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Optimal mineral composition of artificial saliva for fermentation and methanogenesis in continuous culture of rumen microorganisms
Animal Feed Science and Technology 79 (1999) 43±55
Optimal mineral composition of artificial saliva for fermentation and methanogenesis in continuous culture of rumen microorganisms Laurent-Philippe Broudiscou1,a,*, Yves Papona, Anne F. Broudiscoub a
DeÂpartement Elevage et Nutrition des Animaux, Institut National de la Recherche Agronomique, Paris, France b LPRAI, 31190, Miremont, France Received 13 July 1998; accepted 28 December 1998
Abstract In dual outflow continuous fermenters on a 75 : 25 hay : barley diet, fermentation and gas production ÿ ÿ by mixed rumen microbes were tested in relation to the concentrations of HPO2ÿ 4 , HCO3 and Cl and the Na /K ratio in artificial saliva, by applying a 17-run Franquart design, and by fitting second-order ÿ ÿ polynomial models. The HPO2ÿ 4 , HCO3 , Cl concentrations and Na /K ratio ranged from 0.1 to ÿ1 ÿ1 ÿ1 ÿ1 4 g l , from 0.5 to 7 g l , from 0.1 to 0.5 g l and from 0.5 to 15 g g , respectively. The major factor ÿ 2ÿ was the concentration of HCOÿ 3 , followed by HPO4 and Na /K , while the concentration of Cl had negligible effects. The volatile fatty acid (VFA) production rate was mostly influenced by changes in 2ÿ pH, from 6.7 to 7.2, mediated by HCOÿ 3 and HPO4 concentrations. The analysis of the fermentation ÿ pattern confirmed the predominant action of HCO3 . Butyrate (C4) and branched chain VFA production rates, as well as C4 molar proportion in the fermentation broth were also lowered by high or low values of the Na/K ratio. The action of minerals on gas production and protozoa population density was complex and involved to an equal degree several experimental factors with a non-linear behaviour. Protozoa numbers, which varied from 14 to 38 mlÿ1, were favoured by high Na/K ratios. Using response surface analysis, the composition of a mineral base was optimised to simultaneously promote protozoa numbers and methanogenesis in vitro, at 39 mlÿ1 and 1.20 mmol hÿ1, respectively. The ÿ1 ÿ1 HCOÿ Clÿ, and had a resulting artificial saliva contained 1.65 g lÿ1 HPO2ÿ 4 , 4.19 g l 3 , and 0.22 g l ÿ1 Na /K ratio of 14.2 g g . # 1999 Elsevier Science B.V. All rights reserved. Keywords: Rumen; Methanogenesis; DOE; Fermentation; Micro-organism; Fermenter * Corresponding author. Tel.: +33-(0)-144-081-756; fax: +33-(0)-144-081-853. 1 Present address: Laboratoire de Nutrition et Alimentation, I.N.A.P.G., 16 rue Claude Bernard 75231, Paris cedex 05, France. 0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 0 0 2 - 4
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1. Introduction In ruminants, the minerals supplied by the diet or salivary secretion directly act on rumen fermentation by providing micro-organisms with enzymatic cofactors and essential elements for biosynthesis, and by modifying rumen pH and osmolality. They may, also, affect the host digestive physiology, e.g. rumen contents dilution rate and absorption rates of metabolites, with indirect consequences on rumen microbes. Most studies on the action of salts on rumen metabolism have been performed in husbandry conditions, and did not differentiate between the ways in which minerals influenced the microbes or take account of their actual influx. In vitro continuous cultures offer another experimental technique allowing the quantitation of the direct response of rumen microbes to a change