cells per tube of medium. The standard inoculum consisted of 3 X 105 cells obtained from the water of syneresis of cultures

Transcription

1 NUTRITIONAL CHARACTERISTICS OF A BUTYRIVIBRIO' JAMES W. GILL2 AND KENDALL W. KING Department of Biochemistry and Nutrition, Virginia Agricultural Experiment Station, Virginia Polytechnic Institute, Blacksburg, Virginia Received for publication January 27, 1958 Very little is understood about the nutritional characteristics of the cellulose-decomposing bacteria indigenous to the rumen with the exception of the volatile fatty acid requirement of Bacteroides succinogenes (Bryant and Doetsch, 1955). The present report considers certain of the nutritional characteristics of a cellulolytic bacterium belonging to the genus Buiyrivibrio as described by Bryant and Small (1956). This genus embraces the "less-cellulolytic rod" described by Hungate (1950). MATERIALS AND METHODS The organism was obtained from a well-isolated colony in a roll tube containing 10-7 ml of fluid from the rumen of a fistulated steer maintained on a hay-grain ration. The procedures used were essentially those of Hungate (1950) with minor modifications described by King and Smith (1955). The initial isolate was strongly cellulolytic and showed uniform colonial and microscopic morphology. Through two subsequent dilution series in roll-tube cultures no evidence of contamination could be seen and the cellulolytic capacity was retained. Lyophilized cultures stored at room temperature and cultures frozen in glycerol according to the method of Howard (1956) were still viable when last tested one year after preservation. The basal medium described in table 1 supplemented with 1.5 per cent agar, was used to maintain the stock culture. This medium allowed I This investigation was supported in part by a grant-in-aid from the Polychemicals Department of E. I. du Pont de Nemours and Company and contributes to Southern Regional Pasture and Forage Crops Project S-12. A preliminary report of portions of this work was presented at the 56th Annuaj Meeting of the Society of American Bacteriologists, Detroit, Michigan, Present address: Department of Poultry Husbandry, University of New Hampshire, Durham, New Hampshire. initiation of growth from inocula as small as 10 cells per tube of medium. The standard inoculum consisted of 3 X 105 cells obtained from the water of syneresis of cultures incubated for 18 to 28 hr at 39 C in the basal agar medium. The cell suspension was taken up in sterile 0.05 M potassium phosphate at ph 7 containing 0.2 mg of cysteine per ml. After determining the concentration of cells using the Klett- Summerson colorimeter, the suspension was diluted further in the same sterile, poised buffer to a final concentration of 3 X 106 cells per ml. Inoculation was then carried out using 0.1 ml aliquots. Under these conditions the carry-over of stock medium was estimated to be 2 to 4 x 10-5 ml per ml of experimental medium. Growth was measured using a Klett-Summerson colorimeter with the no. 42 blue filter. In establishing requirements for B-vitamins, the basal medium was modified to contain the following compounds at the concentrations indicated (,ug per ml): riboflavin, 1.0; biotin, 0.01; p-aminobenzoic acid, 0.20; folic acid, 0.05; pyridoxal hydrochloride, 1.0; thiamin, 1.0; niacin, 1.0; cobalamine, 0.001; and calcium pantothenate, 1.0. Final growth was determined in a series of media each lacking only one vitamin; then growth in media containing only the three essential vitamins was compared with that obtained in the presence of all of the vitamins tested. In investigating amino acid requirements, the casein hydrolyzate in the basal medium was replaced by mixtures of the pure iisomers of amino acids at final concentrations of either 0.20 or 0.40 mg per ml of each amino acid. As in the studies of vitamin requirements, the growth response to deletion of single amino acids was reexamined using more limited synthetic mixtures of amino acids. The rumen fluid used in growth-stimulation experiments was prepared by heating whole rumen ingesta in a sealed flask for 15 min at 121 C in an autoclave. After cooling, the clear supernatant obtained by centrifugation at 15,000 x 666

