identification of slowly metabolized sugars and sugar derivatives that could be used to establish...

CURRENT MICROBIOLOGY Vol. 19 (1989), pp. 271-274 Current Microbiology �9 Springer-Verlag New York Inc. 1989

Identification of Slowly Metabolized Sugars and Sugar Derivatives that Could Be Used to Establish New or Modified Microbial Species in the Rumen

R. John Wallace

Rowett Research Institute, Bucksburn, Aberdeen, UK

Abstract . For identification of compounds that could potentially be used to sustain a population of new or genetically modified organisms in the rumen, the rates of metabolism of several sugars and sugar derivatives were measured in ovine rumen fluid in vitro. Several sugars and sugar alcohols, including sorbitol, xylitol, dulcitol, arabinose, ribose, and maltitol, were degraded slowly and were therefore identified as candidates for use in this new manipulation strategy. None of the rumen bacteria, Bacteroides amylophilus WP91, Bacteroides ruminicola M384, Butyrivibrio fibrisolvens J W l l , Lactobacillus casei LB17, Megasphaera elsdenii J1, Seleno- monas ruminantium Z108, or Streptococcus boris C277, grew on the above sugar alcohols, and only B. ruminicola M384 and L. casei LB 17 grew significantly on the pentoses. The non-rumen strain, Escherichia coli ML308, grew rapidly on dulcitol and sorbitol; this suggests a possible role for E. coil in manipulation of rumen fermentation.

The advances that have been made in molecular genetics during the last decade have prompted rumen microbiologists to speculate as to how this technology might be used to manipulate rumen microorganisms and thereby rumen fermentation [6, 8, 14, 16, 17]. Enhanced cellulolytic activity, improved tolerance of cellulolytic bacteria to low pH, and an improved amino acid composition of rumen bacteria are among the many new properties that have been suggested to be possibly useful.

Once it has been decided which new property should be introduced, two main obstacles must be overcome. The first is the genetic manipulation of the microorganism chosen to carry the new property. Although the development of systems for the introduction of stable new genetic information into these organisms will undoubtedly take time, the advances already evident with rumen and other gut bacteria [3, 5, 11, 15] suggest that molecular genetics will not limit the success of such a venture. There is less indication, however, of how a new or modified strain could be introduced and then survive in the rumen, given that most new properties would almost certainly cause the organism to be at a selective disadvantage compared with the wild type or other indigenous species [14].

Address reprint requests to: Dr. R.J. Wallace, Rowett Research

One potential way of overcoming the latter problem may be to create an artificial ecological niche in rumen contents that enables the new organism to survive. The present paper identifies some sugars and sugar derivatives that are slowly metabolized and may be useful for this purpose, and de- scribes the growth of some of the major rumen bacterial species on the most promising of the compounds.

Materials and M e t h o d s

Metabolism of sugars in vitro. Samples of rumen contents were removed from an adult sheep via a rumen cannula 3 h after feeding a 67% grass hay/33% barley-based concentrate diet. The rumen fluid was strained through four layers of muslin, and 5 ml was added to test tubes containing 5 mg of sugar or sugar deriva- tive. Samples (0.5 ml) were added to 7.0 ml of 0.25 M HzSO 4 immediately or after incubating the tubes at 39~ for 1 h, and supernatants were prepared by centrifugation at 11,600 g for 10 min. Most sugars were assayed by a phenol-sulfuric acid method [13], and sugar alcohols by the colorimetric determination of formaldehyde formed by oxidation with periodic acid [19]. Su- pernatants containing fructose, sucrose, dulcitol, adonitol, and xylitol were neutralized with bicarbonate form ion-exchange resin IR-400 (BDH) and evaporated to dryness under reduced pressure. Trimethyl silyl oxime derivatives were prepared and quantified by GLC [12]. All compounds were D-isomers except for rhamnose.

Institute, Bucksburn, Aberdeen AB2 9SB, UK.