in mineral supply under controlled conditions, without the confounding effect of absorption and secretion in vivo. An immediate application of this procedure is in the compounding of a balanced mineral based buffer solutions. In the present study, we investigated the effects of mineral salts on fermentation and gas production rate by mixed rumen microbes maintained on a standard diet, by applying response surface methodology. The resulting polynomial models were used to optimise the chemical composition of an artificial saliva which favours the in vitro maintenance of two sensitive rumen microbial populations, protozoa and methanogens. Some preliminary results have been published in abstract form (Broudiscou et al., 1997a). 2. Materials and methods Dual outflow continuous fermenters were used to run these experiments (Broudiscou et al., 1997b). In these culture systems, the fermentation broth was characterised by a differential turnover of particulate and liquid phases owing to the presence of an overflow and an outlet equipped with a filter (in our system, a 100 mm pore size stainless steel gauze). The design and operation of our fermenters differed from the original apparatus (Hoover et al., 1976) in the daily collection and analysis of fermentation gases, the supply of solid substrates in separate meals rather than on a semi-continuous mode, and the use of a marine impeller and baffles rather than flat-blade turbine impellers for fermentation broth mixing. The working volumes of vessels were 1100 (10) ml. 2.1. Experimental strategy The major inorganic components of natural and artificial salivas are potassium or sodium salts of bicarbonate, of hydrogen- and dihydrogen phosphate, and of chloride. In vitro, the pH may be adjusted by the addition of KOH or NaOH. The main characteristics of any saliva can, thus, be defined by four variables: (1) the amount of phosphates, expressed as hydrogen phosphate equivalent (HPO2ÿ 4 ), (2) the amount of carbonates ), (3) the amount of chloride (Clÿ), and (4) expressed as bicarbonate equivalent (HCOÿ 3 the ratio of sodium to potassium amounts (Na /K ). These variables were taken as experimental factors in our study, and we aimed at determining their quantitative effect ÿ ÿ on rumen microbial metabolism. The values assumed by HPO2ÿ 4 , HCO3 and Cl in our
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Table 1 Limits of the experimental domain Independent variables
Symbols
Levels
Coded
Natural
Amount of hydrogen phosphate
HPo
HPO2ÿ 4
Amount of bicarbonate
HCo
ÿ1 HCOÿ 3 (g l )
Amount of Chloride
Cl
Clÿ (g lÿ1)
Ratio of sodium : potassium
NaK
Na/K (g gÿ1)
ÿ1
(g l )
Coded
Natural
ÿ1 1 ÿ1 1 ÿ1 1 ÿ1 1
0.1 4 0.5 7 0.1 0.5 0.5 15
trial (Table 1) reproduced physiological variations around the average values reported by Clarke (1977) and Durand and Kawashima (1980). A distinctive feature of factor Na/K was the location of the values of greatest interest at the periphery of the experimental domain rather than at its centre, since the Na/K ratios in most salivas were close to either 14 or 2 (McDougall, 1948; Bowie, 1962; Gray et al., 1962; Rufener et al., 1963; Aafjes and Nijhof, 1967; Weller and Pilgrim, 1974; Hino et al., 1993). We thus dimensioned the interval of variation of this factor to include both sets of values. Published data have clearly demonstrated linear or quadratic responses of bacterial growth (Sistrom, 1960; O'Brien and Stern, 1969; Caldwell et al., 1973; Ingram and Thurston, 1976; Iijima et al., 1977; Atlas and Bartha, 