Alkaline Protease Production from Brevibacterium ...

Appl Biochem Biotechnol DOI 10.1007/s12010-016-2341-z

Alkaline Protease Production from Brevibacterium luteolum (MTCC 5982) Under Solid-State Fermentation and Its Application for Sulfide-Free Unhairing of Cowhides R. Renganath Rao 1,2 & M. Vimudha 1 & N. R. Kamini 3 & M. K. Gowthaman 3 & B. Chandrasekran 4 & P. Saravanan 1

Received: 16 September 2016 / Accepted: 24 November 2016 # Springer Science+Business Media New York 2016

Abstract Enzyme-based unhairing in replacement of conventional lime sulfide system has been attempted as an alternative for tackling pollution. The exorbitant cost of enzyme and the need for stringent process control need to be addressed yet. This study developed a mechanism for regulated release of protease from cheaper agro-wastes, which overcomes the necessity for stringent process control along with total cost reduction. The maximum protease activity of 1193.77 U/g was obtained after 96 h of incubation with 15% inoculum of the actinomycete strain Brevibacterium luteolum (MTCC 5982) under solid-state fermentation (SSF). The medium after SSF was used for unhairing without the downstream processing to avoid the cost involved in enzyme extraction. This also helped in the regulated release of enzyme from bran to the process liquor for controlled unhairing and avoided the problem of grain-pitting. Unhairing process parameters were standardized as 20% enzyme offer, 40% Hide-Float ratio at 5 ± 1 rpm, and process pH of 9.0. The cost of production of 1000 kU of the protease was calculated as 0.44 USD. The techno-economic feasibility studies for setting up an SSF enzyme production plant showed a high return on investment of 15.58% with a payback period of 6.4 years.

Electronic supplementary material The online version of this article (doi:10.1007/s12010-016-2341-z) contains supplementary material, which is available to authorized users.

* P. Saravanan [email protected]

1

Leather Process Technology Department, Central Leather Research Institute, Adyar, Chennai 600020, India

2

Anna University, Chennai 600025, India

3

Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai 600020, India

4

Central Leather Research Institute, Adyar, Chennai 600020, India

Appl Biochem Biotechnol

Keywords Solid-state fermentation . Actinomycete . Protease . Unhairing . Grain-pitting . Techno-economic analysis

Introduction The leather industry holds a prominent place in global as well as Indian economy. Leather industry uses a variety of chemicals to process raw animal hides and skins to make leathers. Each phase of leather making uses a certain chemical that leads to environmental pollution. Unhairing of animal hides and skins uses chemicals such as lime and sodium sulfide, which are found to be problematic to the environment [1]. Lime, being sparingly soluble in water, comes out as such in the wastewater. Sulfide’s action on hair makes it a pulp that adds to the pollution load. Researches have been carried out to develop chemical-free eco-friendly unhairing methods. Enzymes from a variety of origins have been tried and tested for the purpose of hair removal. Pancreatic proteases were used for unhairing of hides since 1910 [2], but pancreatic proteases have been replaced by microbial proteases, since the latter were found to be genetically flexible, economical, and could be produced in huge amounts for large-scale industrial applications. Among a large number of microbial enzymes, proteases occupy a pivotal position owing to its wide application. Proteases from microbial origin such as bacterial and fungal origins have been reported earlier. Thanikaivelan et al. [3] reviewed the emerging novel enzymatic methods in leather processing. Dettmer et al. [4] researched the application of protease from Bacillus subtilis aiming at the replacement of chemical method of unhairing by complete enzyme-based method. Reports from Council for Scientific and Industrial Research—Central Leather Research Institute (CSIR-CLRI) presented a system of unhairing using crude protease from Bacillus pumilus (MTCC 7514) [5], which was found to be effective in removing the hair completely. Among mold/fungal proteases, proteases from Aspergillus flavus and Aspergillus terreus [6], Conidiobolus brefeldianus [7], Aspergillus oryzae, Aspergillus fumigatus, Aspergillus effusus, Aspergillus wentii, and Aspergillus parasiticus [8] exhibited unhairing potency. Actinomycetes are a group of bacteria that resemble fungi and have properties of both bacteria and fungi. Actinomycetes are morphologically similar to filamentous fungi, which have a vegetative thallus during the least part of their life cycle [9]. The actinomycetes have many bacterial properties as well. Actinomycetes produce a large number of proteolytic enzymes, which have high substrate specificity. Proteases from actinomycetes find application in leather industry for unhairing of hides and skins. Among the actinomycete, proteolytic enzymes from Streptomyces sp. were reported to effectively dehair hides and skins. Enzymes from Streptomyces sp. such as Streptomyces moderatus NRRL 3150, Streptomyces froadiae [10], Thermoactinomyces sp. RM4 [11], Streptomyces griseus [12], and Streptomyces nogalator [13] were found to be effectively dehairing the skins and hides. Although there has been many researches on protease production for unhairing process, there are no literature pertaining to the production of proteases from microbial culture sourced from the tannery hair-waste dump yard. Such proteases are thought to be much more appropriate for the unhairing process. Proteases are produced in either submerged fermentation (SmF) or solid-state fermentation (SSF) methods. SSF involves the use of low-cost easily available substrates that are wastes generated from other industries. Since the fermented substrates could be used directly without any downstream processing unlike in SmF, this further cuts down the total cost involved in production of enzymes. Agro-industrial wastes, such as rice bran, rice straw, cotton, bagasse, and wheat bran, have been studied for the induction and biosynthesis of


protease [14]. Wheat bran is a better carbon source and can be utilized well for protease production involving actinomycetes. The present work dealt with the production of protease using actinomycete Brevibacterium luteolum (MTCC 5982) on low-cost agro-industrial wastes for the unhairing application. Use of moistened agro-wastes for the production of protease and using the fermented bran directly for unhairing of cowhides have minimized the cost of enzyme production as well as ensured better unhairing without any grain damage. Direct use of fermented bran for unhairing instead of using extracted enzyme from the bran marks the novelty of the work.

