nov | Sewage sludge | Chen et al. [22] |
| Bacillus cereus 146 | Soil of a wood factory | Shafee et al. [106] |
| Bacillus amovivorus | Degraded pulse sample | Sharmin et al. [111] |
| Bacillus megaterium | Thai fish sauce | Yossan et al. [140] |
| Xenorhabdus nematophilaBA2 | Nematode Steinernema carpocapsae | Mohamed [79] |
| Bacillus circulans BM15 | Mangrove sediment | Venugopal and Saramma [133] |
| Bacillus sp. | Dog dung | [110] |
| Bacillus lincheniformis N-2 | Compost containing dead animal’s remnants | Nadeem et al. [85] |
| Bacillus firmus MTCC 7728 | Soil from leather factories | Rao and Narasu [100] |
| Bacillus cereus MCM B-326 | Buffalo hide | Zambare et al. [143] |
| Bacillus proteolyticusCFR3001 | Fish processing waste | Bhaskar et al. [18] |
| Bacillus laterosporus-AK1 | Agro waste storage compost | Arulmani et al. [13] |
| Bacillus circulans | Vegetable waste | Jaswal et al. [55] |
| Bacillus sp. | Alkaline salty soil | Darani et al. [28] |
| Bacillus sp. HUTBS71 | Water from hot spring | Akel et al. [10] |
| Bacillus sp. SAL 1 | Tannery waste | Almas et al. [11] |
| Bacillus sp. | Tannery industry effluent | Srinivasan et al. [123] |
| Bacillus subtilis KO | Molasses | Younis et al. [141] |
| Pseudomonas fluorescens | Meat waste contaminated soil | Kalaiarasi and Sunitha [60] |
| Pseudomonas thermareumGW1 | Soil from poultry waste site | Gaur et al. [39] |
| Bacillus pumilus SG2 | Food processing industrial effluent | Sangeetha et al. [104] |
| Pseudomonas aeruginosaMCM B-327 | Vermicompost pit soil | Zambare et al. [144] |
| Geomicrobium sp. EMB2 | Sambhar Salt Lake | Karan et al. [63] |
| Bacillus sp. AS-S20-I | Soil | Mukherjee and Rai [82] |
| Fungi | ||
| Penicillium sp. | Soil from soy meal manufacturing industry | Agrawal et al. [5] |
| Beauveria felina | Soil from soy meal manufacturing industry | Agrawal et al. [6] |
| Mucor sp. | Herbivorous dung | Alves et al. [12] |
| Engyodontium albumBTMFS10 | Marine sediment | Chellappan et al. [21] |
| Aspergillus ustus | Sediment samples from Central Indian Basin | Damare et al. [27] |
| Penicillium chrysogenumIHH5 | Soil | Haq et al. [50] |
| Aspergillus clavatus ES1 | Wastewater | Hajji et al. [48] |
| Penicillium godlewskii SBSS 25 | Soil | Sindhu et al. [117] |
| Penicillium sp. | Soil | Hamzah et al. [49] |
| Aspergillus flavus and Aspergillus terreus | Soil around leather industry | Chellapandi [20] |
| Beauveria sp. MTCC 5184 | Rabbit dung | Shankar et al. [108] |
| Actinomycetes | ||
| Streptomyces nogalator Ac 80 | Soil from leather and hair dumping areas | Mitra and Chakrabartty [78] |
| Streptomyces sp. | Soil | Mehta et al. [76] |
| Streptomyces roseiscleroticus | Sediment sample of an estuarine shrimp pond | Vonothini et al. [135] |
| Nocardiopsis prasina HA-4 | Limestone quarry | Ningthoujam et al. [88] |
| Streptomyces sp. A6 | Intertidal zone 2 km way from the sea coast | Singh and Chhatpar [118] |
| Streptomyces ambofaciens | Soil | Bajaj and Sharma [15] |
3. Alkaline protease production under submerged fermentation
The nature of the fermentation, solid or submerged, influences various aspects of the growth of the microorganism as well as enzyme production [49]. The use of the submerged culture is advantageous because of the ease of sterilization and process control is easier to engineer in these systems [134]. Proteases are generally produced using submerged fermentation due to its apparent advantages in consistent enzyme production characteristics with defined medium and process conditions and advantages in downstream in spite of the cost-intensiveness for medium components [97].
