Polysaccharide degradation by human intestinal bacteria during growth under multi-substrate limiting conditions in a three-stage continuous culture system

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Polysaccharide degradation by human intestinal bacteria during growth under multi-substrate limiting conditions in a three-stage continuous culture system Sandra Macfarlane *, M.E. Quigley, M.J. Hopkins, Dorothy F. Newton, G.T. Macfarlane Medical Research Council Dunn Clinical Nutrition Centre, Hills Road, Cambridge CB2 2DH, UK Received 28 October 1997; revised 27 April 1998; accepted 5 May 1998

Abstract Human faecal microorganisms were grown on mixtures of chemically diverse polymerised C-sources (starch, pectin, xylan, mucin, arabinogalactan, inulin, guar gum) in a three-stage continuous culture model of the colon. The effects of retention time (R = 27.1 h, R = 66.7 h) on bacterial populations, their expression of hydrolytic enzymes involved in substrate depolymerisation, carbohydrate utilisation and short chain fatty acid formation were investigated. Eleven bacterial marker groups were studied in the fermenters. Strictly anaerobic bacteria predominated including bacteroides, bifidobacteria, clostridia and anaerobic Gram-positive cocci. Changing system retention time from 27.1 to 66.7 h did not significantly affect the predominant bacterial populations in V1, however, enterobacterial cell numbers increased in V3, while saccharolytic anaerobe recoveries declined markedly, reflecting their greater dependence on polymerised carbon sources in the fermentation system. The majority of polysaccharide degrading activity in the colon model was cell-associated, under all culture conditions. Increasing R from 27.1 to 66.7 h did not substantially change overall polysaccharidase (amylase, polygalacturonanase, xylanase, arabinogalactanase, galactomannanase) profiles, however, synthesis of some glycosidases was enhanced (e.g. Kglucosidase, N-acetyl-L-glucosaminidase, neuraminidase), whereas reduced expression of other enzymes such as Lgalactosidase, N-acetyl-K-galactosaminidase, K-fucosidase and K-arabinofuranosidase occurred. These observations demonstrate that catabolite regulation is an important control process in the colonic microbiota, with respect to the induction and repression of enzyme synthesis, and that substrate availability plays a major role in regulating bacterial metabolism. Measurements of carbohydrate utilisation demonstrated that while all polysaccharides in the feed medium were digested extensively by bacteria growing in the fermentation system, specific rates of carbohydrate utilisation were maximal at R = 27.1 h. These data also provided evidence of bacterial substrate preferences in the colon model, particularly in relation to xylan and inulin digestion, demonstrating that catabolite regulatory mechanisms were also involved in controlling the assimilation of carbohydrate in the microbiota. Short chain fatty acid measurements showed that fermentation was more efficient at R = 27.1 h compared to R = 66.7 h, with putative conversion of carbohydrate to short chain fatty acids being approximately 60% and 40%, respectively. This was probably due to increased maintenance energy requirements at low bacterial growth rates. Differences were also observed with respect to short chain fatty acid molar ratios, with more propionate, branched chain and longer chain

* Corresponding author. Tel.: +44 (1223) 415695; Fax: +44 (1223) 211273; E-mail: [email protected] 0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 3 9 - 7

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fatty acid production at R = 66.7 h, demonstrating the increasing importance of amino acid fermentation under these culture conditions. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Colonic bacterium; Large intestine ; Polysaccharide; Carbohydrate fermentation ; Continuous culture

1. Introduction Quantitatively, the major sources of carbon and energy for bacteria growing in the human large intestine are dietary starches, plant cell wall polysaccharides (¢bre) and oligosaccharides, together with host mucopolysaccharides that are associated with gastric, small intestinal and colonic mucins, and exfoliated epithelial cells [1,2]. While many di¡erent types of (limiting) carbohydrate are present in the large intestine at any given moment, concentrations of individual substrates are continually changing as they are broken down, replenished or replaced [3]. Together with dietary and endogenously produced proteins and peptides [4], these substances support a complex microbiota in the large bowel, comprising several hundred di¡erent bacterial species [5]. In the large intestine, polymerised carbohydrates are degraded by a wide range of bacterial polysaccharidases and glycosidases to smaller oligomers and their component sugars, which are subsequently fermented to short chain fatty acids (SCFA), H2 , CO2 , and a variety of other acidic and neutral end products [6]. Either through SCFA formation, or gas production, the fermentative activities of intestinal bacteria impact on many facets of host metabolism [7,8]. It is therefore important to understand the ecology and physiology of the colonic microbiota, and how the microbiota interacts with the host, especially in relation to substrate availability, since this can be manipulated to a large extent through diet. We have previously demonstrated that fermentation of di¡erent polysaccharides gives rise to speci¢c patterns of SCFA production, for example, butyrate and lactate are primarily associated with starch breakdown, while pectin fermentation mainly yields acetate [9,10]. However, nothing is known of how the microbiota deals with several substrates simultaneously, and simple fermentation experiments in the laboratory, using single carbohydrate sources, may