2 19,581 NUTRITION OF BUTYRIVIBRIO 667 G for 15 min was decanted and refrigerated until used. Fatty acid analyses were conducted as described by Smith, et al. (1956). RESULTS Cultural characteristics. The organism conformed very closely with the genus Butyrivibrio as described by Bryant and Small (1956). It was a monotrichous, gram-negative, curved rod vary- TABLE 1 Composition of the basal medium Component Amt per L KH2PO g K2HPO g NaHCO g MgSO g CaCl g MnCl2 *4H g FeSO4*7H g CoC12*6H g (NH4)6Mo702,14H2O g ZnCl g CuSO4c5H g Glucose g (NH4) 2SO g Acid hydrolyzate of casein* ml Pyridoxal hydrochloride mg Folic acid mg Biotin mg Resazurin mg Cysteine... O.50 g * Vitamin-free grade from Nutritional Biochemicals, Inc., was used after neutralization with NaOH, to ph 6.9 to 7.0. This amount was equivalent to 5.0 g of casein. ing between 0.3 to 0.4 by 1.7 to 2.6 A in size, and it occurred both singly and in short chains. Oxygen prevented growth but killed only slowly, for cultures reduced with cysteine 4 hr after heavy inoculation into aerobic broth grew as rapidly as those reduced immediately after inoculation. Several heavy inocula left in resazurine-oxidized media for 27 hr produced good growth upon reduction. When standard inocula were prepared in dilution fluids lacking the cysteine, an increase in the lag phase was evident in the later tubes to be inoculated. Conclusive identification of the isolated organism with the genus Butyrivibrio lies in the analysis of the fatty acids produced by glucose fermentations. Table 2 compares the analysis of several fermentation (media) with the results reported by Bryant and Small (1956) for their type strain, Butyrivibrio fibrisolvens. The strong butyric acid production and small propionic acid production are obvious for both cultures in media containing rumen fluid, and confirm the identification of the culture as a member of the genus Butyrivibrio. The dependence of high butyric acid production on the presence of rumen fluid, and the shift in fermentation products upon addition of the basal medium nutrients to the rumen fluid medium are worthy of special attention. Bryant and Small (1956) concluded that the cellulolytic ability of Butyrivibrio is a variable characteristic. There is no doubt that the organism isolated for this investigation digested the cellulose and the cellulodextrins used in the isolation procedure. However, when suspensions of cellulose in broth media containing traces of glucose and cellobiose were inoculated, digestion TABLE 2 The effect of rumen fluid on the nature of the acids produced from fermentation of glucose by a Butyrivibrio Organism Organism Acids Produced (meq/100 ml) Mediumn. Butyric Propionic formic lactic ~ ~~~~~~utri PoponcAcetic plus Succinic plus B. fibrisolvens Basal minerals and buffers plus 40 per (Bryant and cent of rumen fluid Small, 1956) Present isolate Basal minerals and buffers plus 40 per cent of rumen fluid Present isolate 80 Per cent complete basal medium plus per cent rumen fluid Present isolate 100 Per cent complete basal medium

3 668 of the cellulose was consistently too slight to measure reliably. Viscosimetric assay of cellulase, as described by King (1956), gave positive evidence of at least a partial cellulase system in the cell-free supernatant of a 0.5 per cent glucose-0.12 per cent cellobiose broth culture. In view of the similar experiences of Hungate (1950) with his "less-cellulolytic rod" and of Bryant and Small (1956) with their strains of Butyrivibrio regarding loss of cellulolytic ability by glucose-grown cultures, no further efforts were made to demonstrate hydrolysis of cellulose. A consistent property of the organism was the production of a viscous material during the late stages of growth in media rich in glucose. Negative staining of a heat-fixed smear of such a viscous culture with aqueous nigrosine solution after a simple stain with crystal violet showed definite encapsulation. The cell-free centrifugal supernatant fluid from a viscous culture was itself viscous. The ph requirements for the organism were determined in a carbon dioxide atmosphere which made accurate control of the ph difficult. Consequently, no one set of media could be used to show both ph optima and limits. Several such sets of media gave the following values without confficting with each other. The optimum ph for initiation of growth was 6.9 to 7.0; beyond a range of 6.3 to 7.2 growth was strongly inhibited. The basal medium allowed initiation of visible growth about 10 hr after inoculation, and full growth in about 24 hr. The ph at 24 hr was CONCENTRATION OF PYRIDOXAL(pig/mi) Figure 1. Growth response of Butyrivibrio to pyridoxal. GILL AND KING to 6.5 and dropped to a terminal value of 6.0 after 48 hr. Ascorbic acid was found to be a satisfactory reducing agent for broth media, although it did not prevent the media from developing a slight yellow color upon heat sterilization as did cysteine. Sodium sulfide and sodium thioglycolate at approximately equivalent reducing concentrations, 100 and 50 mg per 100 ml, respectively, did not support as much growth and were much less reliable for reducing media by the procedure used. In addition, the sodium sulfide formed a black precipitate and the sodium thioglycolate produced an intense yellow color. Both ascorbic acid and cysteine reduced sterile media when sterilized separately in an autoclave and then added