Introduction

Cellulose and keratin are the two most abundant biopolymers in nature, providing structural stability to living creatures. While origin of cellulose is mainly from plants, keratin is of animal origin. Cellulose, a linear polysaccharide having thousands of β-d-anhydroglucopyranose units linked by β(1,4)-glycosidic bonds, is chiral and insoluble in water, whereas keratin is a fibrous and recalcitrant protein constituting major structural component of feather, horns, hooves, skin, hair, etc [1]. Common protease cannot degrade keratin due to its intense cross-linking of cysteine disulfide bonds, hydrogen bonds and hydrophobic interactions [2]. However, being highly abundant components, accumulation of cellulose and keratin-based organic wastes is common due to natural and anthropogenic activities. Pollution and related health problems are thus apparent in the areas like feather dumping sites (slaughter houses). Further, urban garbage dumping grounds receive a mixture of both cellulose- and keratin-containing wastes. Therefore, microbial application to biodegrade such composite material through microbial enzymes is a challenge to the scientific community as typically same bacteria does not act upon both the polymers. Physical separation is a cumbersome task for the cellulose–keratin mixed wastes and conventional methods for degrading them involve combustion and chemical treatments [3]. Approximately 20% of total insects harbor intracellular endosymbiotic bacteria in their gut, and in turn, these endosymbionts help in the digestion of food and supply essential nutrients to their host [4]. Both cellulose- and keratin-degrading bacteria are reported to be present naturally in the gut of different organisms [5, 6]. According to the feeding habits, gut of different insects like moths (Tineidae), chewing lice (Mallophaga), Dermestidae trogidae and Scarabaeidae beetles harbor keratin-degrading bacteria [7]. Furthermore, cockroaches (Periplaneta sp. order: Blattaria or Blattodea) are significant omnivores and have diverse hindgut microbiota encompassing hundreds of microbial species. [8, 9]. Moreover, due to its unique niche, the gut of cockroach is likely to be the habitat for unique bacteria, degrading different substrates like cellulose and keratin. The present study deals with the screening and characterization of bacteria collected from the gut of cockroach inhabiting a feather dumping site at Tezpur area, Assam, India. Associated soil samples were also collected from the same feather dumping site where cockroaches were collected, to assess the possibility of finding bacteria having dual nature of both cellulose and keratin degradation properties. All the strains were assessed for their caseinase and keratinase activities and filter paper assay (for total cellulase activity and endoglucanase activity). Additionally, dehairing properties of keratinase positive isolates were also studied. The present work will be helpful in developing microbial enzyme-based ecofriendly strategy for management of complex urban waste.

Material and Methods

Sample Collection and Isolation of Bacteria from Collected Sample

Soil samples from feather dumping site (slaughter houses) at Tezpur, Assam (26°39′4.3848″N and 92°47′1.7268″E), were collected from the top surface (up to 3 cm) having feather waste. Simultaneously, cockroach specimens (Periplaneta americana) from the same site were collected in live condition. Subsequently, insect specimens were knocked out by chloroform and surface-sterilized by ethanol, gut was dissected out, and isolation of culturable bacteria was done according to method described previously [10, 11]. On the other hand, soil sample (1.0 g) was suspended in 9 ml sterilized sterile PBS, and culturable bacterial colonies were isolated in nutrient agar [12].

Protease Activity and Caseinase Activity (Primary Screening)

Protease-secreting bacteria were screened by using skim milk agar medium (HiMedia, M530-500G) from collected samples. The index of hydrolysis of clear zone was recorded and calculated as ratio of diameter of clearing zone and colony [11, 12]. In addition, caseinase activity was determined by modified protocol of El-Ayouty et al. [13]. Further, the amino acid released due to breakdown of casein was estimated by the method of Lowry et al. [14] with bovine serum albumin as a standard. One unit of caseinase activity was defined as the amount of enzyme that liberates 1 µM of tyrosine per hour under optimal assay conditions.

Secondary Screening of Keratinolytic Bacteria and Keratinase Estimation

Proteolytic activity positive isolates were then secondary-screened for keratinolytic activity. For this, feathers were collected from dumping site and processed according to Sekar et al. [12]. Bacterial preparations (2 × 107 cells/ml) were incubated with single feather (carbon and nitrogen source for keratinase production) in test tubes containing 10 ml of minimal media (gm/l) modified basal salt medium according to Burtt and Ichida [15], at 37 °C on a rotary shaker at 160 rpm for 7 days. Degradation of feather was examined visually. After incubation, enzyme extraction was performed by centrifugation of the media at 2000 g for 20 min at 4 °C and the supernatant was used to estimate keratinase activity. For keratinase estimation, keratin powder was used according to Mazotto et al. [16]. One unit (U/ml) of keratinase activity was defined as the amount of enzyme required to produce an absorbance increase of 0.01 under the described assay conditions.

