BIODEGRADATION OF FEATHER KERATIN BY BACILLUS CEREUS IN PURE CULTURE AND COMPOST

Anna Rodziewicz, Wojciech Łaba
Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

The cultures of Bacillus cereus B5esz strain in feather keratin-containing media showed, that proteinceaous substrate was gradually degraded and next used as a carbon and nitrogen source. This strain biosynthesised extracellular enzymes, such as: keratinases, proteases and others. As a result, increases in soluble protein and amino groups concentration were observed, together with the release of inorganic sulphur compounds and medium alkalinization. This coincided also with increasing concentrations of thiol compounds. An excess of cystine-derived sulphur was accumulated in inorganic sulphates (IV and VI) or thiosulphates. The investigated bacterial strain in twelve-day cultures decomposed 70 % of feather keratin. Inoculation of feather-containing compost with B. cereus, initially resulted in predominance of spore-forming and thermophilic bacteria. After six weeks of composting, yeast or filamentous fungi were eliminated. Bacterial inoculum slightly accelerated the mineralization of feathers and increased the fertilizer value of the compost. Electron microscopy visualization of feather keratin fibres after composting, showed enhanced degradation of the feather structures. The degradation of feather keratin structures was conspicuous both in the medium and compost. The feathers were covered with a complex microbial matrix.

Key words: Bacillus cereus, keratinolysis, feather keratin, composting.

INTRODUCTION

Bacteria of Bacillus cereus species possess high proteolytic potential, expressed by their ability to syntesise proteolytic and keratinolytic enzymes. This particular property can be used for degradation of highly stable fibrous proteins, e.g. keratins, primary constituents of poultry feathers. A family of keratins is the largest and most complex group of intermediate filaments (IFs). Inside epithelial cells of vertebrates, keratins form IFs, which are a part of a flexible cytoskeletal scaffold. Keratin monomers of types I and II assemble obligatorily into coiled-coil heterodimers. Two dimers aggregate into parallel tetramers and the latter associate into protofilaments by aligning in antiparallel way, thus creating a superhelical structure. These three-level structures are additionally stabilized by disulphide bridges of cystine [2,18]. Moreover, keratins of skin appendages are included into two-phase structures, where microfibrils of 8 nm in diameter are submerged in amorphic proteinceaous matrix. B. cereus species is commonly found in the environment. This spore-forming, mesophilic and relatively anaerobic rod is potent in terms of biosynthesis of various hydrolytic enzymes, including proteases, alpha- and beta-glucanases or lipases. Proteolytic enzymes containing serine proteinases [19], Ca²⁺- and Zn²⁺- dependent metaloproteinases [3], keratinases [18] and collagenases [10,25] are known to be produced by B. cereus. Of glucanohydrolases, beta-amylases, alpha-D-glucosidses, pullulanases, glucosylotransferases [3,15] and chitinases [24] are described. Due to the properties of B. cereus, especially the ability to form biofilm and synthesise extracellular hydrolases, this species can be applied in animal and plant wastes utilization [2]. The composition of those wastes is dominated by fibrilar proteins, represented by keratin (the major constituent of feathers), collagen and elastin (fibrilar proteins of dead chickens) and plant polysaccharides from straw and poultry litter.

The aim of the study was to evaluate the degree of feather keratin degradation in submerged cultures of B. cereus B5esz bacteria and in feather compost inoculated with a bacterial culture.

MATERIALS AND METHODS

The strain of B. cereus, designated as B5esz, was isolated from keratinous wastes in our earlier study [17]. Bacterial cultures were carried out in 250 cm³ Erlenmayer flasks, containing 50 cm³ of mineral medium, at 30-45°C and 170 rpm, for 4-15 days. Nutrient broth culture of absorbance A₅₅₀ = 0.2 served as inoculum, used in 1 cm³ volume per flask. Composition of the mineral medium (g·dm^-3): MgSO₄ (1.00), CaCl₂ (0.10), KH₂PO₄ (0.10), FeSO₄·7H₂O (0.01), supplemented with yeast extract (0.5); pH 7.0. The basic carbon and nitrogen source were uncut, defatted, white chicken feathers (10.0 gdm^-3). Optionally, the medium was enriched with beech xylan (10.0 g·dm^-3) or beet pulp (15.0 g·dm^-3) in order to induce xylanases.