3.1. Optimization of fermentation conditions under SmF
Media composition plays a significant role in enzyme production by microorganisms. Apart from this environmental factors such as temperature, pH, incubation time also influence microbial metabolism up to a large extent [1]. These factors are important to promote, stimulate, enhance and optimize the production of proteases [99]. In order to obtain high and commercially viable yields of protease it is essential to optimize fermentation media for the growth and production of protease [109]. No defined medium has been established for the best production of alkaline proteases from different microbial sources. Each organism or strain has its own special conditions for maximum enzyme production.
3.1.1. Source of growth media
The carbon and nitrogen are major media components which act as essential stimulant for growth of microorganism and enzyme production as well. Maximum enzyme production by a bacterial strain AKS-4 was observed when glucose at a concentration of 1% (w/v) was used as carbon source in the growth media. Protease production was 59.10 U/ml when glucose was used [109]. An increased level of protease production by Bacillus pseudofirmus AL-89 has been observed upon addition of glucose whereas for Nesterenkonia sp. AL-20 protease production was suppressed in the presence of glucose. In the latter case, the glucose may act as catabolic inhibitor [41]. Protease production by Aspergillus oryzae 637 increased steeply with increase in glucose concentration up to 0.6% (w/v) and further increase up to 1.2% (w/v) showed only marginal increase [124]. The maximal production of alkaline protease (2450 U/ml) by Bacillus licheniformis was obtained in medium supplemented with 60 g/l glucose and further increase in glucose concentration resulted in slight reduction in enzyme production [35]. Glucose at high concentration inhibited the enzyme production by Streptomyces sp. and 0.5% (w/v) concentration was optimum for enzyme production while growth was optimum at 1% (w/v) concentration [76]. This is probably due to the catabolic repression mechanism suggesting that in the absence of glucose; the protease plays a role in supplying peptides or amino acids as the carbon or energy source in addition to being a nitrogen source. Consequently, protease synthesis could be repressed when the energy status of the cells is high in the presence of glucose. It is now known that the catabolite control protein (CcpA) is responsible for the regulatory mechanisms of glucose catabolism, and acts as a signal for the repression in protease synthesis [128]. Protease production by Geobacillus caldoproteolyticus sp. nov. was repressed by glucose but enhanced by supplementation of basal media with sucrose [22]. Protease production by Pseudomonas aeruginosa MCM B-327 in soyabean-tryptone media was suppressed by 95% and 60%, when supplemented with glucose and fructose respectively [144]. On the other hand, fructose and glucose proved to the best carbon sources for improving the productivity of protease from Aspergillus flavus and Aspergillus terreus respectively [20]. Kumar et al. [70]used various carbon sources like glucose, lactose, galactose and starch for protease production by Bacillus aryabhattai K3 and reported maximum protease production (622.64 U/ml) with lactose (10 g/l) as carbon source in the medium. Similarly Dodia et al. [33] while studying eight isolates for protease production found that for most of the isolates enzyme secretion was optimum with lactose. B. licheniformis BBRC 100053 exhibited higher productivity of protease in culture media containing lactose as carbon source [42]. Use of 5% (w/v) starch as carbon source led to maximum protease production by Bacillus sp. 2–5 [28]. The Bacillus clausii strain No. 58 grew well on various starch-based carbon sources. Corn starch at a concentration of 0.5% (w/v) gave the highest productivity of protease, followed by wheat flour and wheat bran. However, supplementation of potato starch caused a decrease in protease titre, which possibly may be due to the presence of protease inhibitors in potato [67]. The highest protease production by S. roseiscleroticus was obtained when starch was used as the carbon source while it was minimum with dextrose [135], whereas, Jaswal et al. [55] reported that maltose and glucose were equivalent and significantly better than starch and fructose for production of protease by Bacillus circulans. Sucrose at 5% (w/v) was found optimal for enzyme production by actinomycete Nocardiopsis prasina HA4 [88]. Dextrose and sucrose were found as the best two sources for both biomass and protease production by L. mylittae especially when these two were combined together in a ratio of 1:1 in the medium [145]. Dextrose was also reported to be the suitable carbon source for protease producing Bacillus subtilis strain [29]. Among carbon sources used for protease production by Pseudomonas fluorescens, wheat bran and maltose were found to support protease production [60]. Wheat flour was observed to be most effective for protease production by a Bacillus sp. [23]. Usharani and Muthuraj [131] reported Bacillus laterosporus to be capable of utilizing a wide range of carbon sources; the best carbon sources for protease secretion were soluble starch, trisodium citrate, citric acid and glycerol.