1993; Cote and Gherna, 1994) or enzymatic activity (Halpern et al., 1973; Atlas and Bartha, 1993) to physiological changes in mineral supply, in accordance with the law of ecological tolerance formulated by V.E. Shelford in 1913. We thus modelled the relationships between experimental factors and microbial metabolism parameters by second-order polynomial equations. Response surface methodology, initiated by Box and Wilson (1951) and extensively reviewed in the statistics literature (Cochran and Cox, 1957; Box et al., 1978), appeared to be suited to our study by providing efficient experimental strategy and analysis to determine the relationships between the four independent variables and the responses relative to microbial metabolism. We explored the spherical domain comprised within the values given in Table 1, using an experimental design published by Franquart (1992). This design has been found using simulated annealing with D-optimality for criterion. Rather than selecting a central composite design for this trial, we opted in favour of Franquart design, which also led to a valid estimate of the response surface model, though with a much smaller number of experiments. As the Franquart design has not been published in the agronomic or statistical literatures yet, we have compared the main characteristics of both designs in Table 2, as summarised by Peissik (1995). Maximal inflation factors are equally satisfactory (below 4), and the Franquart design is close to orthogonality. Using this design leads to an adequate prediction of dependent variables (or responses), with a maximal variance function dMax of 1. The Franquart design is almost rotatable, as shown by the Khuri index which is above 98% (Khuri, 1988). Our experimental worksheet is developed in Table 3. All the runs but No. 16 are evenly distributed at the edge of the four-dimensional experimental domain. Run No. 16, at the
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Table 2 Characteristics of Franquart and central composite designs
Number of runs Factor levels Trace (X0 X)ÿ1 Fi Fii Fij Khuri Index (%) G-efficiency (%)
Franquart
Central composite
16 7; 7; 7; 7 18.34 1 1.62 1.01 99.6 93.75
25 5; 5; 5; 5 15.22 1 1 1 89.2 33.74
X: design matrix. Fi, Fii, Fij: maximal inflation factors for the terms Xi, Xi2 and Xij. Table 3 Experimental worksheet Run No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Experimental factors ÿ1 HPO2ÿ 4 (g l )
ÿ1 HCOÿ 3 (g l )
Clÿ (g lÿ1)
Na/K (g gÿ1)
1.3597 1.3597 2.7403 2.7403 0.5875 0.5875 3.5125 3.5125 0.6714 3.4287 2.0500 2.0500 2.0500 2.0500 2.0500 2.0500
1.7610 5.7390 1.7610 5.7390 2.3428 5.1573 2.3428 5.1573 3.7500 3.7500 0.9355 6.5645 3.7500 3.7500 3.7500 3.7500
0.4224 0.1776 0.1776 0.4224 0.2134 0.3866 0.3866 0.2134 0.3000 0.3000 0.3000 0.3000 0.1268 0.4732 0.3000 0.3000
10.3165 10.3165 10.3165 10.3165 9.5625 9.5625 9.5625 9.5625 2.6243 2.6243 4.1250 4.1250 4.1250 4.1250 15.0000 7.7500
See Table 1 for symbols.
centre, was applied three times to give an estimation of experimental error. The 18 runs were randomly assigned to six independent fermenters, identically assembled, each operated for three 7-day contiguous experimental periods. This randomisation was justified by a preliminary trial, where the fermenters were run identically for two 11-day experimental periods with no statistically significant differences between periods or between fermenters for most measurements (Broudiscou, unpublished data). 2.2. Experimental procedure Two wethers, fed on 1000 g per day chopped hay and 200 g per day ground and pelleted barley, were used as donors of rumen contents. 4 l of rumen contents were