Materials and Methods Chemicals All the chemicals used in this work were analytical grade chemicals obtained from M/s SigmaAldrich and M/s Merck. The microbiological media were obtained from M/s Hi-Media. Wet salted cowhides were obtained from a local slaughterhouse.

Culturing of Microorganism B. luteolum (MTCC 5982) previously isolated (GenBank accession KU925858) in the laboratory, from the soil sample of tannery hair waste dump yard in Erode, Tamil Nadu, India, was used for the current study. The culture was maintained in nutrient agar slants (pH 7.4) that contained peptic digest of animal tissue (5 g/L), sodium chloride (5 g/L), beef extract (1.5 g/L), and yeast extract (1.5 g/L) at 4 °C and sub-cultured once in every 2 weeks.

Solid-State Fermentation for Enzyme Production The pre-inoculum for the SSF enzyme production was prepared by transferring a loop-full of culture from the nutrient agar slants into the nutrient broth medium and kept at 37 °C for 18 h at 150 rpm in a rotary shaker. Wheat bran was ground to a particle size of approximately 1 mm in order to maximize the performance of SSF for enzyme production. SSF was carried out in 250-mL Erlenmeyer flask containing 10 g of sized down wheat bran and 0.5 g rice flour as inducer was moistened with distilled water in the ratio of 1:0.7 (10 g bran with 7 mL distilled water) and autoclaved at 121 °C, 15 psi pressure for 15 min. This medium was then inoculated with 10% (1 mL per 10 g) preinoculum and kept in an incubator for 48 h at 37 °C. The incubation time and temperature were maintained at 48 h and 37 °C, respectively, to compare between the results obtained from the optimization process. The fermentation medium was thoroughly mixed once in every 12 h with a sterile glass rod in order to avoid the anaerobic condition, which affects the microbial growth [15].

Enzyme Assay Determination of Protease Activity For estimating the protease activity of the product from the SSF, the enzyme had to be extracted from the SSF medium. Tris buffer was used as the medium of extraction of the enzyme for estimating the activity. To 10 g of fermented bran, 100 mL of 1.0 M Tris buffer


(pH 7.4) was added and the enzyme was extracted by rotating it in a shaker for 2 h at 150 rpm. The extract was then centrifuged at 10,000 rpm for 10 min at 4 °C, and the cell-free supernatant was used for enzyme assay. It is necessary to centrifuge at 4 °C in order to avoid the denaturation of proteins by heat. Sigma’s non-specific protease activity assay using casein as substrate was followed as the standard protocol for estimation of protease activity [16]. Casein (0.65%) in 50 mM Tris–HCl pH 8.0 was used as the substrate solution. To 5 mL of casein solution, 1 mL of the enzyme (extracted in buffer solution) was added and incubated at 37 °C for 10 min. The reaction was then terminated by adding ice-cold 110 mM trichloroacetic acid (TCA) and kept at 37 °C for 30 min. Blank was prepared by adding 1 mL of enzyme (extracted in buffer solution) after the addition of TCA. Samples were then centrifuged at 10,000 rpm for 10 min at 4 °C. To 2 mL of these supernatant, 5 mL of 500 mM sodium carbonate solution and 1 mL of Folin-Ciocalteu reagent were added and incubated at 37 °C for 30 min. The absorbance of the samples was measured at 660 nm using a Jasco UV-Visible Spectrophotometer (Model V-660). Tyrosine at a concentration of 10 to 100 μg was used as standard calibration curve. One unit of protease enzyme activity is defined as the amount of enzyme required to liberate 1 μg of tyrosine under standard assay conditions. This activity was divided by fermented bran concentration (weight of fermented bran taken = 10 g, amount of water added = 100 mL; concentration = 0.1 g/mL) to obtain protease activity in terms of units per gram of fermented substrate.

Determination of Protein Content Protein content of the cell-free supernatant was determined using Lowry’s protein estimation. To 0.2 mL of sample, 2 mL of alkaline copper sulfate reagent (analytical reagent) was added. The contents of the test tube were mixed well by inversion. This solution was incubated at room temperature for 10 min. Then 0.2 ml of Folin-Ciocalteu solution was added to each tube and incubated for 30 min. The UV-Vis spectrophotometer was zeroed with blank and the optical density (absorbance) of the solution was measured at 660 nm. The absorbance was recorded and the concentration of the unknown sample was found out. Bovine serum albumin (BSA) of concentration ranging from 0.05 to 1 mg/mL was used as protein standard [17].

Standardization of SSF Enzyme Production Process Parameters Standardization of SSF enzyme production parameters were studied initially using wheat bran and rice flour as the fermentation medium. Process parameters like temperature, initial pH, type of water, moisture content, inoculum size, and fermentation time were varied, and its effect on enzyme production was studied. The effect of another major agro-waste, rice bran, was also considered for the fermentation study. The combination of wheat bran and rice flour along with rice bran in varied concentrations was taken as fermentation medium, and the yield was recorded by measuring the protease activity of the produced enzyme. All the experiments were conducted in triplicates and their mean values were taken.