272 CURRENT MICROBIOLOGY Vol. 19 (1989)

Table 1. Utilization of sugars and sugar derivatives by mixed rumen microorganisms in vitro

Proportion of compound used

Mono-, di-, and Addition ~ Mean SD trihexoses Hexitols Pentoses Pentitols Deoxysugars Other

Mannitol 0.98 0.02 Sucrose 0.94 0.09 Fructose 0.94 0.10 Glucose 0.87 0.15 Maltose 0.81 0.22 Mannose 0.79 0.10 Salicin 0.78 0.09 Cellobiose 0.78 0.15 Galactose 0.77 0.16 Raffinose 0.68 0.10 Trehalose 0.66 0.04 Xylose 0.50 0.13 Sorbitol 0.49 0.06 Melibiose 0.46 0.11 Palatinose 0.29 0.10 Arabitol 0.24 0.15 Dulcitol 0.15 0.11 Ribose 0.11 0.08 Arabinose 0.09 0.06 Maltitol 0.09 0.12 Adonitol 0.06 0.09 Rhamnose 0.06 0.07 Lyxose 0.04 0.03 Fucose 0.02 0.04 Glucoheptose 0.02 0.05 Xylitol -0 .04 0.08

a Rumen fluid was taken from a sheep receiving a mixed roughage/concentrate diet, strained, and added to the compounds to give an initial concentration of 1 mg/ml. The mixture was then incubated in a shaking water bath at 39~ Samples were removed at zero time and after 1 h for carbohydrate analysis. Results are the means and SD of six incubations.

Table 2. Growth of pure cultures of bacteria on glucose and some compounds metabolized slowly by rumen contents

Growth at 24 h (0D650)"

Bacteria b Glucose Arabinose Dulcitol Maltitol Ribose Sorbitol Xylitol

Bacteroides ruminicola M384 0.83 0.15 c - - 0.20 - - - - Butyrivibrio fibrisolvens JW11 0.83 - - 0.02 . . . . Escherichia coli ML308 0.81 0.08 0.80 0.01 0.25 0.84 - - Lactobacillus casei LB17 0.70 0.07 - - - - 0.25 0.02 - - Megasphaera elsdenii J1 1.01 0.02 0.03 0.01 0.01 0.01 - - Setenomonas ruminantium Z108 0.70 0.02 - - - - 0.11 - - - - Streptococcus boris C277 1.10 0.02 0.02 - - 0.02 0.03 - -

a Bacteria were grown under CO2 in medium containing the compound to be investigated as sole energy source at 4 mg/ml. Results are expressed as net growth at 24 h, obtained by subtracting the OD650 of a culture containing no added energy source from the 0D650 of the culture on medium to which the compound had been added. Results are the averages of two cultures. An OD650 of 1.0 is equivalent to a cell density of 0.67-0.86 mg protein/ml, depending on the species. Results are not maximum growth yields, since most cultures had entered a stationary phase within a few hours, and declines in optical density had occurred. Furthermore, the quantity of sugar not fermented was not determined. b Ruminobacter (formerly Bacteroides) amylophilus WP91 was included in this study, but failed to grow on any of the above compounds, including glucose. c - - , no growth.

R.J. Wallace: Establishment of New Species in the Rumen 273

Bacterial growth on slowly metabolized sugars. For comparison of the anaerobic growth of several major species of rumen bacteria on selected, slowly metabolized compounds, the same com- plex medium as medium 2 of Hobson [9] was used, except that the compound was the only energy source present, at a concentration of 4 mg/ml, and the pH was adjusted to 7.2-7.6 by the addition of NaOH before autoclaving. Tubes containing appro- priate medium were inoculated from fresh cultures grown on complete medium 2, and were grown overnight at 39~ in Hungate tubes. They were then subcultured with a 5% (vol/vol) inoculum, and growth was measured turbidimetrically at 650 nm in a spectrophotometer (Novaspec, LKB) adapted to read Hungate tubes. The initial growth rate was determined from a semi-log plot of optical density against time. Protein was determined with the Folin reagent on cell pellets digested by NaOH [7]. Escherichia coil ML308 (ATCC 15224) was maintained on nutrient agar. The other rumen strains were isolated at the Rowett Research Institute and were maintained as described by Hobson [9]. The gas phase used throughout was O2-free CO2.