not provide realistic information on their metabolism in vivo. The aims of this study were to investigate processes involved in the depolymerisation and fermentation of complex carbohydrates by mixed populations of intestinal microorganisms, particularly in relation to substrate utilisation patterns. A three-stage continuous culture model was used in the investigation, which was designed to reproduce in vitro the disparate nutritional and environmental conditions (carbohydrate availability, pH, retention time) that a¡ect the growth and activities of bacteria growing in the proximal and distal colons [2]. This system has previously been used to investigate bacterial breakdown of pancreatic endopeptidases, and the relationship between sulfate-reducing bacteria and mucin fermentation by other intestinal microorganisms [11,12], and has been validated in conjunction with bacteriological and chemical measurements made on intestinal contents obtained at autopsy [13].

2. Materials and methods 2.1. The continuous culture system The colon model consisted of three glass fermentation vessels (V1, V2, V3) arranged in series, with working volumes of 220, 320 and 320 ml, respectively. pH and temperature (37³C) were controlled as described previously [12]. Culture pH in the three fermenters was maintained at 5.5 (V1), 6.2 (V2) and 6.8 (V3) to re£ect pH in the proximal, transverse and distal colons. The vessels were stirred and kept under an atmosphere of CO2 . The sterile culture medium was continuously sparged with O2 -free N2 and was fed by peristaltic pump to V1, which sequentially supplied V2 and V3. Several di¡erent polymerised carbon sources were included in the growth medium to enhance species diversity in the fermentation system, and their chemical compositions are shown in

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Table 1. The culture medium consisted of the following (g l31 ) in distilled water: starch (BDH), 5.0; porcine gastric mucin (Sigma type III), 4.0; xylan (oatspelt), 2.0; pectin (citrus), 2.0; guar gum, 1.0; arabinogalactan (larch wood), 2.0; inulin (chicory root), 1.0; yeast extract, 4.5; peptone water, 5.0; tryptone, 5.0; casein (BDH), 3.0; bile salts No. 3, 0.4; FeSO4 W7H2 O, 0.005; NaCl, 4.5; NaHCO3 , 1.5; KCl, 4.5; KH2 PO4 , 0.5; MgSO4 W7H2 O, 1.25; CaCl2 W6H2 O, 0.15; cysteine, 0.8; Tween 80, 1.0; haemin, 0.05. The system was initially operated at a retention time (R) of 27.1 h, followed by an increase to R = 66.7 h. Retention time was calculated as the reciprocal of dilution rate (D). System retention constituted the sum of individual R values in each fermenter. Dilution rates of individual culture vessels at R = 27.1 h were 0.14, 0.10 and 0.10 h31 , respectively. Corresponding values at R = 66.7 h were 0.06, 0.04 and 0.04 h31 . Each vessel was inoculated with 100 ml of a 20% (w/v) faecal slurry from a healthy nonmethanogenic donor. The cultures were allowed to equilibrate overnight, before medium was introduced to the system, which was operated for at least 336 h at each retention time to ensure steady state conditions had established, before material was taken for enzyme measurements, and chemical and bacteriological analyses. Steady state conditions were assessed by monitoring SCFA formation. At each steady state, two samples were taken either 48 h apart for chemical analyses and bacteriology, or 96 h apart for enzyme measurements. 2.2. Bacteriology Samples from individual fermentation vessels were