Effect of Salt Concentration and Surfactants on Enzyme Activity Leather processing involves use of salt and surfactants in its unit operations. The enzyme’s stability in the presence of such salts and surfactants was ensured prior to the unhairing process. The enzyme activity was tested in the presence of various surfactants such as SDS,


Tween 80, cetyltrimethyl ammonium bromide (CTAB), MS Powder (commercial leather application wetting agent from M/s Stahl Chemicals), and Triton X-100. The salt concentration (NaCl) was varied between 0.2 and 1.2 M, and its effect on enzyme activity was studied.

Standardization of Unhairing Process Parameters The unhairing process parameters like enzyme offer level (fermented bran without any downstream processing), drum rotation speed, and the Hide-Float ratio (amount of water offered during unhairing process based on the weight of the cowhide) were standardized by visually analyzing the extent of hair removal from cowhides. The fermented bran was shade dried and stored at 4 °C prior to use. The process pH was optimized by measuring the activity of the enzyme at various pH values. All the experiments were conducted in triplicates, and the corresponding observations were made.

Enzyme Leaching Phenomenon and Application of Enzyme for Unhairing Process To study the leaching phenomenon of enzyme into unhairing process liquor from fermented bran, the standardized process was carried out using a Rotospin rotary mixer without the cowhide to mimic the unhairing process. Five milliliters of enzyme leached into the process liquor was harvested for every 30 min and was centrifuged at 10,000 rpm for 20 min at 4 °C. The protein content of the cell-free supernatant was measured to determine the total time taken for the complete leaching out of enzymes and its availability for the unhairing process. Lowry’s protein measurement with the Folin-Phenol reagent was followed as the protocol to estimate the protein content. Application trials were conducted using cowhides. Hides were washed using 300% w/w water for 3 h in a drum rotating at a very low rpm of 2–3. After 3 h, the water was drained out and the hides were soaked in a pit containing 300% w/w water, 0.01% w/w biocide (Formaphen A), and sodium carbonate 0.01% w/w for 12 h (based on the raw weight of the cowhides). The water was drained out and hair removal was carried out in the drum using 0.3% sodium carbonate (pH 9.0), 0.01% w/w biocide (Formaphen A), and 20% w/w (based on the soaked weight of the cowhides) of SSF enzyme along with 40% w/w water for 6 h at 5 ± 1 rpm. Loosened hair was removed by washing with 100% w/w water and 0.1% w/w nonionic wetting agent for 10 min and drained out. All percentage was calculated based on the soaked weight of the cowhides.

Techno-economic Analysis The total capital cost (TCC) required for setting up an SSF enzyme production plant was calculated by the sum of the fixed capital investment (FCI) and working capital cost (WCC). The FCI consisted of the total cost of the equipment purchased and the other direct and indirect plant costs. The annual production cost (APC) was calculated by summation of the cost involved in raw materials (CRM), utilities (CU), operating labor (COL), maintenance and repair (CMR), laboratory cost (CL), and other necessary costs (CO). The unit cost of enzyme produced was calculated by dividing the APC by total amount of enzyme produced in a year. The annual profitability of the SSF enzyme production plant was assessed, and the payback period was calculated to demonstrate the economic feasibility of the project.


Results and Discussion Effect of Various Process Parameters on SSF Enzyme Production The need for producing protease from SSF of B. luteolum (MTCC 5982) was realized due to the fact that the unhairing process required the use of fermented bran as direct enzyme source, which was commercially unavailable. This also led to the need for optimizing the parameters of production of enzyme using bran in SSF.

Effect of Temperature and pH on Enzyme Production The effect of temperature on enzyme production was studied by varying the temperature from 24 to 39 °C with an increment of 3 °C by keeping other parameters constant. It was found that protease production was maximum (397.19 U/g) at a temperature of 33 °C (Table 1). Also, from the results, it was found that the enzyme production was favorable at a temperature ranging from 30 to 39 °C, which showed that the organism was capable of producing protease enzyme over a wide range of temperature. Keeping the temperature constant as 33 °C, the initial pH of the fermentation medium was varied from 6.0 to 10.0 with an increment of 1 U and the maximum activity (577.17 U/ g) was obtained at pH 9.0 (Table 1). The organism was efficient in protease production at alkaline pH rather than the neutral pH range.

Effect of Type of Water as Moistening Agent and Moisture Content on Enzyme Production Tap water, double distilled water, and Millipore water were used as moistening agents. A maximum protease activity of 593.60 U/g was observed when tap water was used as moistening agent among aforementioned ones (Table 1). It was evident due to the fact that the presence of mineral ions in tap water enhanced the production of the enzyme. Similar results were observed with Bacillus sp. RRM1 and Bacillus sp. JB-99 [18, 19] under SSF when tap water was used as moistening agent. The ratio of substrate to moisture during fermentation was varied from 1:0.7 to 1:1.5. It was found that the saturation in protease activity (860.72 U/g) was obtained with the ratio of wheat bran to water ratio of 1:1.3 (Table 1).

Effect of Inoculum Size and Fermentation Time on Enzyme Production The inoculum size varied from 5 to 25% (Table 1), and the protease activity of 944.57 U/g was obtained with 15% inoculum. Divakar et al. reported that the higher protease activity was obtained at inoculum levels of 20% with wheat bran as substrate using Thermoactinomyces thalophilus PEE 14 strain [20]. With the above-optimized conditions, the fermentation process was carried out to optimize the time taken for fermentation process to complete. The fermented medium was assayed for every 24 h, and it was found that the maximum activity of 1132.72 U/g was obtained after 96 h (4 days), after which there was no significant increase in enzyme production (Table 1). Vijayaraghavan et al. found that the alkaline protease produced under SSF using cow dung and other agro-wastes showed highest activity just after 72 h of incubation. [21]

Appl Biochem Biotechnol Table 1 Effect of temperature, pH, type of water, moisture content, inoculum size, and fermentation time on protease production Process parameter

