Metabolic Characteristics of Beggiatoa Alba in Thiosulfate Medium and Porcine Colon Contents

Metabolic Characteristics of Beggiatoa alba in Thiosulfate Medium and Porcine Colon Contents

Shigeki KOBAYASHI and Hiroomi SHIBATA

School of Agriculture, Meiji University, Tama-ku, Kawasaki-shi 214-8571, Japan

(Received September 1, 1998; Accepted June 14, 1999) Abstract The strain Beggiatoa alba DSM-1416 is a filamentous, gliding, colorless, sulfur bacterium. In order to get the basic information on deodorizing stinking odor in animal feeding, metabolizing characteristics of the strain were examined in the defined liquid medium and porcine colon medium. Carbon sources such as alcohols of low molecule (except methanol) and many short chained organic acids were positively utilized, but a number of saccharides were not utilized. Nitrogen sources such as ammonium salt and some amino acids (glutamate etc.) were positively utilized. In the liquid medium, acetate was utilized first even in the presence of thiosulfate. The thiosulfate was degraded to elemental sulfur and sulfite, and to sulfate ultimately. Also in the porcine centripetal colon medium, the cells of DSM-1416 could propagate. Hydrogen sulfide and methanethiol were utilized, which was not enough to deodorize the medium. In both media, the elemental sulfur stored in periplasm of the cells was gradually consumed by itself when carbon sources in the medium was exhausted. The phenomena demonstrated a possibility of disassimilating stinking odors remaining in animal intestine by sulfur bacteria. Animal Science Journal 70 (5): 349-355, 1999 Key words: Beggiatoa alba, Hydrogen sulfide, Methanethiol, Porcine colon medium, Stinking odor

The stinking odors originating from animal produc- Beggiatoa alba, which is found rather in microaerobic tion, especially in swine feeding, are troublesome and circumstances in soil9,14,22) is anticipated to diminish should be removed in any feeding districts. As prac- stinking odors remaining in animal intestines. tical means for diminishing stinking odors, the follow- In this study, metabolic characteristics of one of B. ings have already been proposed; a forced mixing of alba freshwater strain that is mixotrophic and can feces and a fermentation acceleration by aerobic grow under both anoxic and microoxic conditions4,15) microorganisms3), a direct slurry injection into the were examined. Defined liquid media and centripetal farmland, a suppression of stinking odors from feces colon media from animal were prepared. The colon by direct bacteria dosage to animals7), and others. media were prepared from the contents of porcine The means of direct bacteria dosage to animals have centripetal colon, because sulfur compounds of low already been proposed by a few corporations, but the molecules were contained in relatively high levels in details of biological species and substantial effects are porcine colons8) scarcely reported21). Materials and Methods One of the filamentous, gliding and colorless bacteria, the genus Beggiatoa, is known to oxidize reduced Strain and medium sulfur compounds10-12)and to store elemental sulfur in Beggiatoa alba DSM-1416, which was purchased periplasm of the cell17). It is useful in degrading from Deutsche Sammlung von Mikroorganismen, was sulfur compounds remaining in animal intestine. maintained and used for the examinations. Medium Corresponding: Shigeki KOBAYASHI (fax: +81 (0) 44-934-7902, e-mail: koshige@isc. meiji.ac.JP)

Anim. Sci. J. 70 (5): 349-355, 1999 349 KOBAYASHI and SHIBATA

ATCC183 was used for the maintenance. Medium samples were diluted with 5 folds deaerated tap water,

AT was used for the cultivations, which was modified and were centrifuged (20,000×g, 15min). To the

from the medium LT21) and composed of (per 1l) 0.50 supernatants was added solution of hydrogen sulfide,

g of CH3COONa・3H2O, 0.50g of Na2S2O3 the concentration and volume of which were adjusted 5H2O, 0.50g of (NH4)2SO4, 0.11g of K2HPO4, 0.085 to become ca. 1.0mg/l after the subsequent autoclav-

g of KH2PO4, 0.10g of MgSO4 7H2O, 0.05g of ing procedure. The colon media, which were divided CaCl2・2H2O, 0.002g of FeCl3・6H2O, 2.38g of to 55ml, were inoculated with DSM-1416 preculture HEPES, and 0.003g of EDTA・2Na (pH7.2). fluid in AT medium, and were incubated for 15 days. Medium A (T) which is same as medium AT but Chemical analyses lacking in thiosulfate, was used for 3 days- In order to determine acetate, sulfite and sulfate in

