World Journal of Microbiology & Biotechnology 16: 551±554, 2000.
Ó 2000 Kluwer Academic Publishers. Printed in the Netherlands.
551
Feather degradation by Kocuria rosea in submerged culture L. Vidal1, P. Christen2 and M.N. Coello1; * 1 Laboratorio de Procesos BiotecnoloÂgicos, Instituto de BiologõÂa Experimental, Universidad Central de Venezuela, AP 47279, Caracas 1041-A, Venezuela 2 Laboratoire de Microbiologie IRD, IFR-BAIM, UniversiteÂs de Provence et de la MeÂditerraneÂe, ESIL, Case 925, 163 Avenue de Luminy, 13288, Marseille cedex 9, France *Author for correspondence: Fax: +58 2 753 5897, E-mail:
[email protected] Received in revised form 1 August 2000; accepted 15 August 2000
Keywords: Biodegradation, feather, Kocuria rosea, submerged cultures
Summary A strain of Kocuria rosea with keratinolytic activity was studied. In batch culture, the optimum temperature for feather degradation, bacterial growth and protease secretion was at 40 °C. A speci®c growth rate of 0.17 h)1 was attained in basal medium with feathers as fermentation substrate. Under these conditions, after 36 h of incubation, biomass and caseinolytic activity reached 3.2 g/l and 0.15 U/ml, respectively. Extracellular protease secretion was associated with the exponential growth phase. In batch fermentation, feather degradation up to 51% in 72 h was obtained with a conversion yield in biomass of 0.32 g/g. No organic acids were detected in the fermentation broth in signi®cant amount.
Introduction Feathers are a poultry by-product rich in protein (mainly keratin), generated in very large amounts as a waste product from the poultry-processing industry. For many years, feathers have been subject to nutritional studies in order to incorporate them as a nitrogen supplement in animal feedstock. This allows the poultry industry to take advantage of their usefulness and eliminates the environmental problem of their accumulation. In the native state, feathers have two important nutritional limitations: an amino acid imbalance and a poor digestibility (Lin et al. 1992). Industrially, a great part of the feather waste is cooked under high pressure and temperature, producing a feather meal, which can be incorporated into poultry feedstock as a protein supplement. This product has been evaluated extensively and the results showed that it is not very digestible and the destruction of certain amino acids during the process, such as cysteine, magni®es the imbalance of essential amino acids (Bhargava & O'Neil 1975; Baker et al. 1981). Thus, there is a growing interest in alternative methods for the treatment of feathers to improve the nutritional quality of the feather meal and to develop new and useful added value products. Their nutritional value might be improved by microbial action, which results in a modi®cation of the structure of keratin, altering the resistance to the enzymes of the digestive tract (Williams et al. 1991). The bacterial cells incorpo-
rated into the fermented feathers may contribute to some amino acids, hence improving their balance. Also, feathers as a fermentation substrate may be useful to obtain other microbial products such as proteolytic enzymes, organic acids, etc. This research has been conducted on a strain of Kocuria rosea with keratinolytic activity isolated from soil to determine microbial growth and optimum feather degradation conditions in submerged fermentation. Materials and Methods Microorganism The strain of K. rosea, designated as LPB-3, was isolated from soil in aerobic conditions. It was selected for its capacity to grow in a medium containing feather powder as the organic substrate. Materials Yeast extract and nutrient agar were from Difco Laboratories (Detroit, MI, USA). Ninhydrin, ethylene glycol, casein (C-5890) and organic acids were from Sigma Chemicals (St. Louis, Mo, USA). White feathers were obtained from a poultry-processing plant. They were washed extensively with raw water, dried, and sterilized by autoclaving at 121 °C for 15 min and then stored at room temperature in sealed bags until used.