R e s u l t s a n d D i s c u s s i o n

Metabolism of sugars and sugar derivatives in rumen contents. The mos t rapidly fermented compounds in rumen fluid f rom a sheep receiving a mixed diet comprised a group containing predominant ly hex- oses and their di- or tr isaccharides (Table 1). Since most or all of these compounds were metabolized within 1 h, they would be unlikely to be of use in enriching for new organisms added to the rumen. Presumably these compounds reflect the natural substrates entering the rumen, and the indigenous populat ion is therefore well adapted to ferment them.

Pentoses and pentitols were metabolized more slowly (Table 1), so that an added concentrat ion of 1 g/liter might normally be expected to persist for be tween 2 and 10 h. Xylitol was particularly resistant. The deoxyhexoses , rhamnose and fucose, were also slowly fermented. Thus any of these compounds could potentially be used to sustain new species with the ability to ferment them, with per- haps larger concentrat ions of some pentoses and pentitols being required than for xylitol and the deoxyhexoses .

The hexitols and maltitol varied in their rates of breakdown. With the exception of mannitol, which was degraded rapidly, they could be considered as potentially useful (Table 1). Their relative cheap- ness would be at t ract ive compared with most other resistant compounds . Sorbitol and dulcitol (galacti- tol) can be produced at a similar cost to their parent aldoses.

The resis tance of xylitol and arabitol to microbial degradation in the rumen has been observed

before [10], as has the faster, but still slow relative to glucose, b reakdown of sorbitol [10, 18]. The reason for the earlier interest in sugar alcohols was for their beneficial effects on hepatic function.

Sorbitol was one of the compounds selected at this stage for further study. It is very much cheaper than any of the other compounds in the resistant groups, so although it was by no means the most resistant compound and little if any sorbitol would escape fermenta t ion in the rumen [18], it was chosen mainly for this economic reason. Selective en- r ichment for a sorbitol-fermenting organism for even a few hours could be exceedingly useful for manipulat ion purposes , depending on the other propert ies of the strain. The five next cheapes t compounds in the resistant group were xylitol, dulcitol, arabinose, r ibose, and maltitol, in that approximate order, and they were also included in a survey of their ability to support growth of several major species of rumen bacteria.

Growth of rumen bacteria and E. coli on slowly metabolized compounds. None of the selected compounds supported growth of any of the species of rumen bacter ia to a degree similar to glucose (Table 2). Bacteroides ruminicola M384 grew weakly on arabinose and ribose, consistent with the growth responses on these pentoses observed by Caldwell and N e w m a n [2], and Lactobacillus easel showed a similar pattern. In contrast , Escherichia coil ML308 grew well on dulcitol and sorbitol, and less well on the pentoses. The initial specific growth rate of E. coli ML308 on both dulcitol and sorbitol was 0.72 h -t , compared with 1. I0 h J for growth on glucose. Thus, if these growth rates could be sustained in vivo, considerable enr ichment of E. coli might be expected to occur.

Potential usefulness of slowly metabolized compounds in manipulation of rumen fermentation. The compounds identified here could be used in differ- ent ways to improve rumen fermentat ion by the in- t roduction of new or modified microorganisms. They could be added to the rumen in combinat ion with a non-nat ive or atypical ( to the rumen) organism, which was able to fe rment the compound and which either possessed naturally or was engineered to express a desirable proper ty or activity for the rumen fermentat ion. Thus the rumen would need to be inoculated once with the new organism, and that organism would then be maintained by the addition of the slowly fermented compound to the feed. Escherichia coil ML308, maintained by the addi-