Variation of process parameter

Temperature (°C)

24

181.96 ± 10.56

27

253.92 ± 9.32

30

317.67 ± 11.95

33

397.19 ± 9.66

36

362.07 ± 10.93

pH

Type of water

Moisture content (Wheat bran/tap water)

Inoculum size (%)

Fermentation time (h)

Protease activity (U/g)

39

353.05 ± 10.78

6.0

288.78 ± 9.88

7.0 8.0

395.87 ± 10.36 473.00 ± 9.54

9.0

577.17 ± 11.18

10.0

531.71 ± 10.47

Tap water

593.60 ± 10.61

Distilled water

555.83 ± 12.94

Millipore water

533.17 ± 11.86

1:0.7

591.49 ± 9.87

1:0.8 1:0.9

620.75 ± 11.79 675.05 ± 11.58

1:1.0

764.81 ± 10.55

1:1.1

799.52 ± 11.71

1:1.2

851.40 ± 12.58

1:1.3

860.72 ± 10.99

1:1.4

863.88 ± 9.39

1:1.5

865.75 ± 10.26

5 10

631.98 ± 10.38 824.54 ± 12.54

15

944.57 ± 9.89

20

926.36 ± 11.23

25

919.78 ± 10.75

48

965.22 ± 12.97

72

1052.34 ± 13.88

96

1132.72 ± 10.64

120 144

1135.64 ± 11.49 1140.40 ± 10.66

Optimization of Fermentation Medium Five compositions of media using wheat bran and rice bran were studied to identify the suitable combination. No other carbon and nitrogen sources were supplemented to the fermentation medium in order to reduce the cost of enzyme production. Thus, the wheat bran, rice bran, and rice flour acted as the sole source of carbon and nitrogen in the fermentation medium for enzyme production. The protease production was enhanced in presence of 25% rice bran along with 75% wheat bran (1193.77 U/g) (Table 2). The activity obtained using rice

Appl Biochem Biotechnol Table 2 Optimization of wheat and rice bran concentration for maximum protease production SSF medium


Rice bran

Wheat bran

Rice flour

(%)

(%)

(%)

100

0

5

739.25 ± 11.3

75

25

5

871.25 ± 13.56

50

50

5

25

75

5

1193.77 ± 9.71

0

100

5

1125.15 ± 11.89

947.45 ± 10.55

bran as sole source of enzyme production was very low (740 U/g) which was due to the tight packing of rice bran particles when moistened with water resulting in anaerobic conditions. In addition to this, the higher fat content (18–23%) of the rice bran compared to that of wheat bran (4–5%) had a negative impact on protease production [22]. In case of wheat bran, the optimum particle size of 1 mm helped in maintaining the porosity of the fermentation medium [15], which in turn, increased the protease enzyme production even as sole fermentation medium which is evident from its protease activity of 1125.15 U/g. The porosity and the fat content of the wheat bran make it a unique solid substrate for the enzyme production under SSF. Wheat bran showed highest protease activity among other solid substrates such as soy cake, coconut cake, and rice bran when B. subtilis strain was used for SSF production. [23]

Effect of Salt Concentration and Surfactants on the Activity of the Enzyme The activity of the enzyme increased in the presence of NaCl concentration of up to 1.2 M. Interestingly, it was found that there was 22% increase in the activity in the presence 1.0 M NaCl. The enzyme also showed increased activity in the presence of surfactants (0.1%) Tween 80, CTAB, and MS Powder, while SDS and triton X-100 affected the protease activity in negative manner (Table 3). The activity of the enzyme with no NaCl and no surfactant was taken as 0% (base value). This shows that the enzyme was tolerant towards the presence of salts and surfactants making it more suitable for use in detergent and leather industries.

Standardization of Unhairing Process Parameters The case in solution was prepared in different buffer solutions of pH ranging from 5.0 to 10.0. The enzyme (extracted from fermented bran in Tris buffer as mentioned in the BDetermination of Protease Activity^ section) exhibited better activity at pH range of 7.0 to 10.0 with maximum activity of 1182.97 U/g at pH 9.0 (Table 4). Thus, the unhairing process pH was standardized as 9.0. A set of 15 experiments was conducted in which the parameters like enzyme offer (10 to 30%), Hide-Float ratio (20 to 60%), and drum rotation speed (4 to 6 rpm) of the process were varied, and its effect on unhairing process was studied (Table 5). Inadequate hair removal was observed throughout the hide in trial 1, while the hair removal was improved in trial 2 with inadequate hair removal only at neck and backbone regions of the cowhide. Complete hair removal was observed in trial 3 after 8 h of unhairing process. Though the complete hair removal in trial 4 and trial 5 was observed after 7 h of unhairing process, microabrasions were formed on the grain surface after 6 h. Thus, an

Appl Biochem Biotechnol Table 3 Effect of presence of NaCl and surfactants on protease enzyme activity NaCl concentration (M)


Percentage increase or decrease in protease activity

0 (control)