552 Fermentation media and culture conditions The LPB-3 strain was cultivated in 500-ml Erlenmeyer baed ¯asks, containing ®nely cut feathers and mineral medium as described by Williams & Shih (1989). This medium contained (g/l): NH4Cl, 0.5; NaCl, 0.5; K2HPO4, 0.3; KH2PO4, 0.4; MgCl2 á 6H2O, 0.1; yeast extract, 0.1 and ®nely cut feathers, 20. The pH was adjusted to 7.5 with 10% (w/v) KOH. The medium was sterilized by autoclaving at 121 °C for 15 min. For the seed culture, strain LPB-3 was grown at 40 °C in a 500ml Erlenmeyer baed ¯ask containing 30 ml of mineral medium previously described with 5 g ®nely cut feathers/l, shaken at 75 rev/min for 72 h. The seed culture (5% v/v) was then used to inoculate 40 ml mineral medium containing 20 g of feathers/l in a 500-ml Erlenmeyer baed ¯ask. Fermentation was carried out at 40 °C for 96 h with a constant agitation (75 rev/min). Analytical determinations Samples were taken at ®xed time intervals and ®ltered through Whatman No. 4 ®lter paper to retain nondegraded feathers. An aliquot of the ®ltered sample was used to measure cell density by turbidity (600 nm). The OD was then transformed into biomass as dry cell weight (g/l) using a calibration curve. For this, cells were centrifuged at 5000 ´g for 20 min at 10 °C, washed twice with water and dried for 24 h at 105 °C. Another aliquot of the ®ltered sample was centrifuged (5000 ´g for 10 min at 10 °C) and the supernatant recovered for enzyme assay, free amino group determination and organic acids analysis. Free amino groups were estimated using a ninhydrin method as described by Rosen (1957). Cultures on nutrient broth without feather were used as control to obtain the basal levels of free amino groups. For the determination of organic acid content, supernatant samples were clari®ed and deproteinized by mixing two volumes of supernatant with one volume of K4(FeCN6)3H2O (36 g/l) and one volume of Zn(SO4)7H2O (72 g/l), centrifuged (12,000 ´g for 5 min). The supernatant was then ®ltered through a 0.45 lm Millipore membrane, and 20 ll were injected into an HPLC apparatus (Waters 600, Milford, MA, USA) equipped with an u.v. detector (k 210 nm) and a reverse phase column SHODEX (Rspack) K-811. Phosphoric acid 0.8% (v/v) was used as the mobile phase and column temperature was 45 °C. To determine the extent of feather degradation, the whole culture contents of an Erlenmeyer ¯ask were taken at ®xed time intervals and ®ltered through Whatman No. 4 ®lter paper. The residue remaining on the ®lter was used to measure the extent of feather degradation by the weight loss method. The dry mass was obtained by washing the residue twice with distilled water to remove remaining cells and by drying on pre-weighed Whatman ®lter paper at 100 °C for 12 h. Feather degradation was determined by subtracting the weight of keratin in
L. Vidal et al. non-inoculated ¯asks, used as control, from the weight of keratin left over after each fermentation. The presence of possible contaminants was determined by culture of the cells on nutrient agar plates and also by microscopic observation of Gram stained cell samples from broth. Protease activity assay Proteolytic activity was estimated by a modi®ed method of Kunitz (1947). The conditions were: for 1 ml of 0.25% (w/v) casein in 100 mM phosphate buffer (pH 7.5), 0.05 ml of centrifuged culture broth was added, and incubated for 25 min at 40 °C. Proteins were precipitated with 2.5 ml of 0.5% (w/v) trichloroacetic acid (TCA). After 15 min in ice bath, the supernatant was separated by centrifugation at 10,000 ´g for 10 min and the absorbance at 280 nm was determined. A 2.5-ml TCA solution was added before incubation and used as blank. Each assay was triplicated. One unit of proteolytic activity is de®ned as the amount of enzyme which produces an increase in absorbance of 1.0 unit per min. Results and Discussion Only limited information regarding K. rosea is currently available. This gram-positive micro-organism, which was classi®ed in the past in the genus Micrococcus and reclassi®ed recently in the genus Kocuria (Stackebrandt et al. 1995) has been mainly studied in relation to the structure and properties of the carotenoid pigments it produces (Cooney & Berry 1981; Jagannadham et al. 1991). Until now, its feather degradation capacity has not been reported in the literature. The keratinolytic activity of LPB-3 strain was evidenced by visual observation of feather disintegration in the culture ®ltrate, and the high levels of free amino groups detected in the growth medium. Eect of culture temperature on the extent of feather degradation and growth of LPB-3 The optimum temperature growth of K. rosea on ®nely cut feather medium was observed at 40 °C (Figure 1). Similarly, maximum feather degradation measured as generation of free amino groups, occurred between 35± 40 °C. In both cases, free amino groups and biomass decreased at culture temperatures less than 35 °C. For this reason, 40 °C was chosen as the optimum temperature for cultivation. Another strain of K. rosea has been reported to grow at lower temperatures (psychrotrophic), compared with LPB-3 strain (mesophilic) (Jagannadham et al. 1991; Chattopadhyay et al. 1997). Characterization of fermentation At 40 °C, we found a lag phase of nearly 3 h in batch fermentation, followed by an exponential growth phase
Biodegradation of feathers by K. rosea
553
Figure 1. Measurement of the growth of Kocuria rosea (s) and free amino group concentration (r) present in culture supernatant as function of temperature in feather-containing medium (20 g/l). Values are means of determinations on three fermentation replicates.