The requirement for a specific nitrogen supplement differs from organism to organism and different workers have used different organic nitrogen sources (simple or complex), inorganic nitrogen sources and amino acids for enhancing protease production. The effect of different nitrogen sources, such as peptone, tryptone, potassium nitrate and yeast extract phosphate at a concentration of 1%, on the production of protease using Rheinheimera strain (KM459533) was studied and the enzyme production was reported to be 433.63 U/ml [128]. (NH4)2HPO4 was also found to be the best nitrogen source for protease production by A.oryzae 637 [124]. Ammonium sulphate has been reported to be the best nitrogen source for protease production by Bacillus sp. strain AS-S20-I [82]. On contrary, ammonium chloride and ammonium sulphate have been reported to suppress alkaline protease production by Bacillus sp. 2–5 [28]. Neither ammonium chloride nor urea was beneficial for biomass or protease production by L. mylittae; however corn steep liquor enhanced the production of protease [145].
The highest level of protease production by Bacillus cereus strain 146 was observed in presence of beef extract as nitrogen source in the growth media. The presence of yeast extract, peptone and tryptone increased growth but resulted in low protease production in this case [106]. The best nitrogen source for protease production by another Bacillus sp. was beef extract while yeast extract and tryptone were also comparable [86]. Also, the highest protease production by Streptomyces roseiscleroticus [135], A. flavus [20] and A. terreus[20] was obtained when beef extract was used as nitrogen source. Tryptone was found to increase protease production for a Bacillus strain [123]. Among nitrogen sources (beef extract, tryptone, peptone, glycine, casein) used for protease production by Pseudomonas fluorescens, maximum titre was reported with peptone [60]. Peptone was also found optimal for protease production by N. prasina HA4 [88] and B. licheniformis BBRC 100053 [42]. Yeast extract showed maximum influence in enhancement of enzyme production by Bacillus sp. [97]. For Streptomyces sp. growth and enzyme production was optimum with yeast extract [76]. A number of workers [95], [1], [113], [42] have reported a combination of peptone and yeast extract to be optimum for protease production. In case of Bacillus sp. APP1, among all the organic nitrogen sources used, soyabean meal had a significant effect on the production of the extracellular protease [23]. Jaswal et al. [55] also reported that soyabean meal was better than casein, gelatine and peptone for production of protease by B. circulans. Maximum protease production by P. aeruginosa MCM B-327 was obtained with a combination of soybean meal and tryptone [144]. Sharma et al. (2005) reported use of casein, peptone, yeast extract and beef extract as nitrogen source for protease production by bacterial strain AKS-4 s and observed high protease production (49.77 U/ml) in the presence of casein. For protease production by Microbacterium sp. kr10, feather meal was observed to be optimal nitrogen source [129]. Among different organic nitrogen sources, skim milk gave maximum protease yield in case of Bacillus caseinilyticusfollowed by malt extract, peptone and yeast extract. Ammonium chloride as inorganic nitrogen source was found to inhibit the production. MgCl2 and CaCl2induced protease production [81] Mustard cake has been reported as the ideal nitrogen source for protease production by Streptomyces ambofaciens [15].