Determination of Feather Degradation

For feather degradation assay, chicken feather was washed with distilled water, surface-sterilized with 70% ethanol and rinsed twice with distilled water, followed by drying at 45 °C for 24 h in circulating hot air oven. Feather samples were then incubated with isolates (2 × 107 cells/ml) at 37 °C. After 7 days, the total culture was filtered through Whatman No. 3 filter paper, washed twice with distilled water and dried to a constant weight at 105 °C. The percentage degradation was calculated according to the method described in Kani et al. [17].

Dehairing Function of Keratinolytic Positive Isolate

Dehairing capability of the bacterial isolates was performed according to Jaouadi et al. [3] with slight modification. Sterilized bovine, goat and rat skin samples were inoculated with 2 × 107 cells/ml in modified basal salt medium containing: (g/l) 0.25, Na2HPO4; 0.5, NaH2PO4; 0.5, NaCl; 0.01, MnSO4; 0.01, FeSO4; 0.5, MgSO4; 2.0, gelatine; and 1 l distilled water, pH adjusted to 7.0), and the results were recorded according to Jaouadi et al. [3].

Screening of Cellulose-Degrading Bacteria (CMCase Activity Assay)

Qualitative estimation for cellulase positive bacterial isolates was conducted using carboxymethyl cellulose media (CMC) plates, according to Sharma et al. [10]. Isolates capable of degrading CMC were indicated by a clear zone on CMC plates, and the enzyme activity was indexed by measuring diameter of clear zone. Further, hydrolysis activities (HC) of the positive isolates were calculated according to Hendricks et al. [18].

Filter Paper Assay (Total Cellulase) Activity and Endoglucanase Activity

The cellulase activity was estimated according to protocol described by International Union of Pure and Applied Chemistry (IUPAC) [19]. Cellulase activity was expressed in filter paper unit (FPU) per ml of undiluted culture filtrate. One FPU is defined as the quantity (in mg) of reducing sugar liberated in one hour by undiluted enzyme under the standard assay conditions and calculated according to Ghose [19]. Endoglucanase (ß 1-4 endoglucanase-EC 3.2.1.4) activity was assayed by measuring the amount of reducing sugar from CMC. Enzyme activity was estimated in the cellulase positive isolates according to Ghose [19]. One unit of enzymatic activity is defined as the amount of enzyme that releases 1 µmol reducing sugars (measured as glucose) per ml per minute.

Carbohydrate Utilization Pattern

Carbohydrate utilization pattern was performed using the HiCarbo test kit (HiMedia, India). Briefly, 50 µl of freshly prepared culture was added to the test modules, followed by incubation at 37 °C for 24 h, and interpretation of results was done on the basis of color change, as described in the manufacturer’s instruction.

Amplification and Identification of Bacterial Isolates by 16S rRNA Gene Analysis

Total bacterial genomic DNA from each positive isolate was extracted and 16S rRNA genetic region was amplified using universal primers—16S1 5′-gagtttgatcctggctca-3′ (forward) and 16S2 5′-cggctaccttgttacgactt-3′ (reverse) [20]. Amplicons were purified (Sigma-Aldrich, MO, USA) and sent for sequencing in a commercial facility (SciGenom Labs, Kochi, India). The obtained sequences were submitted to the NCBI GenBank and are available under the accession nos. KY619843 through KY619856.

Results and Discussion

A total of 31 culturable bacterial isolates from feather dumping site and cockroach gut were screened for the dual characteristics of keratinase and cellulase production. Out of the studied isolates, 14 showed proteolytic activity on skimmed milk agar. Isolate numbers 4K, 7K, 9K, 11K, 12K, 13K, 14K, 16K, 22K, 24K, 28K, 29K, 30K, 31K showed breakdown of casein into constituent peptides and amino acids in the presence of exoenzyme caseinase, as evident by disappearance of the white color of skim milk media (Fig. S1 and Fig. S2). Varying levels of caseinase activity were exhibited by different isolates, ranging from 2.05 U/ml (isolate 9K) to 6.06 U/ml (isolate 29K) (Fig. 1). Similarly, primary screening of keratin-degrading bacteria of Bacillus species (Bacillus megaterium F7-1 and Bacillus sp. CL33A) was carried out on skim milk agar plates [21, 22]. Besides, caseinase positive isolates were analyzed for keratin degradation by keratinase production and proteolytic activity on feather degradation. Keratinase activity was estimated in different isolates, and the highest activity (201.4 U/ml) was documented in isolate 29K from cockroach gut, 197 U/ml in isolate 13K from soil and 171.1 U/ml in isolate from soil 9K (Fig. 1). Feather degradation potential was also analyzed by calculating percent degradation by different isolates. Nearly complete degradation of feather was visible in 7 days (Figs. 2, 3), and maximum degradation efficiency was reported in isolate 29K with 73.4% degradation; however, the feather, barbules and rachises were found completely degraded and settled at the bottom (Fig. 3). Similar keratinolytic activity in bacterial isolates from feather dumping sites has been reported earlier [12, 23]. Recently, researchers have reported feather degradation by Vibrio sp. Strain kr2 [23], Bacillus sp. Strain kr16 [24] and Chryseobacterium sp. Strain kr6 [25].

