Evolving specific non starch polysaccharide enzyme mix to paddy straw for enhancing its nutritive value

S Senthilkumar, C Valli and V Balakrishnan*

*Department of Animal Nutrition, Madras Veterinary College, Chennai- 600 007. Tamil Nadu, India
drbalakrishnanphd@yahoo.co.in

Abstract

A study was undertaken to assess appropriate fibrolytic enzymes inclusion on in vitro rumen fermentation pattern of paddy straw. Enzymatic release of monosaccharides from paddy straw lead to conclude that mixture of 80 IU of cellulase and 100 IU of xylanase are required per gram dry matter of paddy straw to maximize the nutritive value. The effect of predigestion of paddy straw with these fibrolytic enzymes was examined by allowing enzymes to react with substrate for 24 hours (ETPS) and compared against supplementation of these fibrolytic enzymes (ESPS), a positive control- water spraying alone (WSPS) was included in the experimental design.

In vitro gas production studies were used to evaluate the efficacy of the various treatments of paddy straw. The cumulative gas production measurements made at different hours of incubation revealed that ETPS was producing gas higher than ESPS. The volatile fatty acids and its stoichiometric derivations and fractionation of gas did not yield any statistical difference among treatments experimented. The partition factor derived at 48 hours of incubation suggested a comparable result in ETPS (3.97) and ESPS (3.79) but were significantly (P<0.05) higher than WSPS (3.62) and control (3.57). The measurement on apparent dry matter, true dry matter digestibility and microbial biomass production at 48 hours revealed that ETPS was comparable to UTPS and both were statistically (P<0.05) better than ESPS, while the WSPS or control were inferior on the above parameters studied. The digestibility of fiber fractions measurements made at 48 hours incubation revealed that ETPS was superior (P<0.01) over UTPS. 

From the study it was concluded that maximum monosaccharides can be released from paddy straw when it is treated with cellulase and xylanase (80 and 100 IU per g DM) for 24 hours. Enzyme treatment of paddy straw remarkably improves digestibility of DM and fiber fractions, however enzyme supplementation is less effective compared to enzyme treatment of paddy straw.

Key Words: crop residues, fibrolysis, Oryza sativa, rumen fermentation, urea

Introduction

Cereal crop residues form the staple feed for ruminant livestock in India. Among the various cereal crop residues, paddy straw (Oryza sativa) is the sole roughage source for majority of cattle and buffaloes reared in the southern states of India. The major limiting factor in the use of paddy straw as a roughage is its poor nutritive value. To enhance its nutritive value, various treatments (physical, chemical and biological) have been researched upon over the last two decades.

In this context, treatment of paddy straw with fibrolytic enzymes is gaining researcher’s attention. It has been demonstrated that (Morgavi et al 2000) exogenous fibrolytic enzymes work in synergy with the endogenous rumen microbial enzymes to enhance the digestion of high fibrous feed. Therefore, supplementation of fibrolytic enzymes to ruminant diets or pre-treatment of diets containing high levels of crop residues with fibrolytic enzymes is expected to enhance the digestibility and nutritive value of the diet. In addition use of fibrolytic enzymes would also pave way for effective utilization of paddy straw leading to increased economic benefits for the farmer. Hence, a study with the objective to assess the effect of addition of exogenous fibrolytic enzyme on in vitro rumen fermentation pattern of paddy straw was envisaged.
 

Materials and methods

Enzyme activity assay and fixing of optimal inclusion level of fibrolytic enzymes to paddy straw

Enzyme activity of the enzyme samples used for the study viz., cellulase and xylanase (four replication) were assayed by dinitro salicylic acid (DNSA) reducing sugar method (Miller 1959). The substrate used for cellulase was carboxy methyl cellulose and for xylanase it was oat spelt xylan. One IU of enzyme activity was the micro gram of sugar released at temperature of 40 ºC and pH 5 from one gramme of the respective substrate.

A 3x3 factorial experiment was designed with the objective of finding out the optimum inclusion level of fibrolytic enzymes (viz., cellulase and xylanase) to paddy straw, based on maximum sugar release. The enzyme level for cellulase (40, 60 and 80 IU/g DM) and xylanase (67, 100 and 133 IU/g DM) were considered. The procedure of Nsereko et al (2000) was adopted for the study. 

