Phenylacetic and Phenylpropionic Acids Do Not Affect Xylan Degradation by Ruminococcus albus

Since the addition of either ruminal fluid or a combinationof phenylacetic and phenylpropionic acids (PAA/PPA) has previouslybeen shown to dramatically improve cellulose degradation andgrowth of Ruminococcus albus, it was of interest to determinethe effects of these additives on xylan-grown cultures. Althoughcell-bound xylanase activity increased when either PAA/PPA orruminal fluid was added to the growth medium, total xylanasedid not change, and neither of these supplements affected thegrowth or xylan-degrading capacity of R. albus 8. Similarly,neither PAA/PPA nor ruminal fluid affected xylan degradationby multiple strains of R. albus when xylan prepared from oatspelts was used as a carbohydrate source. These results showthat the xylanolytic potential of R. albus is not conditionalon the availability of PAA/PPA or other components of ruminalfluid.

INTRODUCTION

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Introduction

Ruminococcus albus is a gram-positive anaerobe widely recognizedfor its high cellulolytic activity. A distinguishing featureof R. albus isolates is their dependence on the provision ofmicromolar concentrations of phenylacetic and phenylpropionicacids (PAA/PPA) for optimal rates of growth and cellulose degradation(12, 16, 18, 19, 20). PAA/PPA appear to be necessary for theformation of cell-associated, high-molecular-weight proteincomplexes believed to be cellulosomes (13). Many isolates ofR. albus have also been shown to degrade xylan and the hemicellulosefraction of plant cell walls (3, 9). Greve et al. (11) demonstratedthat R. albus strain 8 produces several enzymes involved inxylan degradation, including ß-1,4-xylanase, ß-xylosidase,and ${alpha}$ -arabinofuranosidase. The strain was also shown to fermentglucose and xylose residues present in alfalfa cell wall preparationsin preference to other sugars (11). However, there are no dataon the possible effect(s) from either PAA/PPA or other componentsof ruminal fluid on xylan degradation and growth of R. albus.Considering that heteroxylans represent a major part of theplant cell wall, it was of interest to determine whether optimalrates of R. albus growth, as well as xylan degradation, wouldbe conditional on the provision of PAA/PPA or ruminal fluid.

Bacterial strains and growth experiments.

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Bacterial strains and growth...

R. albus strains 8, B199, and 7 were obtained from the culturecollection at the National Center for Agricultural UtilizationResearch, U.S. Department of Agriculture, Peoria, Ill. In theexperiments described here the strains were cultured at 39°Cin a semidefined medium, described by Champion et al. (5), containing5% (vol/vol) clarified ruminal fluid (RF) or the same mediumwith ruminal fluid omitted but supplemented with either 25 µMeach of PAA and PPA (PA) or sterile water (WO). Carbohydratesources were included at a concentration of 0.4% (wt/vol). Pebble-milledWhatman No. 1 filter paper was used in cellulose-containingmedia, and the xylan preparations (birchwood and oat spelt)were purchased from Sigma Chemical Co., St. Louis, Mo. The bacterialstrains were passed no less than three times in the respectivemedium before each experiment.

In experiments with R. albus strain 8, the WO, PA, and RF mediawere prepared in duplicate 500-ml anaerobic bottles (BellcoGlass, Vineland, N.J.) fitted with a serum bottle closure thatcan be sealed with a butyl rubber stopper and aluminum seal.At each sampling time the bottles were mixed and a 10-ml samplewas collected anaerobically by using aseptic procedures. Disposablesterile pipettes, with their tips broken off to ensure no impedimentto the collection of the cellulose or xylan, were used to collectsamples. Residual cellulose was measured by the anthrone procedure(10). Water-soluble and -insoluble forms of residual xylan wereprecipitated by the addition of 1 M perchloric acid to culturesamples, and after centrifugation they were measured by theorcinol procedure (10). Bacterial growth was determined by recoveringbacterial cells by centrifugation, washing the pellets twicewith 1% (wt/vol) KCl, and, after boiling in 1% (wt/vol) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonatesolution for 20 min, measuring total protein by the method ofBradford (2), with bovine serum albumin used as a standard.