233

serially diluted in half-strength Wilkins Chalgren broth in an anaerobic cabinet (atmosphere: H2 , 10%; CO2 , 10%; N2 , 80%). Total facultative anaerobes were counted using nutrient agar. Total anaerobes, anaerobic Gram-positive cocci and clostridia other than C. perfringens were enumerated using Wilkins-Chalgren agar, with and without Gram-negative (GN) or non-sporing anaerobe (NS) antibiotic supplements (Oxoid). Bacteroides fragilis group organisms were determined using Bacteroides mineral salts agar [14]. Bi¢dobacteria were counted using MRS agar. Perfringens selective agar with antibiotic supplements, azide blood agar base and MacConkey agar No. 2 were used to enumerate Clostridium perfringens, enterococci and enterobacteria, respectively. Triplicate plates were inoculated with 0.1 ml sample from each dilution tube, and incubated at 37³C for 3 (facultative anaerobes) or 5 days (anaerobes). Facultative anaerobes were identi¢ed according to Cowan [15], anaerobic isolates were identi¢ed to genus level using the methods of Holdeman et al. [16]. 2.3. Polysaccharidase and glycosidase determinations Cell-associated and extracellular enzyme activities were measured. Bacteria (10 ml volumes) were harvested by centrifugation (23 000Ug, 30 min, 15³C), and both cell pellets and cell-free culture supernatants were retained. The bacterial pellets were washed once with anaerobic potassium phosphate bu¡er (50 mM, pH 7.0) and resuspended in the same solution. The organisms were then disrupted by two passages through a French pressure cell (1.1U105 kPa) to produce a crude cell extract. Poly-

Table 1 Composition and properties of polysaccharides used in this study Polymer

Principal monomeric constituents

Enzymes involved in substrate depolymerisation

Starch Mucin Pectin

Glucose Galactose, fucose, N-acetylglucosamine, neuraminic acid, N-acetylgalactosamine Galacturonic acid backbone, arabinose side chains

Guar gum Arabinogalactan

Mannose backbone, galactose side chains Galactose backbone, arabinose side chains

Xylan Inulin

Xylose backbone, arabinose side chains Fructose

Amylase, K-glucosidase L-Galactosidase, K-fucosidase, N-acetyl-L-glucosaminidase, neuraminidase, N-acetyl-K-galactosaminidase Polygalacturonanases (hydrolase, lyase), K-arabinofuranosidase Galactomannanase, L-mannosidase, K-galactosidase `Arabinogalactanase', L-galactosidase, K-arabinofuranosidase Xylanase, L-xylosidase, K-arabinofuranosidase L-Fructosidases

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saccharidase assays were carried out using native polysaccharide substrates (starch, pectin, guar gum, xylan, arabinogalactan), and glycosidase measurements employing a variety of p-nitrophenol substrates were carried out as described previously [17,18]. Neuraminidase activity was determined as follows: Test material (50 Wl), was added to 100 Wl substrate (1 mg ml31 N-acetylneuramin-lactose in 100 mM acetate bu¡er, pH 5.5), which was incubated at 37³C for 2 h. The reaction was stopped by heating at 100³C for 2 min. Released neuraminic acid was determined colorimetrically using the thiobarbituric assay method, as described by Warren [19]. 2.4. Chemical measurements With the exception of neuraminic acid, polysaccharide sugars in the feed medium, and residual carbohydrate in whole culture samples were measured by HPLC [20,21]. Neuraminic acid was determined colorimetrically by the thiobarbituric assay method (see above). Speci¢c rates of carbohydrate consumption (nmol min31 (mg dry wt. cells)31 ) by bacteria growing in di¡erent vessels of the fermentation system were calculated according to McKee et al. [22], as follows: qs = D(So 3S)/(xU60), where D is the dilution rate (h31 ), So is the concentration of substrate entering the fermenters (nmol ml31 ), S is residual substrate in the fermenters, and x represents community dry weight (mg ml31 ). Other samples from the fermentation vessels were centrifuged at 19 000Ug (20 min) to remove bacteria and other particulate substances. SCFA were determined by GC using procedures detailed by Macfarlane et al. [23]. These methods did not distinguish isovalerate from 2-methylbutyrate.

2.6. Chemicals Unless otherwise stated, all chemicals were obtained from Sigma. Bacteriological culture media and associated antibiotic supplements were purchased from Oxoid.

3. Results 3.1. Bacteriological analyses Results in Figs. 1^3 show the distribution of 11 di¡erent bacterial marker populations in V1, V2 and V3, respectively, at both system retention times. Anaerobic species outnumbered aerobes by one to two orders of magnitude, depending on culture con-

2.5. Culture dry weights A representative sample (1.0 ml) of culture taken from the fermenters was centrifuged at 13 000Ug for 10 min. The supernatant was discarded and a further 1.0 ml added and centrifuged. This was continued until bacteria from a total of 5.0 ml of culture had been collected. The bacterial pellet was then washed with distilled water before being dried to constant weight at 130³C.