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Table 4 Composition of feeds
ÿ1
Dry matter (DM g kg ) Composition of DM (g kgÿ1 DM) Organic matter Crude protein (N 6.25) Neutral detergent fibre Acid detergent fibre
Hay
Barley
921
911
913 148 634 342
924 129 157 60
withdrawn after a 24 h fasting period. They were immediately strained through a 1 mm stainless steel screen and kept at 398C under a continuous flow of N2. Within minutes, the vessels, previously filled with 700 ml of artificial saliva (pH 8.0) and 20 g of pelleted feed, were inoculated with 400 ml of strained rumen fluid, flushed with N2, then closed and started up. Each fermenter, maintained at 398C, was continuously infused with one of the 16 tested salivas. All the salivas were supplemented with 0.4 g lÿ1 HCl±Cysteine as a reducing agent. The fermentation broths were separately supplemented with 31.7 mg per day CaCl2, 47.5 mg per day MgCl2 and 0.755 g per day (NH4)2SO4. Ten g of a pelleted diet made of 750 g kgÿ1 orchard-grass hay and 250 g kgÿ1 ground barley was supplied to the fermenters at 10:00 hours and 20 g at 18:00 hours. The composition of feeds is given in Table 4. The saliva and the filtered effluent were pumped so as to obtain dilution rates of 0.03 hÿ1 and 0.06 ( 0.002) hÿ1, respectively for particle and liquid phases. All effluents were separately collected in a container kept at 48C. The fermentation gas volumes and the amount of effluents collected and saliva delivered were measured every day. After a 5-day adaptation period, the displaced and filtered effluents were collected on days 6 and 7, pooled and stored at ÿ208C, prior to subsampling for volatile fatty acids (VFA) and ammonia nitrogen (NH3-N) analysis. On day 6, the fermentation broths were sampled at 11:00 hours for pH, redox potential, osmotic pressure, VFA and NH3-N determinations. The samples for VFA and NH3-N analysis were mixed with 0.1 volume of H3PO4 8.2% (w/w) and stored at ÿ208C until analysis. On days 6 and 7 the fermentation gas composition was determined. At the end of the experimental period on day 7, a sample of filtered fermenter contents was fixed by addition of 3 volumes of a 13.3 ml lÿ1 glutaraldehyde, 666 ml 1ÿ1 glycerol solution and the protozoa population density determined. The osmotic pressure was measured by freezing point depression, VFA were determined as described by Jouany (1982), and NH3-N was determined as described by Davies and Taylor (1965). Fermentation gases were analysed by gas chromatography as described by Broudiscou et al. (1997b). The hydrogen balance was calculated as described by Demeyer and Van Nevel (1975). Phosphorus in feeds was determined by AFNOR standard method NF V 18±106. 2.3. Statistical analysis The results were submitted to response surface polynomial regression by the SAS procedure REG (SAS/STAT, 1990). The following second-order polynomial model was
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fitted to data: Y b0
4 X
bi X i
i1
4 X i1
bii Xi2
3 X X
bij Xi Xj
(1)
i1 j>i
where Xi were the four coded variables presented in Table 1, and b0, bi, bii and bij the 15 coefficients to be estimated. The coded variables Xi, used in multiple linear regression, varied from ÿ1 to 1. They were related to the experimental factors Ni, also called natural variables, by the equation: Xi
2 Ni ÿ Ni0 Ni
(2)