475.36 ± 12.76

0%

0.2

492.06 ± 11.87

+3%

0.4

503.45 ± 13.01

+6%

0.6

530.89 ± 11.52

+11%

0.8

551.83 ± 11.85

+16%

1

581.73 ± 10.98

+22%

1.2

541.49 ± 12.32

+14%

Surfactant (0.1%) Control

473.13 ± 10.67

0%

SDS

464.11 ± 11.03

−2%

Triton X-100

449.27 ± 11.15

−5%

Tween 80

513.55 ± 9.97

CTAB MS Powder

+9%

555.3 ± 10.53

+17%

497.59 ± 12.94

+5%

optimum enzyme offer level was fixed at 20%. Further, to reduce the process time, the effect of other process parameters was studied. Trial 6 and trial 7 had the same results as trial 2 after 3 h of unhairing process with excess microabrasions after 4 h. This is due to the fact that the reduction in Hide-Float ratio significantly increased the enzyme concentration of unhairing process liquor leading to excess damage of skin protein. Also, the reduction in Hide-Float ratio caused excessive impact on the grain surface due to the mechanical agitation of drum. Trial 8 overcame these shortcomings, where the Hide-Float ratio was at an optimum level of 40%. Complete hair removal was observed after 6 h of unhairing process. Thus, the process time was reduced from 8 h in trial 3 to 6 h in trial 8, which is equivalent to 25% reduction in process time. Trial 11 to trial 15 was conducted to find out the optimum drum rotation speed, in which the unhairing process can be carried out without affecting the grain surface. Trial 11 showed complete hair removal after 9 h of processing, as there was not much of mechanical agitation, while trial 15 showed complete hair removal in 5 h with microabrasions. Trial 12, trial 13, and trial 14 (trial 8) showed complete hair removal in 6 h without any damage to the grain surface. Thus, the SSF enzyme-based unhairing process parameters were optimized as 20% enzyme offer with 40% Hide-Float ratio and drum rotation speed of 5 ± 1 rpm at pH 9.0 for complete unhairing process in 6 h. Table 4 Effect of different pH on the protease activity of the enzyme pH of the casein solution in protease assay


5.0

795.15 ± 10.31

6.0

931.86 ± 9.55

7.0 8.0

1056 .72 ± 12.18 1132.43 ± 10.65

9.0

1182.97 ± 10.97

10.0

1096.24 ± 11.86

Appl Biochem Biotechnol Table 5 Standardization of SSF enzyme-based unhairing process parameters Process parameter

Variation of process parameter

Process conditions

Enzyme offer

HideFloat ratio (% w/w) (% w/ w) Enzyme offer 10 (% w/w) 15

Hide-Float ratio (% w/w)

Drum rotation speed (rpm)

a

Experiment legend Drum rotation speed

Extent of hair removala

Impact on grain surfaceb

pH

(rpm)

–

60

6

9.0 Trial 1

3

1

–

60

6

9.0 Trial 2

5

1

20

–

60

6

9.0 Trial 3

7

2

25

–

60

6

9.0 Trial 4

9

4

30 20

– 20

60 –

6 6

9.0 Trial 5 9.0 Trial 6

9 7

5 8

30

20

–

6

9.0 Trial 7

7

6

40 50

20 20

– –

6 6


10 9

3 2

60

20

–

6

7

2

3

20

40

–

9.0 Trial 10 (trial 3) 9.0 Trial 11

7

1

4 5

20 20

40 40

– –


9 10

2 2

6

20

40

–

9.0 Trial 14 (trial 8)

10

3

7

20

40

–

9.0 Trial 15

9

6

Unhairing index 1–10. Higher unhairing index denotes better unhairing with respect to time

b

Degree of grain pitting 1–10. Higher degree denotes major impact on grain surface with respect to time leading to grain pitting phenomenon

Further Insight into Enzymatic Unhairing Process The rate of release of enzyme into solution is another important aspect as the whole fermented medium was used for unhairing. During unhairing, the enzyme needs to be released into solution and then it needs to penetrate into the hide network. The penetrated enzyme then reacts with proteoglycan of the hair bulb and disintegrates the latter [24]. Since the concentration gradient initially was high, the leaching rate of protease from fermented bran was higher during the first 2 h. With progress in time, the enzyme-leaching rate attained a maximum saturation level. The enzyme leaching followed a polynomial trend of Y = −2E−05X2 + 0.0115X + 4.4265 with R2 value of 0.9741, where X = time in min and Y = protein content in milligrams per milliliter. The enzyme release studies showed that the total time of 210 min was required for the complete release (Fig. 1). This regulated release of enzyme ensured the availability of optimal quantity of enzyme for unhairing process, and therefore, the action of enzyme on substrate was not drastic. This facilitates lowering of criticality of process control. Accordingly, the initial loosening of hair was observed after 4 h, and the complete unhairing was observed after 6 h (Fig. 2) with trial 14. This shows the efficacy of the enzyme for unhairing process. It is posited that the problem of grain-pitting, i.e., microabrasions on the grain surface (Fig. 3) that

Appl Biochem Biotechnol Fig. 1 Enzyme leaching profile from fermented bran into unhairing process liquor

was observed during enzymatic unhairing, was due to the excessive loss of PG and collagen leading to the weakening of grain–corium junction. As the grain–corium junction weakens, the hide surface becomes more susceptible to the mechanical agitation caused by the rotating drum. The scanning electron microscope (SEM) micrographs revealed some key information at this juncture. The impact of mechanical agitation on the surface of grain-pitted hide (Fig. 4a) and normal hide (Fig. 4b) was visualized with the help of SEM micrographs. The weakening of the grain–corium junction was verified by comparing the SEM images of cross section of grain-pitted hide and normal hide. Figure 4c shows more opened up structure with void spaces in comparison to that of Fig. 4d. This proved the loss in compactness of the fiber structure of the grain-pitted hide at the grain–corium junction. Thus, to avoid this problem, the drum was run at low rpm. In addition to this, it was found that the hair that comes out of the hide gets adhered to the flesh side of hide (Fig. 5). This phenomenon stops further penetration of enzyme from flesh side, and thus, the penetration of enzymes, thereafter, was predominantly from the grain side. Thus, the excess PGs released are from the grain side of the hide, which leads to the weakening of the grain–corium junction. With trial 14, under the optimized process conditions, there was no visible pitting mark up to 8 h. This was due to

Fig. 2 SSF enzyme-based unhairing process. a Soaked cowhide and b unhaired cowhide

Appl Biochem Biotechnol Fig. 3 Pitting on the grain side of cowhides

the fact that the leaching out of enzymes from bran into water and the transport of enzyme into the hide take place in a controlled manner. Thus, the reduced loss of PG from the matrix resulted in safe grain without any pitting marks.