Fig. 1. Enumeration of marker bacterial populations in vessel 1 of the colon model at di¡erent system retention times. Shaded bars correspond to R = 27.1 h, open bars are R = 66.7 h. Results are means of two separate samplings made 48 h apart, during steady state growth þ S.D.

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3.2. Activities of enzymes involved in substrate depolymerisation Results in Fig. 4 show polysaccharidase activities in di¡erent vessels of the fermentation system. These depolymerases were mainly associated with bacterial cells, however, substantial extracellular activities of some enzymes (amylase, polygalacturonanase, as well as xylanase at R = 66.7 h) were also observed. Amylase was the most active polysaccharide degrading enzyme in all culture vessels, at both retention times, followed by xylanase and polygalacturonanase. In general, bacterial synthesis of these enzymes increased from V1 to V3. Although there was reduced expression of arabinogalactanase at R = 66.7 h, and more galactomannanase was produced,

Fig. 2. Enumeration of marker bacterial populations in vessel 2 of the colon model at di¡erent system retention times. Shaded bars correspond to R = 27.1 h, open bars are R = 66.7 h. Results are means of two separate samplings made 48 h apart, during steady state growth þ S.D.

ditions. The predominant anaerobes were members of the B. fragilis group, bi¢dobacteria, clostridia and anaerobic Gram-positive cocci. Broadly, total anaerobe and total aerobe recoveries increased from V1 to V3 at R = 27.1 h, while the reverse occurred at R = 66.7 h. In V1, changing R to 66.7 h had little e¡ect on the major bacterial communities, although C. perfringens and lactobacillus counts declined markedly, while enterococcal numbers substantially increased. A similar pattern was evident in V2 (Fig. 2). However, total anaerobe counts declined markedly at R = 66.7 h in V3, due primarily to reductions in bacteroides, and to a lesser extent, populations of anaerobic Gram-positive cocci (Fig. 3). Conversely, enterobacterial numbers increased in this culture vessel at R = 66.7 h.

Fig. 3. Enumeration of marker bacterial populations in vessel 3 of the colon model at di¡erent system retention times. Shaded bars correspond to R = 27.1 h, open bars are R = 66.7 h. Results are means of two separate samplings made 48 h apart, during steady state growth þ S.D.

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Fig. 4. Polysaccharidase activities in the colon model at di¡erent system retention times. Open bars indicate cell-associated enzymes, closed bars show extracellular activities. Results are means of two separate measurements made 96 h apart under steady conditions.

increasing system retention time did not profoundly alter overall polysaccharidase enzyme pro¢les in the fermentation system.

Eight di¡erent glycoside hydrolases were measured in the colon model, as well as neuraminidase (Table 2). The vast majority of these enzymes were cell-

Table 2 E¡ect of retention time on bacterial synthesis of cell-associated and extracellular glycosidases Enzymea

K-Glucosidase K-Galactosidase L-Galactosidase K-Fucosidase N-Acetyl-L-glucosaminidase N-Acetyl-K-galactosaminidase L-Xylosidase K-Arabinofuranosidase Neuraminidaseb

R = 27.1 h

R = 66.7 h

Vessel 1

Vessel 2

Vessel 3

Vessel 1

Vessel 2

Vessel 3

747 (46.7) 524 (24.4) 2285 (58.8) 55.0 (1.8) 620 (56.5) 73.2 (15.3) 251 (5.7) 330 (9.1) 30.0 (18.2)

891 (103) 752 (34.7) 2581 (163) 98.2 (4.3) 98.1 (5.6) 85.1 (5.6) 238 (1.9) 308 (2.4) 30.4 (18.3)

556 (161) 1210 (49.5) 2281 (166) 96.6 (8.2) 96.6 (8.2) 77.0 (2.6) 282 (1.9) 302 (15.8) 28.7 (21.2)

1521 (25.5) 461 (28.5) 660 (40.6) 37.7 (2.4) 801 (41.1) 17.8 (5.0) 317 (13.2) 148 (20.0) 58.7 (32.8)

806 (101) 324 (98.5) 694 (72.6) 65.8 (14.9) 1065 (100) 21.9 (5.4) 197 (31.8) 143 (33.5) 58.9 (40.5)