in which Ni0 is the value at the centre of the domain, and Ni is the interval of variation of Ni. For instance, Ni0 and Ni equalled 2.05 and 3.9 g lÿ1, respectively, for variable HPO2ÿ 4 . We assessed the goodness or the lack of fit of the models by visual analysis of the response residuals. Using the resulting polynomials, the mineral composition of the saliva which maximised simultaneously methane production and the protozoa population density was calculated by maximising a desirability function (Derringer and Suich, 1980), using a NEMROD procedure (DESIR/NEMROD, 1995). 3. Results All the regression analyses were performed on 17 runs, as one repetition of experiment No. 16 was stopped due to technical failure. These analyses were still valid because the number of data points was greater than the number of terms in the model by three (Zak, 1984). The estimates of coefficients for daily VFA production are given in Table 5. The VFA production rate ranged from 85 to 121 mmol per day and was poorly described by ÿ the model, with only quadratic effects of HPO2ÿ 4 and HCO3 , and an interaction between ÿ ÿ HCO3 and Cl . It was highest at the centre of the domain. For daily productions of individual VFA, the models provided unequal fits to data. Butyrate (C4) production varied from 10.3 to 17.8 mmol per day. It demonstrated a curvilinear negative effect of HCOÿ 3 , and was lowered by extreme values of Na /K . Acetate (C2) and propionate (C3) productions ranged from 56.9 to 77.1 mmol per day and from 15.1 to 22.2 mmol per day, respectively. C2 production was influenced in a non-linear way, mainly by HCOÿ 3 , then by Clÿ, with an interaction between both factors, and it was decreased by extreme values 2ÿ of HPO2ÿ 4 . The model for C3 production showed a curvilinear negative effect of HPO4 2ÿ inducing a sharp decrease in the response for high values of HPO4 , and a linear negative effect of HCOÿ 3. The fermentation broth variates (Table 6) were satisfactorily modelled except for VFA concentration, only influenced by HPO2ÿ 4 in a quadratic way. The pH ranged from 6.64 to 7.26 and regression analysis showed a linear increase with respect to HCOÿ 3 and a . The redox potential values ranged from ÿ364 to ÿ403 mV, quadratic effect of HPO2ÿ 4 2ÿ and HPO . The VFA molar proportions ranged and were mainly influenced by HCOÿ 3 4 from 59.7% to 68.6% for C2, from 15.8% to 22.3% for C3, and from 12.0% to 15.7% for
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Table 5 Linear regression applied to the production (mmol per day) of VFA, acetate (C2), propionate (C3), isobutyrate (IC4), butyrate (C4), isovalerate (IC5), valerate (C5) VFA R-square Adjusted R2 RSD Terms Intercept HPoa HCo Cl NaK HPo2 HCo2 Cl2 NaK2 HPo HCo HPo Cl HPo NaK HCo Cl HCo NaK Cl NaK
0.92 0.33 6.84
C2
C3
IC4
0.93 0.46 3.23
0.93 0.42 1.60
0.95 0.61 0.079
75.9 ÿ1.4 4.7 4.8 ÿ0.9 ÿ11.2 ÿ15.4* ÿ5.5 ÿ7.3 0.8 ÿ2.5 3.9 10.5 ÿ5.6 4.3
20.9 ÿ1.9 ÿ3.1* 0.3 ÿ0.1 ÿ3.4 ÿ1.5 ÿ2.3 ÿ0.2 1.6 ÿ0.5 1.0 3.1 ÿ1.6 0.2
0.79 0.02 0.03 0.14* 0.03 ÿ0.22* ÿ0.39* ÿ0.06 ÿ0.20* ÿ0.10 0.07 0.06 0.13 ÿ0.12 0.17
C4
IC5
C5
0.97 0.73 1.01
0.96 0.69 0.103
0.97 0.74 0.102
17.7 ÿ0.6 ÿ2.5 ** 0.8 ÿ0.7 ÿ2.7* ÿ4.1* ÿ1.1 ÿ3.1* ÿ1.3 ÿ2.8 2.0 2.4 ÿ2.1 1.6
1.21 ÿ0.01 0.09 0.15* 0.00 ÿ0.41* ÿ0.55** ÿ0.13 ÿ0.36* ÿ0.24 ÿ0.01 0.18 0.24 ÿ0.08 0.18
2.00 ÿ0.10 ÿ0.30 ** 0.10 0.01 ÿ0.39* ÿ0.24 ÿ0.04 ÿ0.23 ÿ0.02 ÿ0.10 0.08 0.24 ÿ0.32 * 0.19
Coefficients 119.3 ÿ4.2 ÿ1.5 6.3 ÿ1.6 ÿ18.6* ÿ22.4* ÿ9.0 ÿ11.6 0.6 ÿ5.7 7.4 16.9 ÿ10.6 6.8
a See Table 1 for symbols. Levels of significance for the null hypothesis: *p < 0.15,
**
p < 0.05.