Six samples of dried, ground paddy straw (60 mg) were weighed into respective test tubes and mixed with 9 ml of 0.1 M citrate phosphate buffer (pH 6.5) containing sodium azide (0.1 mg/ml). Enzyme preparations (cellulase and xylanase at different combinations) were diluted in the same buffer (taking into account the associated activity) and 0.5 ml of each enzyme solution was added to the paddy straw.

A blank containing 10 ml of citrate phosphate buffer (pH 6.5) without enzymes was also prepared. The tube contents were mixed and incubated in a water bath at 39^oC for 24 hours. The tube contents were mixed again and 1.5 ml was pippeted from each tube into eppendorf tubes and the feed particles were removed by centrifugation (12000 x g for 5 minutes).  Samples were then placed in a boiling water bath for 5 minutes to inactivate the enzymes. From the eppendorf tubes, 1 ml of supernatant was pipetted into test tubes, 1 ml of DNSA solution was added and the reducing sugars were measured by DNSA reducing sugar method (Miller 1959). The enzyme combination producing maximum sugar release was identified to fix the optimal dose level of fibrolytic enzymes to paddy straw and was designated “enzyme mix”.

Treatment of paddy straw with enzymes / urea

In order to elicit the difference between enzyme treatment and enzyme supplementation, a study was undertaken by examining the in vitro fermentation and digestibility of “enzyme mix” treated paddy straw and “enzyme mix” supplemented paddy straw. Water being the carrier of enzymes mix, the effect of smoothening due to water was studied by spraying similar quantity of water to paddy straw. Paddy straw without any treatment constituted control group. 

In this study exactly weighed 1600 gram of 4-5 cm long chaffed paddy straw was divided into sixteen lots and each lot was randomly allotted to one of the following four treatments: control, water treated paddy straw (WSPS), enzyme treated paddy straw, enzyme supplemented paddy straw (ESPS). The procedure adopted for each treatment was as follows.

Control

Four lots, each having 100 gram of chaffed Paddy straw, without any treatment constituted the control group. 

Water treated paddy straw

Four lots, each having 100 gram of chaffed paddy straw was sprayed with 10 ml of distilled water alone to each lot. They were stored at room temperature for 24 hours and designated as “water sprayed paddy straw” (WSPS).

Enzyme treated paddy straw

Enzyme treatment of four lots of paddy straw was carried out by spreading 100 gram of chaffed paddy straw on a clean polythene sheet and enzyme mix (cellulase 8000 IU and xylanase 10000 IU/100 g DM in 10 ml of distilled water) was sprayed using chromatography sprayer as per procedure adopted by Dong et al (1999). The enzyme mix treated paddy straw was stored at room temperature for 24 hours. It was designated as “enzyme treated paddy straw” (ETPS). 

Enzyme supplemented paddy straw

The mode of action of enzyme mix supplementation was studied wherein the same enzymes at same concentration were sprayed to four lots of each having 100 g of paddy straw just prior to incubation and were designated as “enzyme supplemented paddy straw” (ESPS).

After 24 hours, the ETPS and WSPS were ground in a Wiley type mill to pass through 1 mm sieve and the samples were used for in vitro fermentation studies immediately

In vitro gas production studies

Samples from each treatment group were analyzed for fiber fractions as per the method of Goering and Van Soest (1970). They were evaluated using Hohenheim gas production test as per the procedure of Menke and Steingass (1988). The rumen liquor for the study was obtained from three cattle maintained on grazing alone to ensure that cellulolysis was optimum. Handling of rumen fluid in the laboratory was carried out by continuous flushing with CO_2­­. Total gas production was recorded at different incubation hours. The net gas volume at each incubation hour was calculated by subtracting the mean gas volume of blank from mean gas volume of syringe containing the sample. The total gas was partitioned as CO_2­­ and CH₄ using saturated KOH solution. A two ml syringe containing saturated solution of potassium hydroxide (KOH) was used to fractionate the fermented gas into carbon di oxide (Co₂) and methane (CH₄) as KOH absorbs Co₂ to form potassium carbonate leaving CH₄ intact. Hence known quantity of fermented gas samples were drawn from the calibrated 100 ml syringe into the two ml syringe containing saturated solution of potassium hydroxide. The residual gas left after one minute was considered to be CH₄ and the difference between the quantity of gas sample drawn and residual gas left after one minute was considered to be Co_2.  The results of gas volume at various time intervals were fitted to exponential equation (Blummel and Orskov 1993) and modified for lag phase as suggested by Krishnamoorthy et al (1991). 