274 CURRENT MICROBIOLOGY Vo1. 19 (1989)

t ion o f so rb i to l o r du lc i to l , w o u l d a p p e a r f rom the p r e s e n t e x p e r i m e n t s to be a p o t e n t i a l l y usefu l or- g a n i s m tha t does not n o r m a l l y a p p e a r in g rea t num- be r s in r u m e n c o n t e n t s [1 ,4] . N o d o u b t m a n y o the r s ex is t . A s t ra in o f E. col i with a des i r ab l e new p r o p - e r t y w o u l d have to be c o n s t r u c t e d , but , g iven the k n o w l e d g e tha t has a c c u m u l a t e d in r e c e n t y e a r s on the m o l e c u l a r b io logy and gene t i c s o f E. col i , find- ing a succes s fu l benef ic ia l p r o p e r t y w o u l d be m o r e diff icult t han its i n t r o d u c t i o n into E. coli .

A l t e r n a t i v e l y , the ab i l i ty to f e rmen t the com- p o u n d c o u l d be t r a n s f e r r e d to na t ive r u m e n organ- i sms , w h i c h w o u l d benef i t the f e r m e n t a t i o n if the i r n u m b e r s w e r e i n c r e a s e d . T h e y w o u l d be i n o c u l a t e d and the n e w p o p u l a t i o n s u p p o r t e d by the add i t i on o f the c o m p o u n d to the f eed as be fo re . Aga in , the de- t e r m i n a t i o n o f w h i c h o r g a n i s m is ac tua l ly benef ic ia l might p r e s e n t the g r e a t e s t p r o b l e m . H o w e v e r , the p r inc ip l e o f us ing some o f the c o m p o u n d s high- l ighted in this p a p e r for man ipu l a t i ng the r u m e n f e r m e n t a t i o n , and c o n c e i v a b l y even the h indgut f e r m e n t a t i o n in m o n o g a s t r i c an ima l s , r e m a i n s val id .

A m a j o r c o n c e r n in the long t e rm is tha t the i nd igenous p o p u l a t i o n m a y a d a p t to u t i l ize the a d d e d c o m p o u n d s and t h e r e f o r e o u t g r o w the intro- d u c e d o rgan i sm. A d a p t a t i o n to xy l i to l and a rab i to l d id o c c u r in the sheep r u m e n , but the i r ha l f - l ives r e m a i n e d g r e a t e r t han 4 h [10]. T h e s e c o m p o u n d s cou ld , t he re fo re , r e t a in the i r u se fu lness for a con- s i de r ab l e pe r iod . A d a p t a t i o n a lso o c c u r r e d wi th so rb i to l , the half- l i fe fal l ing f rom 4.5 h to 1.3 h a f te r 3 w e e k s [10]. A s imi la r deg ree o f a d a p t a t i o n to sor- b i to l was seen in e x p e r i m e n t s done a longs ide the p r e s e n t s tud ies . A d o s e o f 10 g o f sorb i to l admin i s - t e r e d to the s h e e p r u m e n was t a k e n up in 0 . 5 - 2 h a f te r 5 - 8 d a y s o f twice da i ly add i t ion , c o m p a r e d wi th 3 - 4 h in the n o n a d a p t e d an ima l s (R.J. W a l l a c e and H . J . F l in t , u n p u b l i s h e d resu l t s ) . Thus , a l though it w o u l d be e s sen t i a l for l ong - t e rm a p p l i c a t i o n o f so rb i to l tha t the n e w o r g a n i s m was able to o u t - c o m - p e t e the i nd igenous p o p u l a t i o n , shor t - to m e d i u m - t e rm a d v a n t a g e s cou ld c o n c e i v a b l y be ga ined b y its use e v e n i f the i nd igenous p o p u l a t i o n did e v e n t u a l l y adap t .

ACKNOWLEDGMENTS

I thank Margaret L. Falconer for technical assistance, P.J.S. Dewey for GLC analysis, and Drs H.J. Flint and C.S. Stewart for their helpful criticism.

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