As expected, the rate of cellulose degradation by R. albus 8was dramatically improved in PA and RF cultures, the cellulosesolubilization rates being 0.86, 3.05, and 3.11 mg/ml/h forWO, PA, and RF cultures, respectively. Bacterial growth wasalso improved in PA and RF cultures (data not shown) in a mannersimilar to that of previous findings (12). The results verifiedthat R. albus 8 still requires PAA/PPA for maximal rates ofcellulose degradation and growth. In the first experiment withbirchwood xylan-containing cultures, samples were collectedevery 6 h for a total of 72 h. Much of the cell growth and xylandegradation occurred within the first 30 h of incubation (datanot shown), so a second experiment was conducted, with samplingintervals decreased to every 4 h for a total of 36 h. Xylandegradation and bacterial growth from these experiments areillustrated in Fig. 1. The degradation of acid-insoluble xylanwas rapid and largely complete within the first 4 h of incubation,and notably, there was little difference in the rate or extentof xylan degradation among the WO, PA, and RF cultures, norwere there differences in bacterial growth.

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FIG. 1. Time course measurement of R. albus 8 growth (A) and residual xylan (B) in a semidefined medium (5) prepared with either WO ( ${blacktriangleup}$ ), PA ( ${blacksquare}$ ), or 5% (vol/vol) RF ( ${diamondsuit}$ ). Data points represent the averages of the values obtained for samples from two cultures and analyzed in triplicate (n = 6). The lines represent the growth patterns predicted for WO (dashed line), PA (thin solid line), and RF (thick solid line) from using the values fitted to a logistic model (21).

In all three media the concentration of acid-soluble sugarsincreased rapidly, reaching maximal levels within 4 h (Fig.2) but declining over the next 16 h as bacterial growth proceeded.After 20 h of incubation, however, bacterial growth ceased andthe concentrations of the acid-soluble and -insoluble formsof carbohydrate remained largely unchanged for the remainderof the incubation period. The xylooligosaccharide profile inthe three types of cultures was analyzed by thin-layer chromatographyfollowing previously described methods (7). The oligosaccharideswere developed (one ascent) in a solvent of 6:1:1:2 (vol/vol)2-propanol, ethyl acetate, nitromethane, and water, which effectivelyresolved xylose (X₁) through xyloheptaose (X₇), and these werevisualized with an orcinol spray reagent (10 ml of H₂SO₄, 90ml of methanol, 0.2 g of orcinol) followed by heating to 100°C(7). The profiles are shown in Fig. 3. There were no measurablexylooligosaccharides present in the samples prior to inoculation(time zero). The samples collected 8 h postinoculation containedxylose (X₁) to xylohexaose (X₆), and there were no differencesin the profile among the three types of cultures. After 21 hof growth, only xylose and a trace amount of xylobiose wereevident, and neither arabinose nor glucose, which are not presentin birchwood xylan, was detected.

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FIG. 2. Time course measurement of total xylooligosaccharides present in culture fluids following growth of R. albus 8 in WO ( ${blacktriangleup}$ ), PA ( ${blacksquare}$ ), or RF ( ${diamondsuit}$ ) medium. Data points represent the average values obtained from samples of two cultures analyzed in duplicate (n = 4).

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FIG. 3. Thin-layer chromatography analysis of the soluble xylan degradation products produced during growth of R. albus 8 in medium prepared to contain either RF, 25 µM PA, or WO. The samples analyzed were collected at 0, 8, and 21 h after inoculation. Mixtures of xylose (X₁) and xylooligomers (X₂ to X₆) and of arabinose (Ara) and glucose (Glc) were used as standards. The oligosaccharides contained in the samples were identified by comparison to the thin-layer chromatography of authentic standards (Megazyme, Wicklow, Ireland).