The equation used was 

P = a + b (1 – e^–ct).

Where,

P = gas production,
t  = time,
a + b  = potential gas production and
c = rate of gas production. 
a, b and c are constant in exponential equation.

With lag phase:

P = a + b (1 – e^{-c (t-l)}).

Where l = initial lag for one set of fermentation.   

At the end of 48 hours of incubation, the entire contents of the syringes were transferred into 45 ml capped centrifuge tubes. Residues were centrifuged in an ultracentrifuge (HIMAC, model SCR 20BA, Hitachi) at 20,000 g for 30 minutes at 4^oC.  2.5 ml of supernatant was collected for VFA analysis. This was added into centrifuge glass tubes, which already contained 0.5 ml metaphosphoric acid (25 per cent). Total and individual short chain fatty acids concentrations were measured by gas chromatograph method as per the procedure of Chase (1990). Netel make of gas chromatograph model “omega QC” was used in this study.  The internal standard used in analysis was 2 ethyl butyric acid. The concentrations of volatile fatty acids (mmol/L) were calibrated from the standards by using a personal computer attached to the instrument itself. The following formulae were used for arriving at stoichiometric derivations  

Energetic efficiency of volatile fatty acid = 0.622 A + 1.092 P + 1.56 B/A + P + 2B
where A, P and B represent acetic, propionic and butyric acids (mol per cent) (Ørskov et al 1968).

The in vitro apparent digestibility of dry matter, in vitro true digestibility of dry matter and microbial biomass of control and treated paddy straw were studied at 48 hours of incubation as per Blummel et al (1997b).  The microbial biomass was calculated using the equation quoted by Blummel et al (1997a). Microbial biomass = Substrate truly digested – Substrate apparently digested. Partition factor was calculated using the following formula (Blummel et al 1997a).

 Four samples of control, WSPS, ETPS and ESPS  were subjected to in vitro studies as described by Tilley and Terry (1963) to evaluate the digestibility of fiber fractions.

Statistical analysis   

Statistical analysis of the data was done as per the method of Snedecor and Cochran (1980).

Results

Enzyme activity assay and fixing of optimal inclusion level of fibrolytic enzymes to paddy straw

Activity (IU/mg) of cellulase and xylanase used in the study revealed that the cellulase activity was 37.10 ± 2.06 IU/mg.  The cellulase also had a xylanase activity of 22.80 ± 0.78 IU/mg. Similarly the xylanase activity was 37.26 ± 1.76 IU/mg and it also had a cellulase activity of 11.43 ± 0.72 IU/mg. Thus, the level of associated activity of xylanase was 0.62 times of cellulase and the level of associated activity for xylanase with cellulase was 0.03 times of xylanase. The reducing sugars (mg per g DM) released due to the addition of different combination of fibrolytic enzymes (viz., cellulase and xylanase) to paddy straw at 2, 12, 18 and 24 hours of incubation are given in Table 1.

Among the various combinations of enzymes, the enzyme combination of cellulase III and xylanase II had the highest level of release of reducing sugars from paddy straw. The values for 2, 12 and 18 hours incubation were not statistically (P>0.05) significant. However, the monosaccharides release from paddy straw at 24 hours incubation was significantly (P<0.05) higher in cellulase III and xylanase II group than values for various other enzyme combinations.

In vitro gas production studies

The cumulative gas production (ml per 500 mg substrate) and CO₂: CH₄ ratio of control and treated paddy straw at different hours of incubation are presented in Table 2. 