701 (124) 48.0 (123) 582 (130) 45.9 (20.0) 976 (124) 14.0 (7.2) 160 (41.5) 97.3 (37.0) 72.4 (54.9)

a

nmol p-nitrophenol released h31 (mg dry wt. bacteria)31 . Wg N-acetylneuraminic acid released fom N-acetylneuramin-lactose h31 (mg dry wt. bacteria)31 . Results show mean cell-associated glycosidase activities recorded during two separate determinations made 96 h apart under steady state conditions. Values in parentheses are extracellular enzyme activities. b

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Table 3 E¡ect of retention time on carbohydrate utilisation in the fermentation system R (h)

27.1

66.7

Concentration (mg ml31 )

Vessel

Feed medium V1 V2 V3 V1 V2 V3

% Utilised

FUC

ARA

NGA

GAL

NGL

GLU

XYL

MAN UAC

NAN

FRU

Total

0.21 0.12 0.04 0.03 0.05 0.02 ND

0.60 0.37 0.12 0.08 0.19 0.06 0.05

0.21 0.19 0.05 0.04 0.08 0.04 0.02

2.93 1.74 0.28 0.21 1.27 0.20 0.14

0.46 0.21 Ta T 0.06 0.03 NDb

5.43 1.25 0.25 0.10 0.99 0.17 0.07

2.20 1.53 0.11 T 0.04 ND ND

0.85 0.30 T ND 0.10 ND ND

0.51 0.09 0.07 0.03 0.09 0.07 0.03

0.94 0.74 0.20 0.10 0.43 0.10 0.01

15.24 6.71 1.18 0.62 3.24 0.65 0.32

0.90 0.17 0.06 0.03 0.03 0.03 0.03

^ 56.0 92.3 95.9 78.7 95.7 97.9

FUC, fucose; ARA, arabinose; NGA, N-acetylgalactosamine ; GAL, galactose ; NGL, N-acetylglucosamine; GLU, glucose ; XYL, xylose; MAN, mannose ; UAC, uronic acid; NAN, N-acetylneuraminic acid ; FRU, fructose. a Trace ( 6 0.01 mg ml31 ). b Not detected.

bound, with the exception of neuraminidase, where signi¢cant extracellular activities were detected. LGalactosidase, K-glucosidase and K-galactosidase were important enzymes associated with breakdown of plant cell wall and storage polysaccharides (see Table 1). While L-galactosidase and N-acetyl-L-glucosaminidase were the principal mucin degrading enzymes, signi¢cant amounts of K-fucosidase, and Nacetyl-K-galactosaminidase were also formed. Although some glycosidase activities remained relatively constant in di¡erent vessels of the culture system, others varied markedly. Thus, at R = 27.1 h cellassociated K-fucosidase and K-galactosidase increased progressively from V1 to V3. In contrast, activities of N-acetyl-L-glucosaminidase were sixfold lower in V3 compared to V1. At R = 66.7 h, K-glucosidase, K-galactosidase, L-xylosidase and Karabinofuranosidase all declined considerably from V1 to V3.

3.3. Polysaccharide utilisation Although most polysaccharides in the feed medium were rapidly degraded by colonic microorganisms in V1, measurements of residual carbohydrate in spent culture media showed large qualitative and quantitative variations in carbohydrate consumption in individual culture vessels under di¡erent growth conditions (Table 3). Carbohydrate analysis showed that in order of quantitative importance, the main sugars fermented in V1 were glucose, galactose, uronic acid at R = 27.1 h, and glucose, xylose, galactose at R = 66.7 h. The principal substrates fermented in V2 were galactose, xylose, glucose (R = 27.1 h); glucose, fructose, arabinose (R = 66.7 h), and in V3, glucose, xylose, galactose (R = 27.1 h); glucose, fructose, galactose (R = 66.7 h). Less than 5% of polysaccharides in the culture feed medium were recovered in spent culture medium from V3, at either system retention time.

Table 4 Rates of carbohydrate utilisation (qs ) in individual culture vessels at di¡erent system retention times R (h)

27.1

66.7

Vessel

V1 V2 V3 V1 V2 V3

qs (nmol sugar min31 (mg dry wt. bacteria)31 ) FUC

ARA

NGA

GAL

NGL

GLU

XYL

MAN

UAC

NAN

FRU

Total carbohydrate

0.19 0.12 0.01 0.16 0.02 0.02

0.54 0.40 0.06 0.45 0.09 0.01

0.03 0.15 0.01 0.10 0.02 0.01

2.33 1.93 0.09 1.51 0.61 0.04

0.40 0.23 NAa 0.30 0.01 0.02

8.20 1.32 0.19 4.04 0.47 0.07

1.58 2.25 0.17 2.26 0.03 NA

1.08 0.40 NA 0.68 0.06 NA

1.33 0.14 0.04 0.74 0 0

0.48 0.02 0.03 0.22 0.01 0.02

0.39 0.71 0.13 0.46 0.19 0.06

16.55 7.67 0.73 10.92 1.51 0.25

a Not applicable, substrate completely utilised. See Table 3 for legend.

