Enumeration of H2-utilizing methanogenic archaea, acetogenic and sulfate-reducing bacteria from human feces

MICROBIOLOGY ECOLOGY

ELSEVIER

FEMS Microbiology

Ecology

17 (1995) 279-284

Enumeration of H ,-utilizing methanogenic archaea, acetogenic and sulfate-reducing bacteria from human feces J. Dor6 ay*, P. Pochart b9c,A. Bernalier

a, I. Goderel ‘, B. Morvan d, J.C. Rambaud ’

a INRA, CR de Jouy-en-Josas, 78352 Jouy-en-Josas Cedex, France b Chaire de Biologie, Conservatoire National des Arts et Mktiers, 75003 Paris, France ’ INSERM U290, Hipital Saint-Lazare, 75010 Paris, France ’ INRA, CR de Their, 63122 Saint-Genb-Champanelle, France Received

19 January

1995; revised 10 May 1995; accepted

10 May 1995

Abstract Fecal specimens from 19 healthy humans were used to enumerate Ha-utilizing microbial populations of methanogenic archaea (MA), acetogenic bacteria (AB) and sulfate-reducing bacteria (SRB). Eight subjects were methane (CH,) excretors (CH, + > and 11 non CH,-excretors (CH, - ), based on breath methane concentrations. The mean + S.E. of the logarithm of MA per gram wet weight feces were 8.8 + 0.21 and 2.6 f 0.39 for CH, + and CH, - , respectively (P < 0.001). SRB counts were 7.1 + 0.43 and 7.3 f 0.39, respectively (NS), while counts of AB were 4.6 f 0.75 and 6.6 + 0.38, respectively (P < 0.02). Counts of Al3 were negatively correlated with counts of MA (r = -0.53; P < 0.05). These results confirm the potential importance of AB in the human colon, especially for CH, - subjects, and suggest that a much greater competitive interrelation occurs in the human colon between MA and AB than between the former and SRB. We further report on the isolation of representatives of the dominant Hz/CO, acetogenic population. Three strains from two CH, - subjects were characterized from lo-’ -lo-’ dilutions. They all consumed Ha/CO, and several carbohydrates to produce acetate as the sole metabolite. Phenotypically related to the species Peptostreptococcus productus, the strains used Ha/CO, via the acetyl-CoA pathway. Keywords:

Human colon; Interspecies

hydrogen

transfer; Acetogens;

1. Introduction The anaerobic degradation of organic matter by the microbial community present in the intestinal tract of animals is a complex process which requires the contribution of several groups of microorganisms

* Corresponding author. Tel: (33) 1 34 65 27 09; Fax: (33) 1 34 65 23 11; E-mail: [email protected] 0168~6496/95/$09.50 0 1995 Federation SSDI 0168-6496(95)00033-X

of European

Microbiological

Methanogens;

Sulfate reducers

linked in a food chain that brings macromolecules down to short chain fatty acids and the gases CO, and H, [l]. The H,, derived entirely from this process, must be either excreted or consumed by intestinal microorganims, the latter mechanism being the major route of intestinal H, disposal [2]. Three groups of anaerobic H,-utilizing microorganims have been characterized to date in the human colon, methanogenic archaea (MA), sulfate-reducing bacteria (SRB) and acetogenic bacteria CAB). Societies. All rights reserved

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Populations of MA range from undetectable to lOlo per gram feces between individuals, a threshold population of 107-lo8 corresponding to the limit at which CH, becomes detectable in breath [3],[4]. This allows distinction between methane-excretors (CH, + > and non methane-excretors (CH, - >. High sulfate reducing activities have been reported in feces of CH, - individuals compared with very low levels in CH, + individuals [5]. However, controversial results of enumerations of SRB have been reported [3],[5]. Distinct interpretations of the interrelation between SRB and MA in the human colon have therefore been proposed, including a competitive exclusion of SRB and MA [5] and an interspecies H 2 transfer-driven cohabitation [3]. In addition, lactate-utilizing SRB have been reported to out-number HZ-utilizing SRB [5], while characterized lactate-utilizing isolates commonly use H, as well as lactate as electron donor with sulfate [6]. Finally, comparisons of H *-dependent incorporation of [‘3C]C0, into acetate have stressed the potential importance of the reductive acetogenesis process [7],[8], which may account for as much as 27% of the acetate produced in the colon of CH, - [9]. No comparative enumeration data is available for all three groups of microorganisms involved in H, disposal. We herein report on the relation between the CH,-excreting status and populations of MA, H, and lactate-utilizing SRB and AB.

2. Materials

and methods

2.1. Subjects Nineteen healthy human subjects, comprising 8 males and 11 females, aged 19-62 years, volunteered to participate in the present study. All consumed normal western diets, and none had been given antibiotics, laxatives, or enemas during the month preceeding the study. Eight were identified as methane excretors (CH, + >, their breath CH, concentrations exceeding by at least one part per million (ppm) that of room atmosphere [lo]. Eleven were non methane excretors (CH, - >. All gave informed consent to the protocol, which was approved by the local ethics committee.