At 24 hours ETPS showed a cumulative gas production of  38.55 ± 1.05 ml. Irrespective of treatments, there was no significant variation in cumulative gas production (ml) after 24 hours (i.e. 36, 48 and 72 hours) of incubation. However, at 12 hours of incubation the cumulative gas production of ETPS and ESPS was significantly (P<0.05) higher and at 24 hours highly significantly (P<0.01) compared to control and WSPS. The cumulative gas production (ml) as effected by the treatments and fitted through Neway Software (Neway 1992). The cumulative gas volumes from 500 mg of control and treated paddy straw ranged from 62.73 to 68.48 ml at 72 hours of incubation. The rate of gas production (c) ranged from 2.72 to 2.93 per cent per hour. The initiation of gas production (lag phase) varied with control and treated paddy straw [control (6.10 hours), WSPS (5.80 hours), ETPS (4.70 hours), ESPS (5.60 hours) and UTPS (5.50 hours)]. In ESPS also, the lag time was comparatively lower than that of control. The UTPS also had similar lag time.  The UTPS, which produced higher level of gas attained plateau at 72 hours and at that point of time the rest of the treatments also revealed similar gas production level. Irrespective of treatments there was no significant variation in the CO₂:CH₄ ratio at all hours of incubation. The experimental groups viz., ETPS, ESPS and UTPS showed a numerically better CO₂ to CH₄ ratio when compared to control and WSPS.

Dry matter, fibre fractions digestibility and microbial biomass production studies

The data on mean per cent in vitro apparent digestibility of dry matter (IVADDM) of control and treated paddy straw at 48 hours of incubation are presented in Table 3 

The result of the present study indicated that the apparent dry matter digestibility (P<0.01) of WSPS was similar to that of control. The ETPS was highly significant when compared to control at 48 hours of incubation. The apparent dry matter digestibility in ETPS at 48 hours improved to an extent of 13.28 per cent over the control. However, in ESPS it was only numerically higher than that of control and WSPS. The extent of improvement was only 5.21 per cent over control. Similar to apparent dry matter digestibility, the true dry matter digestibility (P<0.01) of WSPS did not significantly differ from that of the control. Among different dietary treatments maximum true dry matter digestibility was observed in ETPS at 48 hours of incubation. The true dry matter digestibility of ESPS was comparable to that of control and were significantly lower (P<0.01) from that of ETPS. The true dry matter digestibility of UTPS was significantly (P<0.01) higher than that of control but comparable to that of ETPS.

The per cent microbial biomass production at 48 hours of incubation of ETPS was significantly higher (P<0.01) than control and WSPS. The microbial biomass production of ESPS did not differ significantly (P>0.05) from that of control and were comparable, but lower than that of ETPS and UTPS.

In vitro digestibility of fiber fractions of WSPS was similar to that of control. In vitro digestibility of NDF, ADF, hemicellulose and cellulose of ETPS was significantly higher (P<0.01) than all the other treatments. In ETPS the digestibility of fiber fractions viz., NDF, ADF, hemicellulose and cellulose were respectively 16.42, 17.27, 37.0 and 28.88 per cent higher than that of control. The treatment effect of ESPS caused a significantly (P<0.01) higher NDF, ADF, hemicellulose and cellulose digestibility compared to control, however digestibility of fiber fractions were significantly (P<0.01) lower when compared to ETPS. The digestibility of NDF, ADF, hemicellulose and cellulose were higher by 8.62, 8.87, 15.21 and 9.52 per cent respectively compared to control. The study thus indicated that ETPS was comparable to UTPS and was superior to ESPS with regard to fiber digestibility.

The mean total volatile fatty acids, individual volatile fatty acids (mmol/L) concentration and stoichiometric derivations [Acetate to propionate ratio, non-glucogenic ratio (NGR), energetic efficiency (%) and partition factor] of control and treated paddy straw at 48 hours of incubation are presented in Table 3. No significant difference was noticed in total volatile fatty acid  concentration and volatile fatty acid fractions among treatments. The results also revealed that there was no significant variation in the stoichiometric derivations (Acetate to propionate ratio , nonglucogenic ratio and energetic efficiency ) of the various treatments from control.

The details of the results of partition factor (mg/ml) as effected by the treatments are furnished in Table 3. The values ranged from 3.57 to 4.07. The partition factor in the present study varied significantly (P<0.05) among the treatments. The partition factor (mg/ml) was significantly higher in UTPS (4.07 ± 0.17) and ETPS (3.97 ± 0.11) than that of control (3.57 ± 0.08) and WSPS (3.62 ± 0.11). The partition factor of ESPS was comparable to both the groups.
 

Discussion

Effect of enzyme addition on release of reducing sugars

Use of fibrolytic enzymes in ruminant diets having good or moderately good quality roughages have been studied (Feng et al 1996, Nsereko et al 2000). The effect of fibrolytic enzyme supplementation in paddy straw had been studied either after pre treatment of it with steam (Liu and Orskov 2000) or in ensiled paddy straw (Nakashima et al 1988). However, in the present study, the paddy straw was neither pre treated nor ensiled.