C4. The molar proportion of C2 was linearly increased with respect to HCOÿ 3 concentration. This factor also acted on C3 in a curvilinear way, with a minimum reached ÿ1 for an HCOÿ 3 amount of approximately 5.7 g l . The effect on C4 was more complex. A canonical analysis, which is a multivariate statistical technique used to analyse secondorder response surfaces (Kettenring, 1971), showed that the proportion of C4 was lowest ÿ 2ÿ at extreme values of HCOÿ 3 and Na /K , central values of HPO4 and low values of Cl , ÿ and was maximised by central values of HCO3 and Na /K and by high values of and Clÿ. NH3-N concentration was always greater than 135 mg lÿ1. It was HPO2ÿ 4 linearly increased with respect to Na/K, but was lowered by the extreme values of 2ÿ HCOÿ and Clÿ and a negative interaction 3 . A positive interaction between HPO4 ÿ between Cl and Na /K was also observed. The osmotic pressure of the fermentation broth ranged from 107 to 296 milli-osmol lÿ1 and was correlated with the osmotic pressure of the saliva. The volume of gases ranged from 2.6 to 3.6 l per day, and the amount of methane produced from 0.85 to 1.58 M hÿ1, respectively. Both responses were satisfactorily modelled (Table 7). The daily volume of collected gases was mainly influenced by ÿ 2ÿ 2ÿ HCOÿ 3 and HPO4 , and to a lesser extent by Cl . It reached a maximum for HPO4 , ÿ ÿ1 ÿ1 ÿ1 ÿ HCO3 and Cl concentrations of 2.4 g l , 4.6 g l and 0.3 g l , respectively. The effects of experimental factors on methane production were more complex, with a ÿ quadratic effect of HPO2ÿ 4 , curvilinear effects of Na /K and HCO3 , and with significant
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Table 6 Results of the stepwise regression applied to pH, redox potential (Eh), VFA concentration, acetate (C2), propionate (C3) and butyrate (C4) molar proportions and ammoniacal nitrogen (NH3-N) concentration in the vessels at 16:00 hours pH R-square Adjusted R2 RSD
0.99 0.99 0.014
Eh (mv)
VFA (mM) C2 (%)
C3 (%)
C4 (%)
NH3-N (mg lÿ1)
0.98 0.87 4.09
0.91 0.30 3.40
0.99 0.99 0.070
0.98 0.84 0.693
0.99 0.95 0.255
0.96 0.71 7.88
Terms
Coefficients
Intercept HPoa HCo Cl NaK HPo2 HCo2 Cl2 NaK2 HPo HCo HPo Cl HPo NaK HCo Cl HCo NaK Cl NaK
7.00 ÿ380.2 0.04** ÿ4.6 0.32** ÿ22.1** 0.06** 1.9 0.02 ÿ1.1 0.14** ÿ14.2* ÿ0.06* ÿ0.2 0.05* ÿ6.7 0.03 ÿ7.7 ÿ0.04 4.4 ÿ0.10** 5.9 ÿ0.06* 6.7 ÿ0.03 8.5 ÿ0.09** ÿ3.8 0.08** ÿ2.0
72.5 ÿ2.8 2.1 ÿ1.8 ÿ0.2 ÿ12.4* ÿ6.1 ÿ6.9 ÿ1.9 ÿ8.9 2.7 3.9 1.9 2.2 ÿ3.0
63.55 0.57** 5.06** 0.48** 0.33** 0.05 0.32* 0.95** 0.13 0.95** 0.83** ÿ1.02** ÿ0.46** 1.09** 2.44**
17.76 ÿ0.97* ÿ2.73** ÿ0.92* 0.30 0.05 2.35* ÿ1.38 1.30 1.04 0.42 0.14 0.20 0.47 ÿ2.07*
14.36 0.34* ÿ1.78** 0.28 ÿ0.78** 0.13 ÿ1.74** 0.32 ÿ0.87* ÿ1.76** ÿ1.42** 0.69 0.35 ÿ0.75 ÿ0.35
175.5 4.2 5.2 9.0 16.3* ÿ16.4 ÿ30.4* ÿ10.7 ÿ4.2 ÿ10.4 27.9* 11.9 9.8 10.3 ÿ19.0
a See Table 1 for symbols. Levels of significance for the null hypothesis: *p < 0.15,
**
p < 0.05.