Collectively, these data support the contention that there isno influence of PAA/PPA (or other components present in ruminalfluid) on xylan degradation, the profile of soluble xylooligomersproduced, or growth of R. albus 8. Xylan degradation was alsoincomplete, suggesting that the carbohydrate composition ofthe residual xylan may be recalcitrant to further hydrolysisand that growth by R. albus 8 is terminated as a result. Tofurther address the reason(s) underpinning incomplete xylandegradation, R. albus 8 was cultured in WO, PA, and RF mediafor 24 h, and then 2-ml samples of each culture were taken formeasurement of residual xylan. The remainder (8 ml) of eachculture was then centrifuged (2,500 x g for 20 min), and thesupernatant fraction was carefully removed with a sterile, stainlesssteel needle inserted through the butyl rubber closure of eachtube. The pelleted bacterial cells and residual xylan were resuspendedin 10 ml of sterile, anaerobically prepared WO, PA, and RF mediathat did not contain xylan. The cultures were reincubated foranother 24 h, and then the residual xylan was determined asdescribed above. After 24 h of incubation, xylan degradationwas 44, 49, and 45% in WO, PA, and RF cultures, respectively.After the addition of fresh medium, 85, 93, and 87% of the xylanwas degraded in WO, PA, and RF media, respectively, showingthat the cessation of xylan degradation and growth is not attributableto alterations in xylan composition but is perhaps due to xyloseaccumulation. Furthermore, neither PAA/PPA nor other componentsof ruminal fluid result in physiological changes that resultin enhanced xylan degradation or bacterial growth.

Enzyme assays.

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Enzyme assays.

Measurements of xylanase and xylosidase activities producedby R. albus 8 are presented in Table 1. Xylanase activity wasmeasured with birchwood xylan as the substrate, which was preparedas a 1% (wt/vol) suspension in 0.1 M NaPO₄ buffer (pH 7). Allassays were conducted aerobically at 39°C, and the linearrange of these assays with respect to protein concentrationand time was first determined. The reducing sugars releasedin 15 min were measured by using the dinitrosalicylic acid procedure(15), and xylose was used to produce a standard curve. One unitof enzyme activity was defined as 1 µmol of reducing sugarreleased per ml of culture. The amount of total xylanase activityproduced by R. albus 8 was similar in all three cultures, althoughmore activity remained cell associated when bacteria were culturedin PA and RF media than in WO medium (Table 1). PAA/PPA andruminal fluid appeared to affect enzyme retention rather thanenzyme production, but these changes did not result in improvedxylan degradation or bacterial growth (Fig. 1). Xylosidase activitywas determined by measuring the release of para-nitrophenol(pNP) from pNP-ß-D-xylopyranoside (pNPX; obtainedfrom Sigma). Total cellular proteins (45 to 60 µg) fromcultures harvested in the late logarithmic phase of growth wereadded to 200 µl of substrate (2.5 mM pNPX in 50 mM NaPO₄buffer [pH 6.8]) and were incubated at 39°C for 30 min.One unit of enzyme activity was defined as 1 nmol of pNP releasedper ml of culture. Xylosidase activity was relatively low inall samples, and it appears that neither PAA/PPA nor ruminalfluid stimulated the production or retention of this enzymeon the bacterial cell surface.

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TABLE 1. Xylosidase and xylanase activities of R. albus strain 8 following growth in xylan-containing cultures containing either no additions (WO), PA, or 5% (vol/vol) RF

Concluding remarks.

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Concluding remarks.