Enzymatic (80 IU cellulose and 100 IU xylanase per 100 g dry matter) release of monosaccharides from paddy straw at 24 hours of incubation was 56.22 mg per g DM was comparatively lower than 112.6 mg per DM  reported by Nsereko et al (2000), from alfalfa hay. The decreased enzymatic release of monosaccharides from paddy straw in the present study could be due to the marked degree of crystallinity within the cellulose polymer. Crystalline regions of cellulose are not readily accessible to endo acting cellulases (Sinitsyn et al 1990). Endoglucanases (EC 3.2.1.4) cleave only the b-1,4 glycosidic bonds of amorphous celluloses, but these enzymes are inactive towards crystalline cellulose. Xylanases (EC 3.2.1.8) are also specific for the internal b,1-4 linkages of xylans. However, exoglucanases or cellobiohydrolases (EC 3.2.1.91) have limited activity on crystalline cellulose (Bhat and Hazlewood 2001). Thus only a limited amount of crystalline cellulose in paddy straw could have been degraded by the fibrolytic enzymes. Another possible contributor to the low release of monosaccharides from paddy straw might have been the presence of lignin and silica. This was corroborated by Chaudhry (1998) that lignin adds rigidity to cell wall structure and renders cellulose and hemicellulose undegradable by entrapping them into various cross-linked cell wall complexes.

In vitro gas production studies

In vitro fermentation studies for 24 hours showed ETPS had a cumulative gas production of 38.55 ± 1.05 ml, which was higher than that reported (27.5 ml) for enzyme treated (cellulase 16 units) paddy straw by Liu and Orskov (2000). The higher value in the present study could be due to the use of tailor made enzyme mixture. At 12 hours of incubation the cumulative gas production of ETPS and ESPS were significantly (P<0.05) higher and at 24 hours highly significantly (P<0.01) than control and WSPS. Similar to the findings in the present study, Liu and Orskov (2000) reported that the gas production at 24 hours increased with addition of enzymes to steam treated paddy straw. The reason for increased gas production during initial hours of incubation in treated straws (ETPS and ESPS) could be due to effect of treatment, causing structural alterations of straw to facilitate higher degradation and gas production (Streeter and Horn 1980 and Nsereko et al 2000).  The degradation of the altered structural polysaccharides in ETPS and ESPS would have been exhausted within the first 24 hours of incubation, leading to similar gas production as that of control. Hence, not much of variation was noticed in cumulative gas production for treated straws compared to control after 24 hours of incubation.

The experimental groups viz., ETPS and ESPS showed a numerically better CO₂ to CH₄ ratio when compared to control and WSPS. The proportionately higher CO₂ produced might be due to increased microbial population in the ETPS and ESPS which in turn could have led to rapid DM disappearance. This is in close agreement with the findings of Srinivas and Singh (1998) that CO₂ production was positively correlated and CH₄ production was negatively correlated with the digestibility of straw substrate. A lower proportion of CH₄ production from treated straws (ETPS and ESPS) in the present study concurs with that reported by Moss et al (1994) who had reported a decrease in methane production per unit of digested feed when cereal straw was treated with alkali. Generally, methane producers are slow growing and last users of organic matter in food chain and scavenge on the indigestible material left after preliminary fermentation (Srinivas and Singh 1998).

Dry matter, fibre fractions digestibility and microbial biomass production studies

The higher apparent dry matter digestibility of ETPS could be due to the action of fibrolytic enzymes viz., cellulase and xylanase, which had acted on b, 1-4 linkages of cellulose and hemicelluloses (xylan) respectively to release soluble sugars (Bhat and Hazelwood 2001). It is quite possible that the released monosaccharides could have facilitated the growth of microbes, which in turn caused an increased apparent dry matter digestibility. Further, synergistic action of exogenous enzymes with endogenous rumen microbial enzymes may also be a probable reason (Morgavi et al 2000). It may also be possible that exogenously added fibrolytic enzymes enhanced microbial attachment and / or improved access to cell wall matrix and increased the apparent dry matter digestibility (Yang et al 1999). Another reasoning could be that colonization of microbes might have increased due to fibrolytic enzymes which in turn increased dry matter digestibility (Cheng et al 1995).