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Fig. 5. In£uence of system retention time on bacterial utilization of individual polysaccharides in di¡erent vessels of the colon model based on HPLC measurements of residual sugars in spent culture media. Closed circles show results from R = 27.1 h, open circles represent R = 66.7 h.

Speci¢c rates of utilisation of individual polysaccharide sugars (qs ) are shown in Table 4. Rates of carbohydrate uptake were greatest at R = 27.1 h, and with the exceptions of xylose, fructose and N-acetylgalactosamine, was maximal in V1. The lowest rates of carbohydrate utilisation were observed with mucin oligosaccharides (fucose, N-acetylgalactosamine, N-acetylglucosamine, neuraminic acid). Based on the known chemical compositions of polymerised carbohydrates in the culture medium (see Table 1), utilisation of individual polymers could be estimated from residual amounts of their component backbone monosaccharides in spent culture £uid. Arabinogalactan metabolism could not be followed by this method, due to the occurrence of its major backbone constituent (galactose) in mucin and

guar gum, and the presence of its side chain sugar arabinose in pectin and xylan. Fig. 5 shows that starch and pectin were fermented most rapidly, while inulin was slowly broken down, especially at R = 27.1 h. The extent of utilisation of individual polymers in V1 was invariably highest at R = 66.7 h, where major di¡erences in the levels of xylan (Table 3) and inulin (Table 4) metabolism were evident. 3.4. Short chain fatty acid formation Acetate, propionate and butyrate were the principal SCFA produced by colonic bacteria in the fermentation system, under all culture conditions (Table 5). Branched chain fatty acids (BCFA) were mainly formed in V2 and V3. Total SCFA produc-

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Table 5 Short chain fatty acids produced by bacteria in the fermentation system at di¡erent retention timesa SCFA

R = 27.1 h

Acetate Propionate Isobutyrate Butyrate Isovalerate/2-methylbutyrate Valerate Isocaproate Caproate Total

R = 66.7 h

Vessel 1

Vessel 2

Vessel 3

Vessel 1

Vessel 2

Vessel 3

38.9 (48.4) 21.7 (27.0) NDb 19.8 (24.6) ND ND ND ND 80.4

56.9 34.2 ND 32.9 4.4 6.0 ND Tc 134.4

61.6 36.8 ND 32.0 4.1 6.0 ND T 140.5

33.3 25.1 ND 17.0 1.6 ND ND ND 77.0

34.2 28.0 ND 20.4 4.2 6.5 0.1 0.1 93.5

37.4 29.0 ND 27.2 5.0 8.0 0.2 0.1 106.9

(42.3) (25.4) (24.5) (3.3) (4.5)

(43.8) (26.2) (22.8) (2.9) (4.3)

(43.2) (32.6) (22.1) (2.1)

(36.6) (29.9) (21.8) (4.5) (7.0) (0.1) (0.1)

(35.0) (27.1) (25.4) (4.7) (7.5) (0.2) (0.1)

a

Values are mM. Molar ratios are shown in parentheses. Not detected. c Trace ( 6 0.1 mM). b

tion was higher in all vessels at R = 27.1 h. Little variation was observed in molar ratios of the predominant fatty acids in individual fermentation vessels at each retention time, however, changes in the relative amounts of di¡erent SCFA formed were observed when R was increased to 66.7 h, where acetate production was reduced in comparison to propionate, BCFA and longer chain SCFAs. Table 6 summarises carbohydrate utilisation and SCFA (acetate, propionate, butyrate) formation in di¡erent vessels of the colon model. The apparent conversion of polysaccharides to SCFA was most e¤cient at R = 27.1 h (60%), reducing to approximately 40% when R was changed to 66.7 h. However, these results are approximate values only, since they do not take into account SCFA formed during amino acid fermentation.