These experiments show for the first time that neither PAA/PPAnor other unidentified compound(s) in ruminal fluid are neededto maximize xylan degradation by R. albus 8 or its growth onxylan degradation products. While PAA/PPA and ruminal fluiddid not greatly affect total xylanase production by R. albus8, it did result in a change in the location of xylanase activity,with more remaining cell associated. The cellulosome from R.albus F-40 has been shown to contain xylanases (17), but thexylanases cloned from R. albus isolates do not possess, to date,dockerin domains, suggesting there are also noncellulosomalforms. Further studies may help identify whether the multiplexylanases present in R. albus are subject to different typesof regulatory control. However, when washed cell suspensionsfrom WO, PA, and RF cultures were used to inoculate xylan-containingmedium, there were still no differences in xylan degradation,suggesting that xylanase per se is not rate limiting to R. albusgrowth.

Birchwood xylan was specifically chosen for these experimentsbecause it is prepared by extraction with ethanol and base,should not be acetylated, and by mass is greater than 90% linearxylose polymers with little or no arabinose present (7). Assuch, it was presumed to be the most homogeneous form of xylanavailable to examine the xylanolytic potential of R. albus.However, the noncellulosic polysaccharides of most plant speciesconsumed by ruminants and herbivores are much more heterogeneousin composition, and it seemed possible that the stimulatoryeffects of PAA/PPA or ruminal fluid might be obscured due tothe xylan source used in these experiments. To address thisconcern, three different R. albus strains were cultured in theWO, PA, and RF media prepared with oat spelt xylan, and xylandegradation was measured from samples taken over 48 h, as describedabove. The results of these studies are shown in Fig. 4, andneither PAA/PPA nor other compounds present in ruminal fluidenhanced the xylan-degrading capacity of any of the R. albusstrains examined. Furthermore, the degradation patterns observedhere are similar to those seen by Dehority with R. albus 7 byusing hemicellulose preparations (8, 9). Based on these findings,we conclude that the findings made with R. albus 8 are typicalof other R. albus isolates with respect to polysaccharide degradationand that the findings with birchwood xylan are not confoundedby either the source or composition of this substrate.

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FIG. 4. Time course measurement of the degradation of oat spelt xylan by R. albus strains 8, 7, and B199 during growth in RF, PA, and WO media. Samples were taken from duplicate cultures at the times shown, and residual xylan was quantified by using the orcinol-based colorimetric assay as described in the text (7).

Although R. albus 8 produces both xylose and xylooligosaccharides,it only utilizes the latter as a carbohydrate source. Two othercellulolytic ruminal bacteria, Ruminococcus flavefaciens andFibrobacter succinogenes, are also xylanolytic, but some strainsof these species do not use xylose per se for growth (1, 13).Like these other cellulolytic bacteria, xylan degradation byR. albus facilitates its access to and use of plant cellulosesas a carbohydrate source, and at least some of the xylan degradationproducts are used by other ruminal bacteria. Cross-feeding betweenruminal bacteria has long been recognized (4), and some strainsof the nonxylanolytic bacterium Selenomonas ruminantium usexylooligosaccharides produced by xylanolytic bacteria such asButyrivibrio fibrisolvens (6, 7). However, given that cellulosedegradation by R. albus is maximal in the presence of PAA/PPA,the preservation of this conditional expression of cellulaseactivity suggests that an additional symbiotic relationship(s)underpins the role of R. albus in ruminal polysaccharide degradation.

ACKNOWLEDGMENTS

This work was supported by research grant US-3106-99C from BARD,The United States-Israel Binational Agricultural Research andDevelopment Fund, and the Ohio Agricultural Research and DevelopmentCenter.

We thank Rhonda Zeltwanger for expert technical assistance withcarbohydrate assays and Estelle Devillard and Ed Bayer for helpfuldiscussions.

FOOTNOTES

* Corresponding author. Mailing address: The MAPLE Research Program, Department of Animal Sciences, The Ohio State University, Columbus, OH 43210. Phone: (614) 688-5399. Fax: (614) 292-7116. E-mail: morrison.234{at}osu.edu.

REFERENCES

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