The extent of apparent dry matter digestibility improvement in ESPS was only 5.21 per cent over control. Similar results were corroborated by Treacher et al (1996). The reason for the reduced apparent dry matter digestibility of paddy straw by ESPS could be due to the fact that in this method, fibrolytic enzymes are only partially active (Hristov et al1998a). The reduced apparent dry matter digestibility could also be due to fluid associated enzyme activity. This theory (fluid associated enzyme activity) was supported by Wang and McAllister (2002). They claimed that efficacy of fluid associated enzyme activity was less than 30 per cent of total enzyme activity in the rumen.  Moreover application of enzymes to the feed by spraying prior to feeding leads to formation of enzyme substrate complex which increases the stability of enzymes (Wang and McAllister 2002).

The higher apparent dry matter digestibility of UTPS obviously was due to increased nitrogen (N) availability for microbes and also due to the alkali (ammonia) effect on the cell wall polysaccharides (Djajanegara and Doyle 1989). The reasons for increased true dry matter digestibility of ETPS might have been due to the action of fibrolytic enzymes on cellulose and hemicellulose to release soluble sugars.  Other reasoning’s as cited for the increase of apparent dry matter digestibility could also be reasoned out here.

The increased microbial biomass production in ETPS of the present study was in agreement with that reported by Nsereko et al (2002). Increase in microbial biomass production of ETPS could have been achieved by removing the many structural barriers of digestion and allowing the bacteria to colonize plant fiber and multiply there (Oellermann et al 1990). The microbial biomass production of ESPS did not differ significantly. The reason for this could be due to the fact that exogenous enzymes are rapidly degraded by an array of proteases produced by ruminal microorganisms, thus bringing down their activity (Kung 1996).

The increased fiber digestion of ETPS could be due to the effect of exogenous enzyme causing partial solubilization of NDF and ADF (Krause et al 1998) and also causing structural changes in fiber, thus making the feed more amenable to digestion (White et al 1993). Addition of exogenous enzymes to feed prior to feeding results in formation of enzyme-substrate complex. This bound enzyme with substrate protects or minimizes enzyme degradation by ruminal proteases and thus makes enzyme supplementation to act at its full efficacy (Kung 1996). The treatment effect of ESPS caused a significantly (P<0.01) higher NDF, ADF, hemicellulose and cellulose digestibility compared to control, however digestibility of fiber fractions were significantly (P<0.01) lower when compared to ETPS. This could be due to the low enzyme stability when cellulases were added directly to rumen fluid due to the proteolytic activity of rumen bacteria (Kopency et al 1987). The findings of this study for ESPS also concurs with that reported by Treacher et al (1996) who had stated that direct addition of enzymes into the rumen actually decreased fiber digestibility. The study thus indicates that ETPS is superior to ESPS with regard to fiber digestibility.

No significant difference was noticed in total volatile fatty acid concentration and volatile fatty acid fractions among treatments at 48 hours of incubation. The results in the present study are similar to the report of Dong et al (1999). Similar to the results obtained in the present study where ETPS had numerically lowered Acetate to propionate ratio than control, Yang et al (1999) also had reported that the Acetate to propionate ratio for enzyme treated diet was only numerically lower than that of control. As in case of Acetate to propionate ratio, the non glucogenic ratio and energetic efficiency of volatile fatty acid fractions of control and treated paddy straw did not differ significantly. Jalc et al (1994) also had observed no significant variation in the energetic efficiency of volatile fatty acid fractions between untreated wheat straw and Polyporous ciliatus treated wheat straw.

The partition factor in the present study varied significantly (P<0.05) among the treatments. The probable reason for significantly higher partition factor of ETPS compared to that of control and WSPS could be due to their higher digestibility brought about due to increased colonization of microbes in ETPS (Cheng et al 1995). In ESPS, the supplemented enzymes might have been partially degraded in the rumen due to the proteolytic activity of rumen microbes (Kung 1996) explaining why the partition factor for this group was comparable to both control and ETPS.

Acknowledgements

The authors acknowledge Tamil Nadu Veterinary and Animal Sciences University for the facilities provided at Department of Animal Nutrition, Madras Veterinary College for the successful conduction of the research.

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Received 21 August 2007; Accepted 26 September 2007; Published 11 December 2007

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