to the broth aseptically and anaerobically. To be sure that the sterilization process did not produce either toxic or stimulatory compounds, a batch of medium was prepared and separate portions of it were sterilized by heating in an autoclave and by filtration through a Morton sintered-glass filter. The filtered medium gave growth quite comparable to that obtained in the heat sterilized medium. TABLE 3 Growth response of Butyrivibrio to deletion of single amino acids from a synthetic medium containing the amino acids in casein Amino Acid Omitted [vol. 75 Turbidity in Klett- Summerson Units (Corrected for Growth in Absence of Amino Acids) 28 hr 72 hr None All L-Glutamic acid DL-Histidine DL-Isoleucine DL-Methionine L-Lysine L-Tyrosine L-Phenylalanine All values are the average of duplicate tubes. The complete medium consisted of the basal medium described in table 1 modified by replacing the casein hydrolyzate with a mixture of 0.2 mg per ml of each of the 18 amino acids in acid hydrolyzates of casein. The observed turbidity of the control medium from which all amino acids were omitted was 50 Klett-Summerson units at both time intervals.

4 19581 NUTRITION OF BUTYRIVIBRIO 669 It was found that media from which both bicarbonate and carbon dioxide were omitted did not support growth. The growth response to graded amounts of sodium bicarbonate under a nitrogen atmosphere was measured and growth was found to be restricted to media prepared with at least 0.02 M NaHCO3 added to the aqueous phase. The carbon dioxide requirement could not be replaced by a mixture of Tween-80, cobalamine, adenine, guanine, thymine, uracil, cytosine, xanthine, and hypoxanthine in physiologically compatible concentrations. Nutritional requirements. Nutritional studies using a series of media from which single vitamins were omitted demonstrated that pyridoxal, folic acid, and biotin were essential. Thiamin, pantothenate, niacin, cobalamine, riboflavin, and p- aminobenzoic acid were not essential, and did not stimulate growth. Growth has been sustained for TABLE 4 Growth response of a Butyrivibrio to deletion of single amino acids from synthetic media containing 8 to 10 amino acids* Amino Acid Omitted Turbidity in Klett- Summerson Units Medium A Medium B (48 hr) (72 hr) None L-Histidine L-Methionine L-Isoleucine L-Cysteine 0 L-Leucine L-Valine L-Tyrosine L-Lysine L-Aspartic acid L-Asparagine.139 L-Trytophan * Dashes indicate deletions which were not tested. Medium A contained 0.25 mg per ml of L-histidine, L-methionine, L-isoleucine, L-cysteine, L-leucine, L-valine, L-tyrosine, L-lysine, L-aspartic acid, and L-tryptophan. Biotin, folic acid, and pyridoxal were the only B-vitamins used. Medium B was like medium A except that it contained L-asparagine but no valine, aspartic acid, or tryptophan. The vitamins included those in medium A plus cobalamine, thiamin, niacin, calcium pantothenate, and p-aminobenzoic acid. Ascorbic acid was used in place of cysteine as the reducing agent in order to allow evaluation of the nutritional role of cysteine. 6 months on the basal medium shown in table 1. A graded response to the essential vitamins was found, with maximum growth occurring at mg of biotin per ml, 0.007,ug of folic acid per ml, and 0.12,ug of pyridoxal hydrochloride per ml. Figure 1 shows the growth-response to pyridoxal which was similar to the curves for biotin and folic acid. Choline and thioctic acid did not stimulate growth in the basal medium. Deletion of single amino acids from a synthetic medium, identical to the basal medium except for replacement of the casein hydrolyzate with pure amino acids, resulted in the growth responses shown in table 3. Histidine, isoleucine, methionine, and lysine seemed to be critical nutrients, although not essential in the strict sense. Those amino acids found in casein which are not listed in table 3 were tested, but like tyrosine and TABLE 5 Growth of Butyrivibrio on basal media containing simple amino acid mixtures* Amino Acid Added to: atu Medium C 60 hr None L-Serine L-Tyrosine L-Lysine L-Tyrosine + L-Lysine + L-serine Medium D 65 hr None L-Asparagine L-Aspartic acid L-Aspartic acid + L-glutamic acid L-Leucine L-Valine L-Leucine + L-valine... 1C L-Phenylalanine L-Asparagine + L-leucine + L-valine + L-phenylalanine * All turbidity data are average Klett-Summerson units of duplicate cultures. Medium C consisted of the basal medium shown in table 1 modified by replacing the casein hydrolyzate with 0.25 mg per ml of L-histidine, L-methionine, and L-isoleucine, and 0.50 mg per ml of L-cysteine. Addenda were at the latter concentration. Medium D was similarly modified by substituting 0.40 mg per ml of L-histidine, L-methionine, L-iSoleucine, L-cysteine, L-tyrosine, and L-lysine for the casein hydrolyzate. Addenda were at the same concentration.