Assays were done in culture medium after centrifugation at 10 000 g and 4°C. The soluble proteins released into the medium were tested using the Lowry’s method [9]. The amino groups release was measured using Snyder’s method [20]. The presence of thiol compounds was assayed according to the Ellman’s method [14]. Sulphates (VI) were determined according to Kolmert’s method [5], sulphates (IV) according to the method described by Pachmayer [4] and thiosulphates were assayed using the Sörbo’s method [21]. The dry matter of the residual feathers was determined after drying at 105°C. Keratinolytic activity was determined on soluble keratin preparation, at 40°C and pH 7.5. One unit of keratinolytic activity (KU) was defined as the 0.01 rise of absorbance of TCA-soluble products at 280 nm, per 1 cm³ of enzyme in 1 minute [11]. Proteolytic activity was determined using the modified Anson method with casein as a substrate, at 30°C and pH 7.5. One unit of proteolytic activity (PU) represented an absorbance increase of 0.01 per 1 cm³ of enzyme in 1 minute. Additional enzyme activities were also tested: 1) collagenases – in reaction with collagen at 35°C and pH 7.4; the unit was 1 micromole of L-hydroxyproline released by 1 cm³ of culture medium in 1 minute, assayed with a standard method: PN-ISO 3496:2000; 2) lipases – in reaction with olive oil at 37°C and pH 8.0; the unit was 1 micromole of NaOH used for neutralization of fatty acids released by 1 cm³ of culture medium in 1 hour [23]; 3) amylases – in reaction with starch at 30°C and pH 6.8; the unit was 1 micromole of reducing sugars released by 1 cm³ of culture medium in 1 minute, assayed with 3,5-dinitrosalicilic acid (DNS) [15]; 4) chitinases – in reaction with chitin at 40°C and pH 4.8; the unit was 1 micromole of N-acetylglucosamine released by 1 cm³ of culture medium in 1 minute, assayed with DNS [13]; 5) cellulases – in reaction with carboxymethylcellulose at 50°C and pH 4.8; the unit was 1 micromole of reducing sugars released by 1 cm³ of culture medium in 1 minute, assayed with DNS [12]; 6) xylanases – in reaction with xylan at 50°C and pH 4.8; the unit was 1 micromole of reducing sugars released by 1 cm³ of culture medium in 1 minute, assayed with DNS. All substrates were of Sigma brand.

Composts were prepared from chicken feathers (20 kg) and bovine manure (40 kg) in static glass reactors, aerated from below (8-10 dm³·min^-1). The compost was inoculated with 24 h culture of B. cereus B5esz obtained in 5 dm³ bioreactor, in mineral medium with feather keratin (28°C; 2 dm³·min^-1; 500 rpm; no pH regulation). The inoculum was added in portions of 2.5 dm³ at the beginning and 2.0 dm³ after 21 days of composting period. In the second stage (after 42 days) 3.5 dm³ of inoculum was added as a ripening agent of compost. The moisture content of composts was maintained at 50 %. The uninoculated compost was a control 1. The compost consisting of inoculated chicken feathers free of manure, was a control 2. The duration of composting was 42 days, with mixing of the compost material on days 19 and 29. The inner temperature and pH, as well as a profile of microflora inhabiting composts, were monitored throughout the whole process. On completion of composting the following assays were carried out: analysis of mineral compounds, after mineralization in a Mars 5 microwave mineralizer (CEM), nitrogen, carbon and sulphur, determined in CHNS EA-1110 elemental analyzer (CEInstruments), nitrate and ammonia nitrogen, assayed colorimetrically. The amount of mineral compounds introduced with inoculum, was taken into account.

Keratinic structure of feathers after degradation in submerged cultures and in compost was visualized by scanning electron microscopy, using a SEM LEO 435 VP microscope, at magnitude of 4000 X. Microbial counts were performed weekly throughout the composting process. 5 grams of compost sample was suspended in 45 cm³ of sterile saline and placed on a rotary shaker for 15 minutes. Afterwards, a series of decimal dilutions followed by inoculation onto solid selective media were made. The total number of bacteria (nutrient broth with glucose), sporulating bacteria (10 minutes of pre-incubation at 80°C), thermophilic bacteria (incubation at 45°C) and the total number of filamentous fungi and yeasts (LAB-agar; in g·dm^-3: yeast extract 5.0, glucose 20.0, chloramphenicol 12.0, agar 12.0) were quantified. Petri dishes with bacteria were incubated at 30°C and those with fungi and yeasts at 20°C. The results were given in cfu·g^-1 of a sample. All assays were carried out in triplicate.