ÿ 2ÿ interactions between HCOÿ 3 , HPO4 , and Cl . A canonical analysis showed that 2ÿ ÿ methanogenesis was highest for HPO4 , HCO3 , Clÿ and Na/K, at 2.6 g lÿ1, 4.7 g lÿ1, 0.48 g lÿ1 and 5.8 g gÿ1, respectively. The protozoa population density comprised between 14 and 38 mlÿ1 and was clearly linked to Na/K by a positive curvilinear relationship. It was lowered by high or low values of HCOÿ 3 , and by an interaction 2ÿ between HCOÿ and HPO . The hydrogen balance was satisfactory in all experimental 3 4 runs, with hydrogen recovery rates higher than 0.9. The simultaneous maximisation of methanogenesis and protozoa numbers maintenance led to the mineral composition of artificial saliva given in Table 8. Fig. 1(a) and (b) show the contour plots of protozoa ÿ ÿ population density and methane production for HCOÿ 3 and Na /K , with HCO3 and Cl held at the values determined for the optimised saliva. Its composition was characterised by a high Na/K ratio, a low Clÿ content and intermediate values for HCOÿ 3 and . The variance function at this point was 0.862, the standard variations of both HPO2ÿ 4 responses are given in Table 8.
4. Discussion Our experimental domain complied with two constraints. It was within physiological limits (Clarke, 1977; Durand and Kawashima, 1980). Furthermore, it contained both
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Table 7 Results of the stepwise regression applied to the daily production of fermentation gases (volume), the production of methane (CH4) and the protozoa population density (Protozoa) CH4 (mmol hÿ1)
Volume (l per day) R-square Adjusted R2 RSD
0.99 0.92 0.089
Terms
Coefficients
Intercept HPoa HCo Cl NaK HPo2 HCo2 Cl2 NaK2 HPo HCo HPo Cl HPo NaK HCo Cl HCo NaK Cl NaK
3.63 ÿ0.30** 0.36** 0.03 ÿ0.03 ÿ0.71** ÿ0.71** ÿ0.40* ÿ0.17 0.05 0.00 0.15 0.28* ÿ0.08 ÿ0.12
a See Table 1 for symbols. Levels of significance for the null hypothesis: *p < 0.15,
Protozoa (mlÿ1)
0.99 0.90 0.0627
0.97 0.76 2.75
1.535 0.001 0.145** 0.078* ÿ0.137* ÿ0.485** ÿ0.379** ÿ0.152 ÿ0.280* ÿ0.314* 0.410** ÿ0.049 0.231* 0.135 ÿ0.043
25.7 0.9 0.6 ÿ3.9* 4.0* ÿ1.9 ÿ11.8* 0.2 7.0* ÿ9.8* ÿ4.6 ÿ5.2 5.5 3.9 ÿ5.3
**
p < 0.05.
Table 8 Characteristics of the saliva optimised by the procedure DESIR Response
SD
Methane production (mmol h ) Protozoa density (mlÿ1)
1.20 38.7
0.058 2.55
Coordinatesa ÿ1 HPO2ÿ 4 (g l ) ÿ1 HCOÿ (g l ) 3 Clÿ (g lÿ1) Na/K (g gÿ1)
1.65 4.19 0.22 14.2
ÿ1
a
See Table 1 for symbols.
artificial salivas concurrently used in continuous cultures of rumen microbes. The first saliva was proposed by Rufener et al. (1963), and adapted from the study of sheep salivary secretion by McDougall (1948). The second one was adapted by Weller and Pilgrim (1974) from the determination of the rumen fluid mineral composition in two cows by Aafjes and Nijhof (1967). The Rufener saliva is characterised by a high Na/K ratio, 13.6 versus 2.1, and provided slightly greater amounts of Clÿ and HPO2ÿ 4 . The rationale for both mineral compositions was essentially the accurate imitation of a natural
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Fig. 1. Contour plots of (a) protozoa population density and (b) methane bicarbonate concentration (HCOÿ 3 and ratio Na/K, at hydrogenophosphate and chloride concentrations of 1.65 and 0.22 g lÿ1.