Cost of Enzyme Production at Lab-Scale Level The total cost involved in production of 5 kg enzyme at the lab scale was calculated. This included the cost of raw materials and the other factors like the power consumption by the equipment used during the enzyme production (Table 6). The total cost for producing 1000 kU of protease was calculated as 0.44 USD.

Analysis of the Cost Factors in Setting Up of SSF Plant An inventory with required equipment and its quantity was prepared based on the process flow as described in Fig. 6. The initial pre-inoculum of 150 mL of B. luteolum (MTCC 5982) in shake flask, which was then transferred to various microbial fermenters of volume 3, 30, and 300 L with approximately 70–75% working volume, respectively. The total equipment cost was calculated to be 61,286.15 as explained in Table 7. In Table 8, the direct and indirect costs based on the total equipment cost were calculated as proposed by Peters et al. [25]. The total direct cost and the total indirect cost were found to be 123,798.02 USD and 77,220.54 USD. As explained in the BTechnoeconomic Analysis^ section, the FCI was calculated by summing up the total equipment cost, direct and indirect costs as 262,304.72 USD. The WCC is total money required for the initiation of the production process. It was assumed as 75% of the total equipment cost (or 15% of TCC), and it was 45,964.61 USD. The TCC was found to be 308,269.33 USD, which is obtained by adding the FCI and WCC. The CRM required for running a single batch of production process was recorded, and the cost was extrapolated for the total raw material cost required for a year of enzyme production. The total time for running a single batch of production was calculated to be 174 h (7.25 days) excluding the time involved in packing the enzyme after shade drying. Total yield of 850 kg dry SSF enzyme was obtained with a single batch of production taking the moisture content at the completion of fermentation as approximately 50%. The total time for one batch of SSF enzyme production, drying, and packing was taken approximately as 10 days. Thus, totally, 3 batches a month and 36


Fig. 4 SEM micrographs showing a surface view of unhaired cowhide with grain pitting, b surface view of unhaired cowhide without grain-pitting, c cross-section view of unhaired cowhide with grain pitting, and d crosssection view of unhaired cowhide without grain-pitting

batches a year with a production capacity of 30,600 kg of SSF enzyme per year were made possible with no break time in production process. The COL was taken as 20,000 USD/annum for six labors, an engineer and a lab assistant. The CMR was calculated as 2623.05 USD/annum (1% of FCI), and CL was calculated as 1600 USD/annum (8% of COL) as reported by Han et al. [26]. The total production cost (TPC) was calculated by summation of CRM, COL, CMR, and CL as 48,515.37 USD/annum, which is just 2550.76 USD/annum lesser than that of the assumed WCC showing the better fitting of the cost estimate model. The APC was found to be 50,941.14 USD after the addition of CO. From these values, the cost of 1 kg of SSF enzyme was found to 1.66 USD (Table 9). Due to non-availability of commercial SSF enzyme (as fermented bran itself), the unit cost could not be compared with any other standard enzymes for depicting the efficiency of the proposed method. The cost of SSF enzyme per kilogram was taken as 3 USD. The annual revenue (AR) generated from SSF enzyme production was calculated by multiplying the total production capacity with the unit

Appl Biochem Biotechnol Fig. 5 Hair adhered to the flesh side of cowhide during unhairing process

cost of the product, which was found to be 91,800 USD. Thus, the total annual profit (TAP) was found to be 40,858.86 USD (AR–APC). The return on investment (ROI) was calculated as suggested by Vlysidis et al. [27]. %ROI was calculated by dividing the TAP by FCI. The ROI was found to be 15.58% in 1 year. Thus, the breakeven on the FCI can be achieved in 6.4 years. Higher ROI proved that the proposed project is economically feasible. Similar studies were conducted in the production of enzymes such as glucoamylase and protease under SSF using wheat flour hydrolysate as substrate by fungi (Aspergillus awamori and A. oryzae) [28, 29]. The obtained enzymes were used to hydrolyze ground down food waste in a bioreactor to produce food waste Table 6 Cost of SSF enzyme production of 5 kg at lab scale Steps

Power Cost of raw consumption material/cost of power consumption

Quantity of raw material required for 5 kg enzyme production

Cost of raw material required for 5 kg enzyme production

–

74.72 USD/kg

0.01 kg

0.75 USD

–

0.03 USD/L

0.75 L

0.02 USD

3 kwh Media Sterilization

0.09 USD/kwh

–

0.27 USD

Incubator shaker

0.09 USD/kwh

–

0.27 USD

Wheat bran –

0.16 USD/kg

3.75 kg

0.60 USD

Rice bran

–

0.19 USD/kg

1.25 kg

0.24 USD

Rice flour

–

0.27 USD/kg

0.25 kg

Tap water

–

0.22 USD/1000 L 6.5 L

Incubator

4.8 kwh

0.09 USD/kwh

Raw materials/ equipment

Pre-inoculum Nutrient preparation broth Distilled water

SSF enzyme production

3 kwh

–

0.07 USD Very negligible 0.43 USD

Total cost for producing 5 kg wet enzyme Total cost for producing 5 kg dry enzyme (moisture ≈ 50%)

2.65 USD 5.3 USD

Total protease units (≈1200 U/g)

6,000,000 U

Total cost for producing 1000 kU of protease

0.44 USD


Fig. 6 Process flow of the SSF enzyme production plant. [The letters in green represent the equipment label, and the time taken for each step is marked in red] (color figure online)

hydrolysate, which was rich in glucose and free amino nitrogen. Then, the produced food waste hydrolysate was used as substrate for biofuels (such as hydrogen) production. The technoeconomic feasibility of the proposed bioprocess has also been evaluated (2016) showing a ROI of 26.75% with a payback period of 5 years [26].