RESULTS AND DISCUSSION

Degradation of feather keratin as an ingredient of culture medium
The bacterial strain of B. cereus B5esz in liquid culture in mineral medium with feather keratin entered a logarithmic growth phase after one day of adaptation. A stationary phase was reached after two days of culture. An increase in concentration of soluble proteins and free amino groups was observed in the logarithmic phase. During four-day culture, the concentration of soluble proteins and peptides reached 1.28 mg·cm^-3 and that of amino groups was 2.71 mM (Fig. 1). Starting from the second day, the activity of proteolytic and keratinolytic enzymes could be detected. The maximum activity of proteolytic enzymes was at the level of 400 PU and the activity of keratinases was at 9.8 KU (Fig. 2).

Keratinolytic microorganisms are capable of keratin decomposition into peptides and amino acids, further metabolized as a carbon and nitrogen source. Keratinases, which belong to endopeptidases of unknown mechanism of catalysis, together with less specific proteinases, are involved in this process. Participation of reductases or reducing agents in cleaving disulphide bridges of cystine to S-sulphocysteine and cysteine is also suggested [7,8].

The highest (1.40 mM) concentration of thiol groups was recorded on the fourth day (Fig. 3). The sequence of secreted metabolites confirms the assumption that cleavage of disulphide bonds is an essential process, parallel to proteolytic degradation [7,8]. Slow accumulation of inorganic sulphur compounds was also recorded from the beginning of the culture. The presence of sulphates (IV and VI) and thiosulphates was detected. Sulphates IV possibly played a role in sulphitolysis and their concentration was constantly rising, reaching a level of 0.8 mM on the fifteenth day. An excess of sulphur was excreted by bacterial cells in oxidized form of sulphates VI and was present at the maximum level of 2.6 mM, on the tenth day. Kunert [6,7] observed similar metabolites in cultures of filamentous fungus Microsporum gypseum and Streptomyces fradiae. According to Kunert’s theory, streptomyces remove an excess of sulphur in the form of thiosulophate. In cultures of the strain used in the present study, the proportions between individual sulphur compounds appeared to be different, mainly with a lower level of thiosulphates.

B. cereus, which is a mesophilic species, reaches its maximum growth rate at temperatures between 20 and 45°C. However, the optimum temperature for the synthesis of proteolytic and keratinolytic enzymes was 30°C (Fig. 4 A, B). Nevertheless, the highest release of proteins was observed at 45°C (unpublished data). The decrease in keratin content after twelve days of the culture performed at 30°C was 70 % (Fig. 5). Such a high metabolic activity of bacteria at relatively low temperature was likely to improve keratinolysis in compost conditions in the mesophilic phase.

Electron microscopy visualization of feather keratin fibres after submerged culture of B.cereus strain showed extensive degradation of feather structure in comparison to a control (Fig. 6). Chicken feathers underwent total fragmentation and were visibly covered with a complex microbial matrix.

The B. cereus strain used in the present study proved to be able to constitutively synthesise extracellular hydrolytic enzymes e.g. lipases, collagenases, alpha- and beta-glucanases and chitinases. The activity of lipases was 15 U, collagenases 0.64 U, amylases 2.9 U, cellulases 0.013, chitinases 0.85 U. Biosynthesis of xylanases was induced in the presence of beech xylan (0.13 U) and beet pulp (0.17 U) (Table 1). The enzymes secreted by B. cereus cells could take a part in antagonistic interactions with other microorganisms and cooperate in decomposition of biopolymers in the compost material.

Biodegradation of feather keratin during composting
The composting process comprises multiple biochemical transformations, in which microorganisms gradually colonize the compost material. It is also closely connected with the formation of biofilms, in which degradation processes are more efficient [2,22,26]. Composting of poultry feathers with manure proceeded similarly in both: compost inoculated with a culture of B. cereus and uninoculated control 1. In contrast, composting of sole feathers in control 2 appeared to be an imbalanced process. The pH of the inoculated compost was initially lower than that of control 1 (pH 6.5 and pH 7.9, respectively), but at the end of the process it was similar (pH 6.5) in both cases (Fig. 7 A). The changes in the pH resulted from biological processes in the compost piles. The temperatures of 46-50°C were achieved right on the first day of the process, in both, the compost and the control 1 (Fig. 7 B). On the next days, the temperature of the inoculated compost was 1-7°C higher as compared to the control 1.