4. Discussion Colonic transit times vary considerably between individuals, ranging for example, from around 20 to 120 h in the UK, with mean values of 60^70 h [24]. Because the rate of movement of digestive materials through the large bowel is an important factor a¡ecting the metabolic activities of intestinal microorganisms [25], we investigated the e¡ect of system retention time on polysaccharide breakdown in the colon model. A complex growth medium containing only polymerised C and N sources was used in these studies, to more realistically represent the types of substrates normally available to large intestinal bacteria [26]. By incorporating a multiplicity of fermentable substrates into the culture medium, more diverse populations of bacteria could be maintained in the gut model [27]. Results showed that complex microbiotas were maintained in all three vessels of the fermentation

Table 6 SCFA (acetate, propionate, butyrate) production in relation to carbohydrate utilisation in the fermentation systema Vessel

1 2 3

R = 27.1 h

R = 66.7 h

SCFA producedb

Carbohydrate utilisedb

Apparent conversion (%)

SCFA producedb

Carbohydrate utilisedb

Apparent conversion (%)

5.68 (5.68) 8.84 (3.16) 9.24 (0.40)

8.53 (8.53) 14.06 (5.53) 14.62 (0.56)

66.6 (66.6) 62.9 (57.1) 63.2 (71.4)

5.36 (5.36) 5.92 (0.56) 6.68 (0.86)

12.00 (12.00) 14.59 (2.59) 14.92 (0.33)

44.7 (44.7) 40.6 (21.6) 44.8 (261)

a Values are results calculated for the fermentation system as a whole. Data in parentheses show SCFA production and carbohydrate utilisation in each vessel of the system. b g l31 .

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system. Calculation of minimum cell doubling times (0.693/D) for bacteria growing in the fermentation system gives values of 5.0 h (V1), 6.9 h (V2) and 6.9 h (V3) at R = 27.1 h, respectively rising to 16.7, 25.0 and 25.0 h at R = 66.7 h. However, cell doubling times derived for V1 are the only signi¢cant values, because spent culture £uid from this fermenter continuously fed V2 and V3, preventing washout of bacterial populations that might otherwise have been unable to compete in these culture vessels. Anaerobes predominated in all of the fermentation vessels, particularly bacteroides and bi¢dobacteria, but clostridia and Gram-positive cocci were also detected in high numbers (Figs. 1^3). Increasing system retention time markedly reduced carbohydrate availability in the colon model, particularly in V2 and V3. Although this had relatively minor e¡ects on cell numbers of the predominant bacterial populations in V1 and V2, more profound e¡ects on anaerobe communities were observed in V3, especially those belonging to the B. fragilis group. The decline in these organisms re£ected their dependence on polymerised carbon sources, while the nutritionally versatile facultative anaerobes could still compete for a variety of other carbon and nitrogen sources in the fermenter, under these strictly carbohydrate-limited culture conditions. Species belonging to the genera Bacteroides and Bi¢dobacterium are ubiquitous in the human large intestine, constituting up to 25% and 30% of the total cultivable gut £ora respectively [28^30]. Both groups of bacteria are saccharolytic, obligate anaerobes that are important in the colonic ecosystem, due to their nutritional versatility, and their ability to digest complex polymers that are resistant to hydrolysis by human digestive enzymes [31]. In view of their high numbers, these bacteria appear to have played a major role in breaking down polysaccharides in the fermentation system. Measurements of hydrolytic enzymes involved in depolymerisation of complex carbohydrate substrates showed that as in the large intestine, the majority of their activities were cell-associated (Fig. 4, Table 2). While polysaccharidase pro¢les were broadly similar at di¡erent system retention times, large changes in enzyme activity were found with some glycosidases. They can be explained in terms of substrate availability and the induction and re-