Conclusions The strain B. luteolum (MTCC 5982) exhibited proteolytic capability. The protease production was maximum at pH 9.0 and temperature 33 °C. The combination of rice bran and wheat bran of weight ratio 1:3 along with 5% rice flour was found to be better for protease production. Effect of kind of water for fermentation on protease activity was studied. It was observed that tap water resulted in better activity than distilled water and Millipore water. Study on effect of moisture content during fermentation indicated that the ratio of moisture 1: 1.3 (Wheat bran to water) was found to be better. The inoculum size of 15% was observed to be better. Fermentation time of 96 h yielded maximum protease activity. Unhairing process parameters using SSF enzyme were standardized as 20% enzyme offer with 40% Hide-Float ratio and drum rotation speed of 5 ± 1 rpm at pH 9.0. The extractability of protein from the fermented bran into unhairing process liquor was studied. It was Table 7 Purchase cost of equipment needed for the setting up of a SSF enzyme production plant Equipment

Volume

Quantity

(L)

Cost (USD)

Incubator Shaker (S1)

35

1

Pre-inoculum fermenter (F1)

3

1

400 2200.70


30

1

7335.68


300

1

22,007.04

Tray type solid-state fermenter (F4)

11,500

1

29,342.72

Total equipment purchase cost

61,286.15

29 12 55

Buildings (including services)

Yard improvements

Service facilities (installed)

4 19 37 126 328 100 428 75 503

Legal expenses

Contractor’s fee

Contingency

Total indirect costs

Direct costs + indirect costs

Equipment purchase cost

Fixed capital investment (FCI) = (direct costs + indirect costs + equipment cost) Working capital cost (WCC)

Total capital cost = FCI + WCC

32 34

Engineering and Supervision Construction expenses

Indirect costs

302

10

Electrical systems (installed)

Total direct costs

39 26 31

Instrumentation and controls (installed) Piping (installed)

308,269.33

262,304.72 45,964.61

61,286.15

201,018.57

77,220.54

22,675.87

11,644.36

2451.44

19,611.56 20,837.29

123,798.02

33,707.38

7354.33

17,772.98

6128.61

15,934.39 18,998.70

23,901.59

61,286.15

USD

% 100

SSF enzyme production plant

Percent of delivered equipment cost for

Purchased equipment installation

Direct costs

Purchased equipment delivered (including fabricated equipment, process machinery, pumps, and compressors)

Total capital cost

Table 8 Calculation of total capital cost for the analysis of techno-economic feasibility


Appl Biochem Biotechnol Table 9 Calculation of the annual production cost and the unit production cost of the proposed SSF enzyme production plant Material/service

Quantity/ batch

Unit cost

Batch cost

Monthly cost (3 Annual cost (36 batches) batches)

kg

USD

USD/ batch

USD/month

204

612

Raw material Wheat bran

1275 0.16/kg

Rice bran

425 0.19/kg

Rice Flour

85 0.27/kg

Pre-inoculum Nutrient broth Distilled water

3.4 74.72/kg 255 L 0.03/L

Tap water Utility Electricity

2040 L 0.22/1000 L 1166 kwh 0.09/kwh

USD/annum

7344

80.75 242.25

2907

22.95

68.85

826.2

254.04 762.14

9145.72

7.65

22.95

0.44

1.34

16.15

104.94 314.82

3777.84

Operating labor Maintenance and repair (1% of FCI) Laboratory charges (8% of operating labor) Total production cost (TPC) Other cost (5% of TPC) Annual production cost (TPC + other cost) Total production per year in kg = [850 (per batch) × 3 (per month) × 12 (per year)] Unit production cost = (annual production cost / total production per year)

275.4

20,000 2623.05 1600 48,515.37 2425.76 50,941.14 30,600 1.66 USD/kg

observed that the maximum release of protein was found to be at 210 min. Concluding, dispensation of downstreaming process reduced the cost of enzyme. The total cost of producing 5 kg dry enzyme at lab scale was calculated as 5.3 USD. This, along with the cost of enzyme-based unhairing wastewater treatment, will substantially lower the cost involved in unhairing process. The technoeconomic feasibility studies for setting up a SSF enzyme production plant showed a high ROI of 15.58% with a payback period of 6.4 years. The regulated release of enzyme into solution favors moderate reaction between enzyme and substrate. Therefore, there is no necessity for stringent process control. From this study, it clearly showed that the use of SSF enzyme could address the two shortcomings of the enzyme-based unhairing system successfully. Acknowledgements This work was carried out by the first author for the partial fulfillment of M.S. (By research) degree under the work titled BDevelopment of Efficacious Unhairing Enzyme.^ The author also acknowledges ZERIS (CSC 0103) for providing funds for carrying out this work.

References 1. Nancy, G., Prakram, S. C., Vivek, K., Neena, P., & Naveen, G. (2014). Approach to ecofriendly leather: characterization and application of an alkaline protease for chemical free dehairing of skins and hides at pilot scale. Journal of Cleaner Production, 79, 249–257. 2. Rohm, O. (1910). Dehairing and cleaning of skins. German Patent, 268, 837. 3. Thanikaivelan, P., Rao, J. R., & Nair, B. U. (2000). Development of a leather processing method in narrow pH profile: part 1. Standardisation of dehairing process. Journal of the Society of Leather Technologists and Chemists, 84, 276–284.