5 670 GILL AND KING [VOL. 75 phenylalanine their omission had little effect on growth. When similar single deletion experiments were conducted with basal media containing less complex mixtures of amino acids, results such as those shown in table 4 were obtained. Using a basal medium containing 10 amino acids (medium A) or 8 amino acids (medium B) the partial requirements for histidine, isoleucine, methionine, and lysine were confirmed and, in addition, the cysteine requirement was seen. Less conspicuous depression of growth by omission of leucine, valine, tyrosine, and asparagine was observed and tryptophan appeared to be nonessential. The inhibitory effect of aspartic acid seen here has been encountered in numerous experiments. Whether the reduction in the methionine requirement indicated in medium B is a result of the presence of added B-vitamins or of the absence of valine, tryptophan, and aspartic acid has not been determined. When the nutritional characteristics of the culture were tested by examining the effect of amino acid supplementation of basal media containing only 4 to 6 amino acids, results such as those shown in table 5 were obtained. The responses to addition of tyrosine, lysine, and leucine were much more convincing than those seen earlier from single deletion experiments. The reversal of aspartic acid inhibition by addition of glutamic acid has been observed repeatedly. Similar interactions in the nutritional response to the TABLE 6 Growth response of Butyrivibrio to variations in the absolute and relative amounts of amino acids* Source of Amino Acids Total Weight of Amino Acids 2.6 mg/ml 5.2 mg/ml Equal weight of each of the amino acids that occur in casein Pure amino acids in proportions in which they occur in casein Acid hydrolyzate of casein Enzymatic hydrolyzate of casein * Figures are average Klett-Summerson uiiits of duplicate cultures at maximum stationary phase. All addenda were added to the basal medium in table 1 at the expense of the casein hydrolyzate. combinations leucine-isoleucine-valine and serine-threonine were not evident. In general, growth was markedly suppressed by substituting pure amino acids for the casein hydrolyzate in the basal medium. The lag period was increased from 8 to 10 hr to 24 to 30 hr by this substitution, and final cell concentrations were usually reduced from 200 to 250 Klett- Summerson units to less than 150 units, the only exception being the experiment shown in table 5, medium D. The suppression of growth did not appear to depend on simple reduction in the amount of organic nitrogen available for two reasons. First, maximum cell yield occurred at a casein concentration of 10 mg per ml and the amino acid media generally exceeded this amount on a nitrogen basis. Second, analysis of cultures for residual ammonia (Johnson, 1941) showed assimilation of up to 70 per cent of that available, and this uptake was not appreciably affected by the amount of casein in the medium. Examination of the data in tables 3 to 5 indicates that the culture responded quite sensitively to amino acid imbalance. This response is specifically posed, but not explained, by the data presented in table 6. Here the effect on growth of both absolute concentrations and relative amounts of the amino acids was examined. 300 _ z.200 c') z 150 I-- a / A /. I.,/. o _ INCUBATION TIME IN HOURS Figure 2. Stimulation of the growth of Butyrivibrio in the basal medium (table 1) by rumen fluid. Curve A, basal medium; Curve B, basal medium plus 1 per cent rumen fluid; Curve C, basal medium plus 5 per cent rumen fluid. C./ B / A I /....4