3.1.2. Environmental conditions
3.1.2.1. Incubation period
Incubation period affects the enzyme production significantly and it varies from 24 h to a week depending upon type of microorganism and other culture conditions such as inoculum size, metabolic state of cell pH and temperature. Protease production by Bacillus pumilus UN-31-C-42 started 16 h after incubation, increased gradually and reached a maximum at about 28 h [52]. For B. subtilis PE-11 [3] and B. licheniformis LBBL-11 [90] maximum growth and enzyme production was observed after 2 days. Bacillus sp. APP1 [23] produced maximum titre of protease after an incubation period of 2 days though the highest biomass yield was recorded after 4 days of incubation period. B. subtilisKO strain exhibited its maximum production of protease within 48 h incubation period [141]. Pseudomonas fluorescens produced maximum protease after 24 h of incubation, the enzyme activity gradually decreased from 48 to 168 h [60]. The maximum protease production from V. pantothenticus [46], B. subtilis [69]and B. licheniformis [69] was recorded after 72 h. Optimum incubation time for protease production by B. licheniformis and Bacillus coagulans has been reported as 96 h [14]. B. subtilis ATCC 633 and Bacillus sp. UFLA 817 CF have been reported to show maximum protease activity after 24 h growth, a period that coincided with the end of the exponential phase [31]. Protease secretion by B. cereusVITSN04 followed a similar pattern as for growth, with the highest enzyme activity in the exponential phase [127]. The protease production by G. caldoproteolyticus sp. nov. [22], B. clausii No. 58 [67], Bacillus brevis SSA1 [4], Bacillus firmus MTCC 7728 [100], Xenorhabdus nematophila BA2 [79] and Geomicrobium sp. EMB2 [63] have been reported to reach maximum level in the stationary phase, after 9, 48, 72, 48, 48 and 72 h of incubation respectively.
The enzyme production by Penicillium chrysogenum gradually increased with the passage of time and the highest enzyme activity was obtained after 72 h of incubation, prolonged incubation decreased the enzyme activity; however the growth of the microorganism was not significantly affected [50]. Also, maximum protease production by A. flavus and Aspergillus terreus was reported after 72 h [20]. Enzyme synthesis by Mucor sp. started in the first 24 h when nutrient consumption was high and there was reduction in the enzyme activity after 120 h of incubation [12]. The synthesis and secretion of the protease by A. terreus IMI 282743 was initiated during the exponential growth phase (7 days after incubation) with a substantial increase near the end of the growth phase towards maximum amounts of protease produced during the stationary growth phase (9 days after incubation) [138]. The optimum culture period for the production of proteases by Botrytis cinerea was 9 days [1]. N. prasina HA4 has been reported to achieve maximal enzyme production in 5 days [88]. Maximum protease production by S. ambofaciens was after 48 h of incubation [15].
3.1.2.2. Medium pH
The pH of culture affects all enzymatic processes and transportation of various components across the cell membrane. However, the molecular basis of pH affecting bacterial metabolism in culture broth is obscure. Since proton motive force in chemiosmosis is affected by the medium pH value, it is possible that under optimum pH range, the relative metabolic efficiency is high [119] Hence it is an important parameter to optimize. A medium with neutral initial pH has been reported for alkaline protease production by P. chrysogenum IHH5 [50], S. roseiscleroticus [135], B. cereus [72] Bacillus polymyxa [72], Bacillus aquimarisVITP4 [113] and P. aeruginosa MCM B-327 [144]. While slightly acidic medium (pH 6.3–6.5) have been reported as optimum for protease production by Bacillussp. MIG [43] and B. cereus SIU1 [119], slightly alkaline medium (pH 8.0–8.5) has been reported to be optimum for protease production by B. licheniformis IKBC-17[89], B. subtilis IKBS 10 [89], Bacillus macerans IKBM-11 [89], Bacillus amovivorus [111] and Aspergillus niger [30]. The reason behind this kind of observation is not yet very clear. For eight isolates studied for protease production, it was found that the optimum pH for growth was 9.0 for the majority of the isolates, while the optimum pH with regard to enzyme secretion varied between pH 8.0–10.0 [33]. A pH 9 has been reported as optimal for protease production by Bacillus sp. [97], Bacillus sp. strain APP1 [23], Bacillus proteolyticus CFR3001 [18], V. pantothenticus [46] and Pseudomonas fluorescens[60]. Higher initial pH, 10.0 for A. oryzae 637 [124] and B. licheniformis TISTR 1010 [132], 10.5 for B. circulans [55] and 10.7 for Bacillus sp. 2–5 [28] have also been reported for maximum protease production.