Figure 3. Free amino groups concentration (r) and feather degradation (h) during feather fermentation at 40 °C and 75 rev/min agitation. Values are means of determinations on three fermentation replicates.
in which K. rosea attained a speci®c growth rate of 0.17 h)1. Protease activity was detected in the culture medium throughout growth, the maximum activity (0.15 U/ml) associated with maximum biomass (3.1 g/l) was achieved at 36 h after inoculation (Figure 2). The lag period observed may be due to the decrease in cell viability in the seed culture, because it was incubated for a long period of time (72 h). Increasing the quantity of feathers in the seed culture and shortening the incubation time may increase both cell density and viability, in this way reducing the lag period and improving feather degradation. The results shown that K. rosea degraded up to 51% within 3 days (Figure 3). This time period is shorter than the reported ones in the literature (at least 5 days) for other micro-organisms with keratinolytic activity for maximum feather hydrolysis under optimum conditions: Streptomyces fradiae (Noval & Nickerson 1959), Bacillus licheniformis (Williams et al. 1990) and Bacillus sp. P-001A (Atalo & Gashe 1993). The free amino group levels, as an evidence of feather digestion, increased up
to 26 mM . The increase of free amino group levels during feather fermentation by LPB-3 strain shows that this bacterium produces a protease(s) capable of digesting keratin. Cultures of the strain carried out on nutrient broth without feather shown that the levels of free amino groups were negligible. This result suggests that feather disintegration was the source/origin of the free amino groups detected in the culture broth during fermentation. Moreover, the maximum feather degradation (%) was achieved in 96 h when the maximum free amino group concentration was detected in the culture broth (Figure 3). Although high levels of free amino groups and feather degradation were found at 96 h, an increase of the incubation time was not useful because these parameters did not increase signi®cantly after 48 h and the energy required for the experiment would increase the operational costs. The increase in the pH of the culture medium from 7 to 8.5 after 48 h of incubation was possibly a result of the proteolysis and the subsequent release of ammonia following utilization of amino acids and soluble peptides as a metabolic fuel for growth and microbial maintenance. Organic acids during the fermentation
Figure 2. Growth curve of Kocuria rosea (s) and protease production (m) during feather fermentation at 40 °C and 75 rev/min agitation. Values are means of determinations on three fermentation replicates and bars indicate ranges of values.
The capacity to produce organic acids as fermentation by-products was evaluated. Under the established conditions, citric, gluconic, malic and propionic acids were too low to be determined accurately. Only acetate and lactate were detected after 48 h of cultivation in the culture medium, in the concentrations of 0.08 and 0.11 g/l, respectively. The data obtained allowed the calculation of the conversion yield of solubilized feather into biomass, Yx/s 0.32 g/g. Even when previous results suggested that 72 h of incubation might be enough to produce a hydrolysed feather meal enriched with bacterial protein, further research is required to evaluate the potential use of the fermentation broth as a food additive. The amino acid content of the meals obtained, besides
554 bioavailability test and digestibility evaluations assayed in animals, are essential, especially since it is known that feather does not provide a balanced amino acid source for animals. The inclusion of bacterial proteins into the additive could help to solve this problem since glutamate and aspartate in feedstock is crucially important. Also, further research is required to optimize the feather degradation process and evaluate other potential industrial applications, such as protease and carotenoid pigment production. Acknowledgements This research was supported by CDCH project No. 09-12-404497 and BTI-88 from Consejo de Desarrollo CientõÂ ®co y HumanõÂ stico of Universidad Central de Venezuela and Consejo Nacional de Ciencia y TecnologõÂ a, respectively. This work was carried out under the collaborative research program between IBE (Instituto de BiologõÂ a Experimenta, UCV) in Venezuela and the IRD (Institut de Recherche pour le DeÂveloppement) in France.
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