6 1958] NUTRITION OF BUTYRIVIBRIO 671 Stimulation by rumen fluid. Supplementation of the basal medium with rumen fluid enhanced both the growth rate and the final cell yield as shown in figure 2. No stimulation was observed when the following were tested in the basal medium at a concentration of 0.1 mg per ml: diglycine, triglycine, ornithine, asparagine, 8- alanine, DI-a, e-diaminopimelic acid, indole, glutamine, norleucine, glutathione, creatinine, betaine, taurine,d-aspartic acid,d-alanine, 3,5- diiodotyrosine, tris(hydroxymethyl)aminomethane, hydroxyproline, norvaline, ribose,; sorbitol, xylose, cellobiose, mannose, i-inositol, fructose, inulin, water-soluble and water-insoluble cellulose dextrins (Dickey and Wolfrom, 1949), lactose, adenine, cytosine, guanine, hypoxanthine, thymine, uracil, xanthine, orotic acid, citric acid, lactic acid, glutaric acid, succinic acid, a-ketoglutaric acid, sodium oxalate, glucuronic acid, calcium gluconate, pyruvic acid, sodium thioglycolate, ascorbic acid, riboflavin, niacin, choline, thioctic acid, thiamin, calcium pantothenate, cobalamine, sodium nitrate, potassium nitrite, urea, and sodium bicarbonate. Several mixtures TABLE 7 Stimulatory effect of several crude preparations when added to the basal medium (table 1) Growth Stimulation Over that on Basal Supplement to Basal Medium* Mediub (pear ent); 14 hr 24 hr None Rumen fluid Whole ribonucleic acid Rumen fluid Basic hydrolyzate of ribonucleic acid Rumen fluid Acidic hydrolyzate of ribonucleic acid Rumen fluid Enzymatic casein digest Rumen fluid * Rumen fluid supplementation was at 5 per cent v/v; other addenda were at 0.1 per cent w/v. Ribonucleic acid hydrolyzates were prepared usingo.1 NHCl oro.1 NNaOH at 55 C for 24 hr. Growth of duplicate cultures was measured against uninoculated controls. The control basal medium had a turbidity of 100 and 240 Klett- Summerson units at 14 and 24 hr, respectively. of propionic, n-butyric, iso-butyric, DLia-methylbutyric, iso-valeric, and n-valeric acids were also tested and found to be either inactive or inhibitory. Rumen fluid ash prepared at 550 C and dissolved in HCI was not active. A mixture of adenine, cytosine, guanine, hypoxanthine, thymine, uracil, and xanthine at final concentrations of 0.05 mg each per ml inhibited growth by 45 per cent. Indole was also mildly inhibitory and KNO2 prevented growth completely. Moderate stimulations (20 to 50 per cent) were obtained when yeast extract (Difco), ribonucleic acid (Nutritional Biochemicals Co.), and enzymatic hydrolyzate of casein (Nutritional Biochemicals Co.) were added to the basal medium at levels of 0.1 mg per ml; positive control media containing 5 per cent rumen fluid in these latter experiments showed approximately 100 per cent stimulation of final cell yield. Data describing the relative activity of enzymatic casein hydrolyzate, ribonucleic acid, hydrolyzates of ribonucleic acid, and rumen fluid are presented in table 7. TABLE 8 Effect of several treatments on the growth-stimulating activity of rumen fluid Treatment of Rumen Fluid* Growth in Per Cent Over that in Unsupplemented Basal Medium; Incubated for: 11 hr 16 hr None Ether extracts: ph < ph > Boiled 30 min in: 2 N HCI N NaOH Dialyzed: Residue Dialysate Effluent from Dowex-50, H+form, ph < Effluent from Dowex-2, OH-- form, ph > * All treated sub-samples were readjusted to ph 6.9 and added to the basal medium in amounts equivalent to 5 per cent v/v of the original rumen fluid sample. Dialysis was carried out against an equal volume of distilled water in the cold. Growth on the basal medium was 120 and 315 Klett-Summerson units at 11 and 16 hr, respectively.