pression of enzyme synthesis. The importance of substrate availability on enzyme induction is particularly evident in comparisons of N-acetyl-L-glucosaminidase activities in V1 with those in V2 and V3 at R = 27.1 h, as well as K-glucosidase, K-galactosidase, L-xylosidase and K-arabinofuranosidase at R = 66.7 h. Conversely, relief of catabolite repression, as a result of substrate depletion, can be seen in relation to K-galactosidase and K-fucosidase at R = 27.1 h, and the mucin degrading enzymes K-fucosidase, N-acetyl-L-glucosaminidase and neuraminidase at R = 66.7 h. Because saccharolytic bacteria exist in a multi-substrate limited environment in the large bowel, particularly in the distal colon [32], they must be able to quickly adapt to changing nutritional circumstances to ensure growth and survival. These organisms may regulate utilisation of polymerised C sources through control of synthesis of depolymerising enzymes and substrate uptake systems, as well as by more responsive and rapid acting mechanisms such as catabolite inhibition and inducer exclusion, which operate at the level of substrate transport into the cells [33]. An important characteristic of catabolite regulation in bacteria is the manifestation of substrate preferences in organisms grown in the presence of a mixture of carbon sources. This is often dependent on the relative availabilities of the substrates, and can be seen when bacteria are cultured on either polysaccharides or sugars. For example, carbon limited chemostats with Bacteroides ovatus grown on either guar gum (mannose backbone, galactose side chains) or xylan (xylose backbone, arabinose side chains) showed that the backbone and side chain sugars were simultaneously utilised [14]. Similarly, when the same organism was grown on a mixture of starch and arabinogalactan, both polymers were co-utilised during carbon limited growth, however, in carbon excess chemostats, starch was preferentially catabolised. This was not due to repressed synthesis of hydrolytic enzymes involved in substrate depolymerisation, because arabinose, and to a lesser extent galactose accumulated in the cultures [17]. Substrate preferences are highly variable in bacteria, even amongst species belonging to the same genus [34]. Measurements of substrate utilisation in this investigation showed that in general, carbohydrate uptake rates were greatest at high substrate

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concentrations in V1, although speci¢c rates of carbohydrate utilisation (qs ), did not always correlate directly with the overall amounts of carbohydrate fermented (Tables 3 and 4). Clear evidence of catabolite regulation of metabolism at R = 27.1 h was shown in regard to xylan and inulin fermentation, together with bacterial utilisation of mucin oligosaccharide constituents, such as N-acetylgalactosamine. However, this was not observed during more C limited growth at R = 66.7 h, where concentrations of repressor substrates were low. The principal SCFA formed during degradation of carbohydrates by bacteria growing in the large bowel are acetate, propionate and butyrate [35]. Other metabolic products include ethanol, lactate and succinate, but these substances are further fermented by cross-feeding species in the large gut, and with the occasional exception of the proximal bowel, do not accumulate to any signi¢cant extent in colonic contents [32]. Reduced SCFA production at R = 66.7 h (Table 5) showed that fermentation and energy generation was less e¤cient at lower speci¢c growth rates (D) in the fermenters, and this was a manifestation of higher bacterial maintenance energy requirements, which increase markedly at low dilution rates in continuous culture [36]. Acetate, propionate and butyrate were used in calculations of fermentation e¤ciency because they were the principal fermentation products in the colon model, and because BCFA and longer chain SCFA are primarily formed by amino acid fermentation [4]. The values in Table 6 should therefore to taken as being very approximate, in that they do not take into account SCFA production arising from dissimilatory amino acid metabolism. This is highlighted by the apparent conversion factor of 261% in V3 at R = 66.7 h, which is explained by the very low levels of carbohydrate being broken down in this fermentation vessel (Tables 3, 4 and 6), and by amino acid fermentation reactions becoming more signi¢cant during extreme carbohydrate limited growth, as occurs in the distal bowel [32]. Indeed, the increasing signi¢cance of these processes in the V3 was shown by BCFA and long chain SCFA production in Table 5. In conclusion, while this study demonstrated that the human colonic microbiota is able to simultaneously degrade complex mixtures of polymerised car-

241

bon sources, clear evidence was obtained for bacterial substrate preferences, and sequential utilisation of some carbohydrates. Since di¡erent bacterial species form distinct patterns of metabolic end products, catabolite regulatory mechanisms ultimately a¡ect the types and amounts of these substances that they can produce from individual substrates [37,38]. These control processes are ecologically important in the large intestine, and are of great physiological signi¢cance to the host, due to the fact that di¡erent fermentation products are metabolised at various body sites. For example, butyrate is the principal energy source for the colonic epithelium, and plays an important role in regulating epithelial and immune cell growth and apoptosis [39,40], while propionate is cleared by the liver and a¡ects cholesterol metabolism [41], and acetate is oxidised in brain, heart and peripheral tissues [42]. SCFA are rapidly absorbed from the large bowel and theoretical calculations indicate that these fermentation products may contribute as much as 10% of the hosts daily energy requirements [43,44]. To provide further information on how bacterial fermentation products are derived from their progenitor substrates, future studies need to investigate the metabolic activities of individual species and groups of intestinal microorganisms, as well as the interactions that occur between microbial communities occupying di¡erent nutritional niches in the colonic microbiota.

Acknowledgments D.F. Newton and M.J. Hopkins were supported by a grant from the European Commission (DG XII) AIR 2-CT94-1095.

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