2.2. Media and enumeration procedures Freshly voided feces were obtained from all subjects, stored at 4°C under anaerobic conditions and processed within 10 h. Samples were homogenized by magnetic stirring to give a ten-fold dilution (wet wt/vol) and serially diluted in anaerobic basal medium containing all the components of the Balch 1 medium except acetate, formate, yeast extract and tryptone [ll]. Serial ten-fold dilutions down to 10-l’ were prepared. All liquid and solid media were prepared using strict anaerobic techniques [ 121. All preparations were carried out under medical grade 100% CO, gas (CFPO, Bonneuil, France). After inoculation, the headspace atmosphere was replaced with HJCO, (4:l) at an initial pressure of 202 kPa for H,-utilizing microorganisms. Pressurized tubes were incubated lying horizontally. All incubations were at 37°C in the dark. Total MA were enumerated by most probable number estimation in triplicate using 30-ml vials [3]. The medium was Balch 1 broth [ll] supplemented with 20% (v/v) clarified rumen fluid from a hay-fed steer and clindamycin-cephalothin as described by Miller and Wolin [4]. Vials with CH, levels above 200 ppm after 15 days of incubation were counted positive. Hydrogen-utilizing SRB were enumerated as described by Morvan et al. [13]. The enumeration methodology was as described above, tubes showing a black FeS precipitate within 8 days being counted positives. Whenever tested, gas phase consumption was consistently associated with FeS formation. Lactate utilizing SRB were enumerated as formerly described [3]. Dilutions from 1O-4 to lop9 were commonly used. Enumerations of HZ-utilizing AB were performed using the method described by Dare et al. 1141. The medium contained, per 1 of distilled water: NH,Cl, 0.5 g; KH,PO,, 0.28 g; K,HPO,, 0.96 g; NaCl, 0.13 g; KCl, 0.16 g; MgSO, .7H,O, 0.02 g, trace metal solution, 10 ml; vitamin solution, 10 ml; yeast extract, 0.5 g; incubated clarified rumen fluid, 100 ml [15]; cysteine-sulfide reducing agent, 20 ml; NaHCO,, 5 g; 0.1% resazurin, 1 ml. The trace metal and vitamin solutions were as described by Greening and Leedle [16]. The cysteine-sulfide reducing agent

J. Do& et al. /FEMS

Microbiology

was prepared under 100% N, as described by Braun et al. [17] to contain 1.25% (w/v) each cysteine . HCl . H,O and Na,S .9H,O. Stoppered, crimpsealed culture tubes (18 X 150 mm; Bellco Glass, Vineland, NJ, USA) were each supplemented with 2% tv/v) of a 2.5 M anaerobic filter-sterilized solution of sodium 2-bromoethanesulfonic acid (BESA) just prior to inoculation. Two series of triplicate tubes were inoculated (0.3 ml in 5 ml medium) for each dilution between 10m2 and lo-“. The headspace of each tube of the control series were brought at 202 Wa initial pressure using N,/CO, (4:1), while those of the test series were treated similarly using H,/CO, (4:l). Acetate was analyzed in the supernatant of each tube (see below) after 15 days of incubation. The number of positive or negative tubes recorded for most probable number estimation was based on the difference in acetate concentration between each tube of a test series and the average acetate concentration in the control tubes of the corresponding dilution [ 141. Total anaerobes were enumerated in duplicate 8 X 400 mm tubes [3] using Wilkins-Chalgren agar (Difco, Detroit, MI). Dilutions between lo-’ and 10-l’ were used. 2.3. Isolation and characterization

of AB

For the isolation of AB, enrichments maintained on the enumeration broth medium by biweekly subcultures were transferred to roll tubes containing the same medium with 20 g/l Bacto agar (Difco, Detroit, MI) under HZ/CO,. Isolates were maintained on slants under the same conditions. The H,-dependent acetogenic metabolism was investigated by comparison of acetate produced with that expected based on consumed CO, equivalents. The tentative assignment at the species level was based on former taxonomic descriptions [ 181. 2.4. Analytical

methods

matic method (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s instructions. 2.5. Data analysis Microorganism counts expressed as log,, microorganisms were reported per gram wet weight of feces. Data were expressed as mean + S.E. Statistical analyses were performed with the PCSM statistical package (Deltasoft, Meylan, France) using Student’s t-tests and linear regression [20]. All tests were two-tailed and the level used to establish significance was P < 0.05.

3. Results 3.1. Counts of Hz-utilizing microorganisms man feces of CH, + and CH, -

from hu-

While populations of total anaerobes were not significantly different between feces of CH, + and CH, - (10.6 f 0.20 vs. 10.5 f 0.18), MA ranged from below the detection level of 2.0 to 9.8 (Table 1) and the average counts were significantly different between CH, + and CH, - . The corresponding SRB population levels were not significantly different between CH, + and CH, - . Counts of H,utilizing AB were significantly higher in CH, than in CH, + and were negatively correlated with counts of MA (Fig. 1).

