pH-dependent cellulose-attachment by Fibrobacter succinogenes monitored by competitive PCR

In vitro fermentation studies were conducted to evaluate effects of initial medium pH on the extent of attachment on pure cellulose by one of major ruminal cellulolytic bacteria: Fibrobacter succinogenes via competitive PCR. After incubation for 10 min, the numbers of F. succinogenes attached to cellulose were 109/g dry matter (DM) of cellulose at all pH levels. Thereafter, the numbers of F. succinogenes attached to cellulose did not change to an appreciable extent until 48 h at higher initial pH. However, at low initial medium pH (5.8), the numbers of F. succinogenes attached to cellulose gradually decreased and drastically dropped at 48 h incubation (105/g DM). This may be the result of higher detachment rate or lower growth when F. succinogenes was exposed to initial medium pH less than 6.0. Higher initial pH resulted in higher DM disappearance. However, changes in the rate of in vitro cellulose disappearance were not synchronized with changes in the number of attached F. succinogenes.


INTRODUCTION
Fibrobacter succinogenes is considered as a representative cellulolytic bacteria in the rumen (Forsberg et al., 1997).Quantitative confi rmation of the rate and extent of fi bre attachment in vivo is necessary in order to estimate the contribution of the representative cellulolytic species to ruminal fi bre digestion.We recently developed competitive PCR assays that facilitate the rapid and accurate enumeration of F. succinogenes, without the need for culturing (not published).
Application of these assays to the analysis of fi bre-associated populations could provide quantitative information on these species in vivo.Moreover, such analysis may confi rm which species play the largest role in plant fi bre digestion in the rumen.
In the present study, we monitored the infl uence of initial in vitro medium pH on the attachment of F. succinogenes to cellulose using competitive PCR assays.

MATERIAL AND METHODS
Ruminal contents were obtained from a rumen cannulated Holstein steer which was fed twice daily with a mixture of 40% concentrate and 60% timothy hay.Ruminal content was homogenized under anaerobic condition, strained through four layers of cheese-cloth, and then mixed with a buffer solution (McDougall, 1948).Half a gram of Sigmacell ® cellulose (Sigma-Aldrich Corporation, USA) was added to the medium and fi nally initial pH was adjusted to 5.8, 6.2 and 6.8 with a sodium carbonate solution.All the tubes in triplicates were incubated at 39 °C for 0, 10 min, 2, 4, 8, 12, 24 and 48 h.
To maximize DNA extraction effi ciencies, physical disruption (Reilly et al., 1998) was used to extract DNA from freeze-dried cellulose samples, which were obtained by centrifugation after respective incubation time.The numbers of F. succinogenes attached to Sigmacell R cellulose (Sigma-Aldrich Corporation, USA) were enumerated by competitive PCR assay.Species specifi c primer sets for F. succinogenes were used to obtain PCR products.Proper internal control was constructed by using restriction enzyme (Ssp I and Sma I).
A detachable pressure transducer and digital readout voltmeter (Laurel Electronics, Inc., CA, USA) were used to measure the headspace gas pressure of fermenting cultures.In vitro DM digestibility was measured by centrifugation method.Data were analysed using the general linear model (GLM) procedure of the Statistical Analysis System Institute, Inc. (SAS) (1985).

RESULTS AND DISCUSSION
Almost maximum microbial attachment was achieved within 10 min of incubation regardless of initial medium pH as shown in Figure 1.There was a slow increase in the number of attached microbes at medium and high initial pH until 4 h incubation, and thereafter the numbers declined to a small extent.However, low initial medium pH (5.8) caused a sharp decline in the numbers of attached F. succinogenes.

pH-DEPENDENT CELLULOSE-ATTACHMENT
Data on DM digestibility (Table 1) study indicated that little disappearance of cellulose occurred at pH values of less than 6.0.On the other hand DM disappearance increased with incubation time at medium or high medium pH.Disappearance of the incubated cellulose (DM digestibility) increased linearly up to 48 h, but the trend was not synchronized with changes in bacterial mass attached to the cellulose.Probably, this is due to the delayed increase in fi brolytic enzyme activities.Similar results have been presented by others (Koike et al., 2003).
The results of this study clearly indicate that attachment of F. succinogenes on cell surface is pH-dependent, and failure of attachment or enhanced detachment of major cellulolytic microbes may explain at least part of reduced fi bre digestion under lower rumen pH, which is a commonly observed phenomenon with high concentrate and low forage diets.

Figure 1 .
Figure 1.Infl uence of initial medium pH on the number of F. succinogenes attached to cellulose

Table 1 .
In vitro DM digestibility (%) as infl uenced by different initial pH SEM, standard error of means a,b,c means in the same row with different superscripts differ (P<0.01) 1