preincubation of the strain DSM-1416. media, the culture fluids were centrifuged (12,000×g, Metabolism analyses 15min), and the supernatants were analysed. Instead of acetic acid in medium AT, other organic Gaschromatograph GL 380 (GL Science Co. Tokyo) acids, such as saccharides and alcohols, were ex- was used for acetate analysis at the following operat- amined to determine whether they were available by ing conditions; column FFAP+H3PO4, carrier gas DSM-1416 as carbon (C) sources. Instead of ammo- N2 30ml/min, temperature 230℃ (injection), 180℃

nium sulfate, amino acids were also examined to deter- (column), 210℃ (detection), FID set 102×24. For mine if they were available as nitrogen (N) sources. analysis of sulfite and sulfate accumulated in the Availability significances of the cultures inoculated media, the SO3/SO4 kit (Kyoritsu Inst. Tokyo) was with DSM-1416 preincubate, were estimated by used based on the suspension formation of barium turbidity formations (OD610) of the cultures17), which sulfate. Elemental sulfur accumulated in periplasm were more than 3 fold compared with those not in- of the cells was analysed by modifying the method of cluding C or N sources. Effects of concentrations of Bartlett and Skoog1). The culture fluid of the cells acetate and thiosulfate on the growth of DSM-1416 was sonicated with supersonic VP-5S (TAITEC, were also examined. In the incubations, medium in- Tokyo), followed by filtration using 0.2μm membrane gredients, except acetic acid or sodium thiosulfate filter (ADVANTEC-TOYO. Tokyo) under aspirator were included similarly in the medium AT. Acetate operation. The residues were dried and weighed. utilization2) and thiosulfate oxidation by DSM-1416 Sulfur was extracted (30±1℃, 24hr) into 15ml were examined by preparing the same culture fluid. ligroin. Six ml of 0.1% sodium cyanide (in 95% And here, media AT (1/3) included a decreased ace- acetone) was added to 2ml of the extracted aliquot in tate contents (1/3 of the initial). For analyzing ele- ligroin, followed by addition of 2ml of 0.1% iron mental sulfur in the bacterial cells, 5ml of the inocu- (III) chloride (in 95% acetone). Optical absorp- lation fluid was added to 150ml of the medium AT, tions (610nm) of the solution were measured in 10 and sulfur stored during 12 days was measured. min. As control, vacant membrane filters treated Preparation of colon media similarly were also measured. For analysis of volatile Sulfur compounds of low molecule, which emit sulfur compounds remaining in porcine colon media , stinking odors, gradually increased in the digestion Gaschromatograph G-3000 (Hitachi, Tokyo) con- process in porcine intestine and were maintained in nected to Chromato-integrator D-2500 (Hitachi, colons and rectum8). Accordingly, a propagation Tokyo) was used at the following operating conditions possibility and metabolic characteristics of the cells ; column ODPN, carrier gas (N2) 70ml/min, temper-

were examined in the medium prepared from the ature 150℃ (injection), 70℃ (column) , 150℃ (detec- contents of porcine centripetal colon. tion), FPD set (750V) 103×23 . Four pigs that were given the formula feed were

slaughtered at 6 months (B.W. 101～106kg), and the centripetal colon contents were taken out. The

Anim. Sci. J 70 (5): 349-355, 1999 350 Beggiatoa alba in Metabolic Colon contents

Table 1. Utilization of organic nitrogen compounds Results by Beggiatoa alba DSM-1416 Utilization of C and N sources in the liquid medium As for C sources for B. alba DSM-1416, mono- saccharides (glucose, fructose, galactose, xylose, mannose), disaccharides (saccharose, lactose, malt- ose), polysaccharides (raffinose, starch), alcohols and organic acids were examined. All of the saccharides were not utilized. Some alcohols (ethanol, n- propanol, n-butanol) were positively utilized. Or- ganic acids, such as acetate, propionate, butyrate, pyruvate, oxalacetate and lactate were utilized. As for N sources in inorganic substrate, ammonium and nitrate were utilized, but not nitrite. Urea was not utilized either. N and C sources in 17 amino acids * +: Positive utilization (It means turbidity formation were analyzed (Table 1). C source only in 2 acids (OD610) of the culture was more than 3 times and N source in 5 acids were utilized. Both sources compared with those that did not include the in asparagine and tryptophan were positively utilized. substrate). Acetate metabolism and reliance on thiosulfate in -: Negative utilization . the liquid medium (C+N): Utilization of both of carbon and nitrogen of When the cells of DSM-1416 were incubated in the the substrate are shown. medium AT (1/3), almost all the acetate in the medium was consumed in 12 days of cultivation. Utilization of acetate was a little higher in the medium with thiosulfate (3mM) than in without it. In the cultivation period, pH in the medium was similarly sustained or a little higher (7.2→7.3) than that in porcine colon contents8). When thiosulfate in the medium AT (1/3) was excluded, the cells continued to grow, but the growths which were estimated by dry matter of the bacterial cells, were significantly lower than those in the presence of thiosulfate in the medium, especially after 6 days of the incubation (Fig. 1). Sulfur storage and the reoxidation in the liquid medium Accompanied by the growth of the cells, the elemental sulfur, which was stored out of cytoplasmic Fig. 1. Medium pH (dashed lines) and the cell membrane18), gradually increased (Fig. 2). It growth (solid lines) in medium AT (1/3) with reached the maximum (72μg/150ml vial) after 9 days (■--- □-)and without (▲--- △-) of incubation. When acetate in the medium was just thiosulfate. exhausted, the sulfur in the cells began to decrease gradually, which coincided with turnabout of the cell sulfate accumulation in the medium linearly increased, weights to lower levels. During the experimental and amounted to 22.9±1.2mg/150ml vial at the 12th period, especially after the 6th day of incubation, day.