Table 1 Distribution of H&king microorganisms in feces of CH,-excretors (CH, +) and non CH,-excretors (CH, -) a

CH, +(n = 8) CH, -(n=

End expiratory samples were obtained using a modified Haldane-Priestly tube [19] and head space gases were analyzed for CH, concentration by gas liquid chromatography [3]. Acetate concentration in the culture supernatant was measured by an enzy-

281

Ecology 17 (199.5) 279-284

P

11)

Methanogenic archaea

Sulfate-reducing bacteria

Acetogenic bacteria

8.8kO.2 (7.69.8) 2.6kO.4 (2Q6.1) < 0.001

7.1 If:0.4 (5.6;9.2) 7.3 k 0.4 (5.7;8.8) NS ’

4.6kO.7 (2.0;7.7) 6.6 * 0.4 (4.8;8.4) < 0.02

a Results are given as means of log,, microorganisms feces (wet wt) f S.E., (range). b NS, not statistically significant (P > 0.05).

per gram

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.I. Dart? et al. / FEMS Microbiology

Ecology 17 (1995) 279-284

SRB lactate. Confidence intervals (95%) for the slope and zero-ordinate were (0.814;1.159) and (- 1.348;1.221), respectively.

10

9t

4. Discussion

f 1

,

,

2

3

,

,

,

,

,‘.,

4

5

6

7

8

9

10

log,, MA per gram laces

Fig. 1. Relationship between counts of methanogenic archaea (MA) and acetogenic bacteria CAB) (log,, CAB) = - 0.308 log,, (MA) + 7.333, r = - 0.530; P.= 0.019).

3.2. Isolation of Hz-utilizing

AB from human feces

AB were isolated from the highest positive dilutions (10-5-10-7) using the roll-tube technique. Three strains were obtained from two CH, - individuals (TLFl, TLN3, VK64). All were strictly anaerobic Gram-positive cocci (0.8-1.0 pm> occurring mainly in pairs or chains. All isolates consumed HZ/CO, as well as glucose, lactose and xylose to produce acetate as major metabolite. On the basis of the above criteria, the hydrolysis of esculin [18] and HJCO, consumption 1211, the human colonic isolates appeared phenotypically related to the species Peptostreptococcus productus. The pathway of HZ/CO, utilization, with regards to the ratio of acetate produced to that expected based on consumed CO, equivalents, was consistent with the acetyl-CoA pathway of homoacetogens (ca. 91.1 + 2.0%, with a corresponding carbon balance of 94.3 f 2.0%, assessed upon third transfer and after 6 days of incubation). 3.3. Correlation utilizing SRB

between

counts

of lactate-or

H,-

A positive correlation was observed between the counts of SRB using H, and the counts using lactate as electron donors. The equation of the regression line (log,, (SRB H,) = 0.987 log,, (SRB lactate) 0.064) was not significantly different to SRB H, =

Former comparative enumerations of the human colonic H ,-utilizing microorganisms had only involved MA and SRB [3],[5]. Consistent with our previous report [3], in the present study SRB were detected in all subjects at similar population levels in both status groups. The metabolic versatility of SRB may explain their ability to maintain significant population levels even in the presence of high numbers of MA [6]. Indeed, in stools from CH, + , SRB may be expected to ferment lactate, transfering H,-equivalents to dominant MA, as reported for interspecies HZ-transfers between rumen strains 1221. Unlike former reports mentioning a smaller proportion of H,utilizing than lactate-utilizing SRB [23], and in agreement with descriptions of isolates [6], we observed a one-to-one relationship between SRB enumerated using H2 or lactate as electron donor. Our results showing that fecal Al3 populations are significantly higher in CH, - than in CH, + are consistent with previous NMR observations of H,incorporation into acetate dependent [ ‘3C]C02 [7],[8],[24]. The negative correlation observed between counts of MA and AB is indicative of a competitive interrelation between these two groups of microorganisms. The contribution of AI3 to acetogenesis may still be significant in the proximal colon where MA are less represented [25]. The versatility of AI3 appears as an ecological advantage for the maintenance of a significant population in the human colon [24]. We also report the selective enrichment and isolation of representatives of the dominant human colonic acetogenic population capable of using HZ/CO, as sole carbon and energy substrates. Two strains of hydrogen utilizing AB were recently isolated from the human colon of a CH, - individual [26]. The population levels were not directly investigated but the dominant strain could be estimated at 7.7 log,, per gram of dry feces, and was a Gram-positive coccobacillus. The isolates described therein appeared phenotypically related to P. productus, a

J. Do& et al. / FEMS Microbiology

species of which sewage sludge isolates have been shown to produce acetate from Hz/CO, [21]. However, none of the dominant P. productus strains characterized so far from gut contents appeared to be able to produce acetate from Hz/CO, [21]. Furthermore, other acetogenic bacterial species such as Eubacterium limosum could contribute to this pathway in the human colon [26]. The importance of reductive acetogenesis in the colonic microbial metabolism, estimated to account for 27% of total acetate production in CH, individuals [9], warrants further investigation. The phylogenetic and biochemical diversity, as well as the ecology of the microorganisms concerned are currently being investigated.

Acknowledgements This work was supported by grant 90.G.0265 from the French Minis&e de la Recherche et de 1’Enseignement Superieur.

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