The total number of bacteria and the number of spores were increasing slowly; from 10⁸ cfuˇg^-1 to 1.2·10¹² cfu·g^-1 in the inoculated compost and 1.6·10¹² in the control 1 (Fig. 8 A, B). The number of thermophilic bacteria (10⁶-10⁸ cfuˇg^-1 in the thermophilic phase) decreased in the mesophilic phase to 1.4·10⁴-1.0·10⁵ cfu·g^-1. In contrast to bacteria, the number of filamentous fungi decreased in the sixth week from 1.1·10⁷ cfu·g^-1 to 1.5·10⁴ cfu·g^-1 in the compost and the control 1 (Fig 8 A, B). The number of yeasts decreased from 1.3·10⁷ to 1.0·10⁴ cfu·g^-1 in the control 1, while in the compost inoculated with bacteria their growth was completely inhibited after six weeks of composting (Fig. 8 A, B). Conducted tests showed that in the compost designated as control 2, consisting of feathers without manure and inoculated with bacteria, the degradation process proceeded incorrectly. Temperature of the compost pile was too low, pH was excessively elevated and filamentous fungi were eliminated after six weeks of composting (Fig. 8 C). The improper course of the process could be a result of an inappropriate C:N ratio, which was considerably lower than optimum. Moreover, the initial insufficient disintegration of feather material, followed by poor aeration, resulted in shortening of thermophilic phase to two days, high alkalinization (pH 8.0) and emission of odors. In the control 2 the total number of bacteria did not exceed 10¹¹ cfu·g^-1 and the number of spores was similar to that present in the inoculated compost and the control 1. The number of thermophiles was similar to that recorded in the inoculated compost. The number of yeasts remained at the level of 10⁶ cfu·g^-1.

The results obtained in the present study show, that inoculation of a compost with spore-forming bacteria did not affect local microorganisms, which suggests domination of resident microflora. The introduction of mesophilic bacteria into the compost environment, was likely to result in the predominance of mesophiles over thermophilic species, during the high-temperature phase. A short period of temperature elevated to 52°C did not eliminate those microorganisms. The growth of thermophilic bacteria was more extensive in the inoculated compost.

Chemical and microbiological changes during composting poultry feathers led to breakdown of the regular structure of keratin fibres, which was visualized using scanning electron microscopy. Feather rachea and barbs were ruptured to smaller pieces, visible in the microscopic picture. The structural composition of keratin fibres was highly disrupted and bacterial aggregates together with an extracellular matrix, adhered to degraded surfaces, were observed (Fig. 9).

Tests of mineral composition of composts confirmed that mineralization was equally effective in the inoculated compost (34.1 %) as in the control 1 (32.1 %), and considerably less efficient in the control 2 (12.0 %). The carbon-to-nitrogen ratio in the inoculated compost was 3.6:1 after the process. The content of minerals was higher than in the control 1 [%]: S- 1.72, K- 2.56, Mg- 0.34, P- 0.37, NH₄⁺- 1.87 (Table 2). Increased level of mineral compounds could enhance fertilizer value of the compost. Fungistatic properties and fertilizer value of composts will be the objective of further investigations.

CONCLUSIONS

The results of the present study show that the strain B. cereus B5esz, besides its specific keratinolytic activity, is able to synthesise such amounts of proteases that are likely to participate in keratinolysis of the substrate. The bacteria taken for the study decomposed feather keratin both in the liquid culture and in the compost. The assays performed in the culture medium showed that the sequence of different extracellular metabolites was linked with the mechanisms characteristic of keratinolysis. During composting of feathers additionally inoculated with bacteria, an imbalance was observed in this biological system, expressed mainly in the suppression of fungi and yeast growth. Thus, the ability of Bacillus cereus to decompose chitin was confirmed. This ability, along with the possible production of antibiotics (tunicamycin) and insecticidal proteins, can find applications in the biocontrol of phytopathogenic fungi and insects in soil. The applied bacterial inoculum caused enhancement of keratin decomposition and slightly increased the mineralization factor. The inoculated compost contained higher amounts of minerals (P, K, Mg) as compared to the control 1. Composting of feathers without plant material additives (control 2) did not give expected results. Inoculation of compost by Bacillus cereus strain may be a promising biological process which accelerates utilization of poultry keratinic wastes.

ACKNOWLEDGEMENTS

This research was supported by a grant from Wrocław University of Environmental and Life Sciences, project No 502/GW/05.

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Accepted for print: 31.03.2008

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