3.1.2.3. Incubation temperature
Temperature is a critical parameter that has to be controlled and varied from organism to organism for maximum cell growth and enzyme production. The optimum temperature requirement reported for alkaline protease production by different microorganisms differs widely. The optimum temperature for protease production by P. aeruginosa PseA [45], B. licheniformis [14], B. coagulans[14], B. cereus [65], P. aeruginosa MCM B-327[144], P. chrysogenum IHH5 [50] and A. oryzae 637 [124] has been reported to be 30 °C. Lower optimum temperature of 25 °C has been reported for B. circulans [55], Microbacterium sp. [129] and 28 °C for B. cinerea [1]. P. fluorescens was capable of producing protease in the range of 27–57 °C with production maximum at 37 °C [60]. A temperature of 37 °C has been reported as optimal temperature for protease production by a number of Bacillus species such as B. amovivorus [111], B. proteolyticus CFR3001 [18], B. aquimaris VITP4 [113] and B. subtilis strain Rand [2]. In contrast to that a temperature of 40 °C has been reported to be best for production of protease by Bacillus sp. 2–5 [28], B. licheniformis GUS1 [105], V. pantothenticus [46] and S. roseiscleroticus [135]. Maximum production of protease by A. niger was obtained at 45 °C [30]. High optimum temperature of 50 °C has been reported for Bacillussp. strain APP1 [23] and B. subtilis BS1 [107]. B. licheniformis IKBC-17, B. subtilisIKBS-10. The optimum temperature of alkaline protease production by B. cereus and B. polymyxa has also been reported as 60 °C [72]. The production of protease using statistical method was studied using B. subtilis K-1 strain. The highest protease activity was detected at 60 °C (312.6 U/ml); however, substantial activity was observed at 70 °C (306.2 U/ml) and 80 °C (301.6 U/ml) [120].
3.2. Alkaline protease production under solid-state fermentation (SSF)
Solid state fermentation (SSF) is defined as the fermentation process which occurs in the absence or near absence of free water [38]. In this process, the solid substrate not only supplies the nutrient to the culture but also serves as an anchorage for the microbial cells. SSF processes are of special economic interest for countries with an abundance of biomass and agroindustrial residues, as these can be used as cheap raw materials. [36]. With regard to cost economics, SSF has been proved to be more efficient than SmF. The product can be recovered in highly concentrated form as compared to those obtained by submerged fermentations [126]. Other advantages include superior volumetric productivity, simpler machinery, use of inexpensive substrate, low energy requirements and low waste water output, simple technique, low capital investment, lower levels of catabolite repression and better product recovery. It therefore becomes very important to determine the environmental conditions of the microorganism for maximum production [139]. However, SSF processes present some limitations, such as the SSF technique is mainly confined to process involving fungi, the restricted range of microorganisms that are able to grow under reduced moisture levels, control of conditions is again very difficult and there is no defined concentration of media components [121]. Also, SSF is usually slower because of the diffusion barriers imposed by the solid nature of the fermented mass [36].