Anim. Sci. J. 70 (5): 349-355, 1999 351 KOBAYASHI and SHIBATA

Characteristics and constituents of porcine colon in the early period of 15 days (Fig. 3). The pH medium values in the medium only showed a small variation, The pH of porcine colon medium was adjusted to which reflected a high buffer potential of the colon 7.1±0.1 (Table 2). Dissolved oxygen (DO) remain- constituents. ing after autoclaving accounted for 1.7±0.9ppm. Elemental sulfur storage in the cells in porcine colon Short chained fatty acids (C=2～4) were abundantly medium included and amounted to ca. 1,180mg/l in total. A Elemental sulfur storage in the cells of DSM-1416 slight stinking sulfur compounds in the medium rapidly increased in the period until the 9th day of remained after autoclaving. incubation, thereafter it decreased (Fig. 4). Dry Cells growth in the porcine colon medium matter of the cell as mentioned above, however, in- The dry matter of the cells of DSM-1416 in the creased continuously also after the 9th day. Mean porcine colon medium increased precisely, especially ratios of sulfur compared with dry matter of the cells in each stage were calculated as 0.974, 0.721, 0.364, 0.556, 0.404 and 0.258% corresponding to the incubation periods of 0, 3, 6, 9, 12 and 15 days, respectively. Under the circumstance of relatively abundant C sources, a successive growth of young cells which were deficient in sulfur storage would have induced the lower storage rate of elemental sulfur in the latter half of the incubation period. Utilization of sulfur compounds in the porcine colon medium A part of H2S and methanethiol (MSH) in the colon medium were utilized by DSM-1416 (Fig. 5). H2S remaining after 9 days differed significantly between cultures innoculated with DSM-1416 and those not innoculated. Our olfactory sense, however, could not recognize the difference of smell emitted from

Fig. 2. Sulfur (dashed line) stored in the cell both cultures. Human olfactory sense is capable of periplasm and sulfate (columns) accumulated in the receiving even a very small amount of the offensive medium AT during 12 incubation days of Beggiatoa substances. A thorough removal of the stinking sub- alba DSM-1416. stances would be demanded. A small amount of

Table 2. Characteristic and amounts of short chained fatty acids in the medium* from the contents of swine centripetal colon

* The contents of the centripetal colon were prepared by diluting to 6 fold with deaerated tap water and by adjusting the pH with Sodium Hydroxide.

Anim. Sci. J. 70 (5): 349-355, 1999 352 Beggiatoa alba in Metabolic Colon contents

Fig. 3. Culture pHs inoculated with DSM-1416

(●-) and not inoculated (○---), and increase of the cell dry matter (■-) in porcine colon medium.

Fig. 5. Hydrogen sulfide (■- inoculated with

DSM-1416 and □--- not inoculated) and

methanethiol (●- inoculated with the same strain and ○-・- not inoculated) remained in the culture

of porcine colon medium. Alphabets show

significant differences (P<0.05) between cultures in

the same period.

dimethyl sulfide (DMS) was also detectd in the colon medium. DMS was hardly utilized directly by B. alba. Sulfite (SO32-)and sulfate (SO42-)accumulation in the porcine colon medium Until the 9th day of incubation, SO32- and SO42- accumulations in the medium were very little, thereafter they increased. The increase of SO42- after the 9th day was rapid. The phenomenum coincided with the increase of cell dry matter and the decrease of sulfur storage in each cell in the latter half of the incubation period. Discussion Fig. 4. Elemental sulfur (columns) stored in the cells of DSM-1416 and the relative weights of the Metabolic characteristic of DSM-1416 in the liquid sulfur (chain line) compared with total dry matter of medium the cells in porcine colon medium. The strain DSM-1416 comes from the freshwater