Fig. 1
figure 1

Caseinase activity and keratinase activity estimated in different isolates

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Fig. 2
figure 2

Percent degradation of feather by different keratinase positive isolates

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Fig. 3
figure 3

Feather degradation by keratinolytic bacteria after 7 days of incubation

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Based on the keratinase enzyme activity, degradation potential of 29K was evaluated using natural keratin source including bovine, goat and rat skin with hairs. Incubation with isolate 29K (Bacillus sp.) resulted in notable digestion of skin. After 24 h of incubation, dislodging of hairs from rat and goat skin was observed, whereas dehairing of bovine skin took 48 h (Fig. 4). Nevertheless, after 72 h of incubation, all the treatments resulted in the total digestion of skin with partial degradation of hairs. Comparatively similar result on dehairing capacity of B. pumilus has been reported by Hunag et al. [26]. Remarkably, 16S rRNA gene sequences revealed keratinase positive isolates as different species of bacteria belonging to the genus Bacillus. Interestingly, isolates 22K and 24K showed maximum sequence homology with Bacillus cereus strain G5 while isolates 30K and 31K with Bacillus kochii strain You8, although the enzyme activities were diverse.

Fig. 4
figure 4

Dehairing activity and degradation potential of 29K after 72 h. a Bovine skin. b  Goat skin. c  Rat skin

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In addition to keratin waste, cellulose waste equally creates aesthetic nuisance due to its slow degradation in nature [27]. To find an enzyme-based approach, keratinolytic positive isolates were further screened for cellulase production and among them 8 isolates (4K, 7K, 12K, 13K, 28K, 29K, 30K, 31K) were found positive on CMC media plates (Fig. S3). Maximum hydrolysis capacity was noted in isolate 29K (3.6 mm), whereas isolates 9K, 11K, 14K, 16K, 22K, 24K did not show notable activity (Figures S3, S4). For isolate 29K, enzyme activity for total cellulase activity was recorded to be 0.11 U/ml, while endoglucanase activity was assayed to be 0.39 U/ml (Fig. 5). Cellulolytic bacteria in our ecosystem play a significant role to degrade cellulose based waste [28]. The present observations are similar to a previous study reporting isolation of cellulolytic bacteria from termite mid-gut [10]. Carbohydrate utilization is one of the important metabolic activities attributed to the cellulase producing bacteria. In the present study, cellulase positive bacteria showed response to different carbohydrates, which are listed in Table 1. All the cellulase positive isolates shared similar biochemical traits for the utilization of dextrose, salicin, mannitol and esculin sugars; however, inability to utilize α-methyl-d-mannoside was observed in all the isolates. All were able to utilize d-cellobiose except 12K, to ferment glucose. Despite sharing common genus, the isolates have shown different biochemical profile. The differences in substrate utilization suggest species diversity among the same genus. Previous studies have shown that Bacillus species having potential hydrolytic activity with high degradation index isolated from different sources [3, 10, 11, 25, 29, 30].

Fig. 5
figure 5

Filter paper assay (total cellulase) activity and endoglucanase activity of cellulase positive isolates

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Table 1 Carbohydrate utilization pattern of cellulase positive isolates from soil of feather dumping site and cockroach from same site
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Conclusion

The present results show that the feather dumping site and cockroach gut are an excellent source of microbes having potent keratinase and cellulase activities, which can be exploited for various biotechnological applications. Remarkably, in this study, eight isolates from cockroach gut show positive keratinolytic activity, which corroborates with their extremely wide range of food habit, as well its adaptability in extreme environments. The present findings further demonstrate the catabolic activity of isolate 29K (Bacillus spp.) against keratin and cellulose and thus represent a potent candidate species for application in management of wastes disposed from slaughter houses and tanneries. In conclusion, formulation of different potential hydrolytic bacteria having cellulase and keratinase activities may provide an efficient ecofriendly solution to the global problem of waste management.