7 672 GILL AND KING [VOL. 75 Certain of the general characteristics of the stimulatory compound, or compounds, in rumen fluid were determined with the results summarized in table 8. DISCUSSION The organism used in this work meets the criteria for the genus Butyrivibrio as described by Bryant and Small (1956) in most respects. Two apparent discrepancies, however, bear on the taxonomy of the genus. Most conspicuous of these is the variability in butyric acid production on media containing different amounts of rumen fluid as seen in table 2. In addition, this culture required quite high concentrations of carbon dioxide when inoculated into chemically defined media. It is possible that this requirement for carbon dioxide was not evident in the media used by Bryant and Small because of either the bicarbonate content of the rumen fluid used in their media or because of an unidentified C02-sparing nutrient in their rumen fluid. Both the requirement for carbon dioxide and the production of butyric acid would bear reinvestigation before the specific taxonomy within the new genus is formally outlined. The inconsistencies between the requirements for amino acids which appear upon comparison of the results of "single deletion" experiments using a complex basal medium (table 3) and the results of stimulation experiments using basal media containing only a few amino acids (table 5) cannot be fully explained with the data at hand. Both approaches, however, indicate that the present culture has only a limited capacity for the synthesis of a number of amino acids and that interconversions between amino acids are carried out with considerable facility. In such a metabolic situation, the "single deletion" experiment exerts a minimum stress on the synthetic capacities of the cell because the deleted amino acid may either be produced from some other amino acid in the basal medium or synthesized from ammonia and the carbon of the energy source. When basal media are designed which contain only those amino acids indicated by the "single deletion" experiments to be necessary (table 5), the opportunities for interconversion are greatly restricted. As a result, amino acids that do not appear stimulatory under the conditions of the less powerful "single deletion" experiment may be seen to be so. It is for reasons such as these that the term "nutritional characteristics" has been used here in preference to "nutritional requirements." ACKNOWLEDGMENTS The authors are indebted to Drs. W. E. C. Moore and R. W. Engel for repeated stimulating discussions and valuable suggestions and to Miss Blanche Ching-yi Wu for analytical assistance. Dr. M. P. Bryant generously collaborated in identification of the culture and in preparation of the manuscript. SUMMARY Certain nutritional characteristics of an unspecified member of the genus Butyrivi brio isolated from the bovine rumen have been investigated using chemically defined media. Maximum growth response to biotin, folic acid, and pyridoxal hydrochloride was observed at approximately 0.005, 0.007, and 0.12,ug per ml, respectively. The other B-vitamins tested were nonessential. Carbon dioxide was essential at concentrations in excess of 0.02 M. No evidence of a requirement for either purines or pyrimidines was seen. The nutritionally critical amino acids included histidine, isoleucine, methionine, lysine, cysteine, leucine, tyrosine, and valine. The requirement for amino acids was, in general, only partial and large amounts of ammonia were assimilated. Rumen fluid and certain other crude preparations were found to contain a substance (or several substances) which was strongly stimulatory to growth. None of 68 compounds commonly stimulatory to microbial growth could replace the active principle in rumen fluid. REFERENCES BRYANT, M. P. AND DOETSCH, R. N Factors necessary for the growth of Bacteroides succinogenes in the volatile acid fraction of rumen fluid. J. Dairy Sci., 38, BRYANT, M. P. AND SMALL, NOLA 1956 The anaerobic, monotrichous, butyric acid-producing, curved rod-shaped bacteria of the rumen. J. Bacteriol., 72, DICKEY, E. E. AND WOLFROM, M. L A Polymerhomogeneous series of sugar acetates from the acetolysis of cellulose. J. Am. Chem. Soc., 71, 825. HOWARD, D. H The preservation of bacteria by freezing in glycerol broth. J. Bacteriol., 71, 625.

8 1958] NUTRITION OF BUTYRIVIBRIO 673 HUNGATE, R. E The anaerobic mesophilic KING, K. W. AND SMITH, P. H Comparicellulolytic bacteria. Bacteriol. Revs., 14, sons of two media proposed for the isolation of bacteria from the rumen. J. Bacteriol., 70, JOHNSON, M. J Isolation and properties of a pure yeast polypeptidase. J. Biol. Chem., SMITH, P. H., SWEENEY, H. C., ROONEY, J. R., 137, 575. KING, K. W., AND MOORE, W. E. C KING, K. W Basic properties of the dex- Stratifications and kinetic changes in the trinizing cellulases from the rumen of cattle. ingesta of the bovine rumen. J. Dairy Sci., Va. Agr. Expt. Sta. Tech. Bull. No. 127, ,