Appl Biochem Biotechnol 4. Dettmer, A., Cavalheiro, J. C., Cavalli, E., Rossi, D. M., Gusatti, C. D. S., Zachia Ayub, M. A., & Gutterres, M. (2012). Optimization of the biotechnological process for hide unhairing in substitution of toxic sulfides. Chemical Engineering and Technology, 35, 803–810. 5. Saravanan, P., Shiny Renitha, T., Gowthaman, M. K., & Kamini, N. R. (2014). Understanding the chemical free enzyme based cleaner unhairing process in leather manufacturing. Journal of Cleaner Production, 79, 258–264. 6. Chellapandi, P. (2010). Production and preliminary characterization of alkaline protease from Aspergillus flavus and Aspergillus terreus. E-Journal of Chemistry, 7, 479–482. 7. Harish, B. K., Sneha, V. M., Kalal, K. M., & Seeta Laxman, R. (2015). Eco-friendly enzymatic dehairing of skins and hides by C. brefeldianus protease. Clean Technologies and Enviromental Policy, 17, 393–405. 8. Gillespie, J. M. (1953). The depilation of sheepskins with enzymes. Journal of the Society of Leather Technologists and Chemists, 37, 344–353. 9. Cross, T. (1968). Thermophilic actinomycetes. Applied Bacteriology, 31, 36–53. 10. Chandrasekaran, S., & Dhar, S. C. (1983). A low cost method for the production of extracellular alkaline proteinase using tapioca starch. Journal of Fermentation Technology, 61, 511–514. 11. Arunachalam, C., & Saritha, K. (2009). Protease enzyme an eco-friendly alternative for leather industry. Indian Journal of Science and Technology, 2, 29–32. 12. Gehring, A. (2002). Unhairing with proteolytic enzymes derived from Streptomyces griseus. Journal of the American Leather Chemists Association., 91, 406–411. 13. Mitra, P., & Chakrabartty, P. K. (2005). An extracellular protease with depilation activity from Streptomyces nogalator. Journal of Scientific and Industrial Research, 64, 978–983. 14. Muthulakshmi, C., Gomathi, D., Kumar, D. G., Ravikumar, G., Kalaiselvi, M., & Uma, C. (2011). Production, purification and characterization of protease by Aspergillus flavus under solid state fermentation. Jordan Journal of Biological Sciences, 4, 137–148. 15. Jarun, C., Sinsupha, C., Yusuf, C., & Penjit, S. (2008). Protease production by Aspergillus oryzae in solid state fermentation using agro industrial waste. Journal of Chemical Technology and Biotechnology, 83, 1012–1018. 16. Cupp-Enyard, C. (2008). Sigma’s non-specific protease activity assay—casein as a substrate. Journal of Visualized Experiments. doi:10.3791/899. 17. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275. 18. Shivasharana, C. T., & Naik, G. R. (2012). Ecofriendly applications of thermostable alkaline protease produced from a Bacillus sp. JB-99 under solid state fermentation. International Journal of Environmental Sciences, 3, 956–964. 19. Rajkumar, R., Jayappriyan, K. R., & Rengasamy, R. (2011). Production and characterization of a novel protease from Bacillus sp. RRM1 under solid state fermentation. Journal of Microbiology and Biotechnology, 21, 627–636. 20. Divakar, G., Sunitha, M., Vasu, P., Udaya Shanker, P., & Ellaiah, P. (2006). Optimization of process parameters for alkaline protease production under solid-state fermentation by Thermoactinomyces thalophilus PEE 14. Indian Journal of Biotechnology, 5, 80–83. 21. Vijayaraghavan, P., Vijayan, A., Arun, A., Jenisha, J. K., & Vincent, S. G. P. (2012). Cow dung: a potential biomass substrate for the production of detergent-stable dehairing protease by alkaliphilic Bacillus subtilis strain VV. SpringerPlus, 1, 76. 22. Kaur, G., Sharma, S., Nagi, H. P. S., & Dhar, B. N. (2012). Functional properties of pasta enriched with variable cereal brans. Journal of Food Science and Technology, 49, 467–474. 23. Prabhavathy, G., Rajasekara Pandian, M., & Senthilkumar, B. (2012). Optimization and production of extracellular alkaline protease by solid state fermentation using Bacillus subtilis. Journal of Academic and Industrial Research, 17, 427–430. 24. Sivasubramanian, S., Murali Manohar, B., & Puvanakrishnan, R. (2008). Mechanism of enzymatic dehairing of skins using a bacterial alkaline protease. Chemosphere, 70, 1025–1034. 25. Peters, M., Timmerhaus, K., & West, R. (2003). Plant design and economics for chemical engineers. Boston: McGraw-Hill. 26. Han, W., Fang, J., Liu, Z. X., & Tang, J. H. (2016). Techno-economic evaluation of a combined bioprocess for fermentative hydrogen production from food waste. Bioresource Technology, 202, 107–112. 27. Vlysidis, A., Binns, M., Webb, C., & Theodoropoulos, C. (2011). A techno-economic analysis of biodiesel biorefineries: assessment of integrated designs for the co-production of fuels and chemicals. Energy, 36, 4671–4683. 28. Han, W., Wang, X. N., & Ye, L. (2015a). Fermentative hydrogen production using wheat flour hydrolysate by mixed culture. International Journal of Hydrogen Energy, 40, 4474–4480. 29. Han, W., Ye, M., & Zhu, A. J. (2015b). Batch dark fermentation from enzymatic hydrolyzed food waste for hydrogen production. Bioresource Technology, 191, 24–29.