3.2.1. Optimization of fermentation conditions under solid state fermentation
The major factors that affect microbial synthesis of enzymes in a SSF system include: selection of a suitable substrate and microorganism; pre-treatment of the substrate; particle size of the substrate; water content and aw of the substrate; relative humidity; type and size of the inoculum; control of temperature of fermenting matter/removal of metabolic heat; period of cultivation; maintenance of uniformity in the environment of SSF system, and the gaseous atmosphere, i.e. oxygen consumption rate and carbon dioxide evolution rate. For optimization of these factors new statistical techniques are now used which are more scientific and less labour intensive [62] Effect of some of these factors on alkaline protease production is reviewed below.
3.2.1.1. Substrate
Substrates from agricultural or industrial wastes (wheat straw or barley, sugar cane bagasse, coffee pulp, grape wastes, copra pasta, among other) or inert materials (as resins of ionic exchange, acrolite or polyurethane foam) can be used. The pre-treatments of these materials are required and generally include milling and washing [7]. Govarthanan et al. [44] used horse gram husk as substrate while producing protease using Bacillus sp. SKK11 and reported maximum protease production (240 U/ml) by using maltose as a source of carbon. Sometimes, a combination of two or even three different substrates gives higher enzyme yields than each of the substrates used individually [126]. There are several reports describing use of agro-industrial residues for the production of alkaline protease, by bacteria [25], [142], [17], [28], [38] and fungus [73], [94], [117], [84]. Cost and availability are important considerations, and therefore the selection of an appropriate solid substrate plays an important role in the development of efficient SSF processes [36]. Pigeon pea waste, pineapple waste, orange peel waste, sugarcane bagasse, wheat bran, rice bran, raw potato starch and raw sweet potato starch were tested for protease production by Bacillus sp. JB-99 and maximum protease activity was obtained with pigeon pea waste [58]. Different agro-industrial waste materials (green gram, chick pea, red gram, black gram husks and wheat bran) were tested for protease production by Bacillus sp. and maximum enzyme production was observed with green gram husk, while minimum protease production was reported with red gram husk [97]. Green gram husk has also been reported to support maximum protease production by B. circulans [96]. Coffee wastes such as pressed coffee pulp, coffee cherry husk, coffee parchment husk, silver skin and coffee spent wastes were tested for protease production by A. oryzaeCFR305 and coffee cherry husk was found to be most suitable [84]. Maximum protease production was observed in watermelon rind among five substrates (melon rind, watermelon rind, rice, lentil and corn husks) used for protease production by Bacillus sp. [38]. High titre of protease activity by T. thalpophilusPEE 14 was obtained in medium containing wheat bran as substrate [32]. Wheat bran has also been used for protease production by Beauveria feline [6] and Engyodontium album BTMFS10 [21]. Rice bran was reported to be the best substrate for protease production by a Bacillus sp. [116]. Broken rice of different varieties i.e. PONNI, IR-20, CR-1009, ADT-36 and ADT-66 was used for protease production by A. niger MTCC 281 and maximum protease production was observed in PONNI [94]. Deoiled Jatropha seed cake was reported to support good bacterial growth and enzyme production when assessed for its suitability as substrate for enzyme production by P. aeruginosa PseA [75]. Defatted soybean cake was used for protease production by a Penicillium sp. [40]. A B. subtilisisolate was shown to be able to produce extracellular protease in solid-state fermentation using soy cake as culture medium [121]. Soybean was used for production of alkaline protease by Teredinobacter turnirae [36]. Among the different agro-industrial waste products and kitchen waste materials, viz. mustered oil cake, wheat bran, rice bran, Imperata cylindrica grass, banana leaves, potato peels and used tea leaves screened as substrates/solid supports for the production of alkaline protease by B. subtilis DM-04, potato peel followed by I. cylindrica grass supported maximum protease production. Potato peel and I. cylindrica grass mixed in a ratio of 1:1 (w/w) significantly enhanced the protease production as compared to individual substrate [83]. A. oryzaeNRRL 2217 was capable of producing maximum protease on mixed substrate coconut oil cake: wheat bran in mass ratio of 1:3 [125]. Wheat bran enriched with fish scales and egg shell in a ratio of 1:2:0.005 (w/w) was used for protease production by Penicillium sp. [49].