Anim. Sci. J. 70 (5): 349-355, 1999 353 KOBAYASHI and SHIBATA strain B 15LD. The utilization of C sources by Deodorizing possibility by means of bacteria admin- DSM-1416 demonstrated similar characteristics of istration strain B 15LD, B 18LD, B 25RD and L 1401-15 It was basically proved in the defined liquid media reported by Mezzino et al.11). N sources in most of that some strains of B. alba are capable of dis- amino acids were scarcely utilized by DSM-1416, assimilating not only sulfur compounds but also some which would be a diagnostic tendency of substrate organic matters of low molecule16). The growth envi- selection in mixotrophic bacteria. ronment in the intestine of animals, however, would In sulfur catabolism of DSM-1416 in the liquid give physical and chemical effects on the bacteria medium AT, thiosulfate would have played an impor- which originated ectosomatically. Osmotic pressure tant part as a sort of catalase in connection with the and proton concentration in the intestine are much reducing action, because some freshwater strains of B. different from those in soil. alba (PD-1, SM-1, 75-2a, U1) are assumed to lack Formation of sulfur globules from sulfide by strain catalase16). In many defined liquid media where B 15LD (and also by B 18LD, B 25RD and L1401- strain ATCC-155512), strain B 15LD, B 18LD, 15)11) has been reported. But the microaerobic bac- 1325RD, L1401-1511), strain 75-2a12), and strain B teria essentially have lower metabolic rates. In order 18LD17) were examined, reductive circumstances to promote the complete disassimilation of stinking generated by constituents would have also offered the substances and to fulfill the expected deodorization, desirable environment. other bacteria/strains and a large quantity of their An autotrophic growth of a marine Beggiatoa (MS- cells should be examined. Simultaneous administra- 81-6) utilizing sulfide as the sole energy source was tions of some strains should be also planned. demonstrated by Nelson and Jannasch13). Another It is likely that very little amounts of stinking sub- marine stain (MS-81-1c) which was obligately stances remaining in the rectum will have an effect on chemolithoautotrophic was recognized to have the emission of stinking odors. It will be effective if adenosine phosphosulfate (APS) pathway5). A Enterobacteriaceae can possess an ability to utilize freshwater strain 75-2a was confirmed to have no APS sulfur compounds of small molecule. In the near activity and to possess sulfite-acceptor future, it would become possible to recombine in- oxidoreductase5). The strain DSM-1416 in this study testine-inhabiting bacteria and to give them an ability apparently grew in microoxic condition, utilized or- to utilize inorganic sulfur compounds and other stink- ganic compounds and stored elemental sulfur in AT ing compounds by means of the tecnology of gene medium. Thiosulfate in the medium would have transduction. In these cases, the absolute majority of been oxidized to sulfate through 2 pathways corre- intestine-inhabiting bacteria will play a part in the sponding to the trophic circumstance as follows12); thorough disassimilation of stinking compounds. -S -SO3-→s0+SO32- It is necessary that the sites and sequences of under acetate exhaustion, chromosomal genes in B. alba and other sulfur- S0+3/2O2+H2O→SO42-+2H+6) oxidizing bacteria participating in disassimilation of Protons released from the process would be con- sulfur compounds should be determined for the pur- sumed for ATP formation. Existence of cytochrome pose of gene recombination. c reductase system in the cells of B. alba in freshwater References strains (B 15LD, B 18LD, B 25RD, PD 1, L 1401-6, ATCC-15551 and B 23SS) has been reported by 1) Bartlett L, Skoog DA. Colorimetric determination of elemental sulfur in hydrocarbons. Analytical Chemis- Strohl and Schmidt19) and Strohl et al.20). Sulfite try, 26: 1008-1011. 1954. which originated from thiosulfate in the medium 2) Burton SD, Morita RY, Miller W. Utilization of would have been oxidized by the sulfite-acceptor ox- acetate by Beggiatoa. Journal of Bacteriology, 91: idoreductase. 1192-1200. 1966. 3) Carr L, Grover R, Smith B, Richard T, Halbach T.

Anim. Sci. J. 70 (5): 349-355, 1999 354 Beggiatoa alba in Metabolic Colon contents

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