3.2.1.2. Particle size of substrate
In solid-state fermentation process, the availability of surface area play a vital role for microbial attachment, mass transfer of various nutrients and substrates and subsequent growth of microbial strain and product production [97]. Smaller substrate particles provide a larger surface area for microbial attack, however, too small particles may result in substrate agglomeration which may interfere with aeration and may thus result in poor growth. At the same time, larger particles provide better aeration efficiency but provide limited surface for microbial attack. Therefore, it may be necessary to provide compromised particle size [93]. The coarse size (2 mm) of soybean was found to be optimal size of the substrate for higher protease production by T. turnirae [36]. Maximum enzyme production by Bacillus sp. was noticed with 1.4–1.0 mm particle size green gram husk material [97]. Wheat bran of particle size <425 µm was found optimal for protease production by E. album BTMFS10 [21]. Protease production by P. aeruginosa growing on animal fleshing size of 0.25 cm was higher than all other substrate sizes [68]. Protease production by A. oryzaeCFR305 was found to be high with 1 mm particle size of coffee cherry husk as compared to 0.5 mm and 2 mm [84].
3.2.1.3. Moisture content
An increase in moisture level is believed to reduce the porosity of the substrate such as wheat bran, thus limiting oxygen transfer, while lower moisture content causes reduction in solubility of nutrients of substrate, lower degree of swelling. Hence, an optimal level of moisture is required for maximum enzyme productivity. High enzymatic titre (240 U/g) was attained when the initial moisture level was 22.4% [112]. It is known that water content of a medium has a profound influence on the production of products by microorganisms. In a solid state fermentation the water content of the substrate is greatly influenced by the absorbing capacity and capillary forces of the substrate, the growth temperature, the amount of metabolic heat generated the quantity of moisture evolved and growth requirement of organisms. The moisture level in SSF has a great impact on the physical properties of the substrate and this factor makes it different from Smf. It is crucial to provide optimized water level that controls the water activity (aw) of the fermenting substrate for achieving maximum product [97]. An increase in moisture content causes a decrease in the porosity of the substrate, thereby decreasing the gas exchange. Low moisture content leads to sub-optimal growth and a lower degree of substrate swelling which also decreases enzyme production [74], [92]. Agrawal et al. [5] reported 50% of initial moisture content to be best for protease production by Penicillium sp. Similar observation has been reported by other workers for A. oryzae NRRL 2217 [125], A. oryzae CFR305 [84] and P. aeruginosa PseA [75]. In case of E. albumBTMFS10 [21] and Penicillium godlewskii SBSS 25 [117] a moisture content of 60% was found to be optimal for protease production. A high moisture content of 80% for T. thalpophilus PEE 14 [32], 120% for Beauveria felina [6], 90–170% for B. circulans [96] and 100% for B. subtilis DM-04 [83] have also been reported.
3.2.1.4. Incubation time
Generally, the time required for the optimum protease production by bacteria or fungus may be as long as 48 h–9 days [8]; [98]. Incubation time of 20 h for P. aeruginosa [68], 24 h for B. subtilis DM-04 [83], 60 h for B. circulans [96], 60 h for Bacillus sp. [97], 63 h for T. turnirae [36], 72 h for Bacillus sp. [38], 72 h for P. aeruginosa PseA [75] have been reported for maximum protease production. The incubation time for maximum protease production in SSF by different fungal species have been reported as, 72 h for Penicillium sp. [5], 168 h for B. felina [6], 48 h for A. oryzae NRRL 2217 [125], 120 h for E. album BTMFS10 [21], 72 h for A. niger MTCC 281[94], 96 h for by P. godlewskii SBSS 25 [117], 120 h for A. oryzae CFR305 [84]. For actinomycete, T. thalpophilus PEE 14, the highest titre of enzyme was attained after 72 h [32]. Protease production by an organism can be both growth and non-growth associated. Protease production profile of B. subtilis was found to be associated with growth, the enzyme production peak occurred at the mid exponential phase (9 h), but enzyme denaturation began to occur before biomass reached its maximum level [121].