state. From this standpoint, both artificial salivas based on either salivary secretion or rumen fluid, were equally justified. The determinations, however, were conducted on one or two animals, which is questionable. Moreover, assessing the sensitivity of rumen microbial metabolism to variations in the mineral content of an artificial saliva is important. A robust saliva would be able to sustain a stable rumen microbial population and activity in vitro, in spite of small changes in its mineral composition. This assessment justified the choice of an intermediate diet composed of ordinary feeds, while the use of synthetic substrates would have allowed a precise control of mineral influxes but would have been poorly representative of livestock feeds. In our experiment, the ions associated with each experimental factor had well defined metabolic roles. Bicarbonates are efficient buffers at pH values of approximately 6.5, and carbon dioxide is used by methanogens as an electron acceptor. Phosphates contribute to the buffering capacity of the medium, and phosphorus is a constituent of nucleic acids, phospholipids and coenzymes. Supplying potassium or sodium chloride merely changes the osmotic pressure. Potassium is a major cation in microbial cells, especially as a cofactor for such enzymes as phosphohexokinases. Sodium is required by a number of slightly halophilic rumen bacterial species (Caldwell et al., 1973; Durand and 2ÿ Kawashima, 1980). In general, the major factor was HCOÿ 3 , followed by HPO4 and ÿ Na /K , while Cl only had noticeable effects on VFA production. Unfortunately, the information available on quantitative effects of minerals on rumen microbe metabolism, with possible interactions, is scarce. The fermentation rate appeared to be mostly influenced by changes in pH mediated by amounts of bicarbonate, with a maximum reached at pH 7.0, and by the supply of phosphates. This could partly be explained by changes in the extent of carbohydrate hydrolysis. In fact, Stewart (1977) reported a similar relationship between pH and cotton degradation by mixed rumen microbes.
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However, in our experiment, the pH was always above 6.3, which is the generally recognised lower limit for optimal fibre degradation (Durand and Kawashima, 1980) and varied within a narrow range. This observation stresses the need for a precise control of pH in continuous cultures, with a tolerance of less than 0.1 pH unit. The analysis of the fermentation pattern confirmed the predominant action of bicarbonates on VFA production rates. A comparison of the present VFA data with in vivo data is of limited value due to the differential absorption of endproducts from the rumen. Phosphates probably also influenced VFA production by means of a change in pH. Phosphorus was supplied in a readily available form which met microbial requirements in all experimental runs, with at least 20 mg gÿ1 OMF (Durand and Komisarczuk, 1988). Moreover, both buffers only modified the redox potential within a narrow range below ÿ350 mV. The production of propionate followed a particular pattern which was characterised by the negative linear effect of bicarbonate. The pH increase may have directly penalised the C3-producing microorganisms, and it may be speculated that, in animals, bicarbonates increase the rumen fluid dilution rate and favour the escape of readily fermentable carbohydrates (Russell and Chow, 1993). The nature of cations was a relatively minor factor. However, it significantly affected C4 and branched chain VFA production rate and C4 molar proportion, probably because of specific requirements for the growth of some of the bacterial strains releasing these endproducts. Bryant et al. (1959) reported that Na and K were essential for the growth of Bacteroides succinogenes in pure culture. The effect of Na/K ratio on C4 production was probably related to bacterial metabolism, rather than to protozoa, which are active butyrate producers (Hungate, 1966; Williams, 1986) but whose numbers in our essay were influenced by the Na/K ratio in a different way. Mackie and Therion (1984) have stressed the greater sensitivity of bacteria to higher concentrations of potassium than sodium. Iwami et al. (1997) reported that Na and K ions had opposite effects on proton excretion in Streptococcus mutans grown in an anaerobic continuous culture system. The pattern of NH3-N concentration tended to mirror the VFA production rate, probably as a consequence of the effect of pH on fermentation, but it was never limiting and nor could it explain many of the changes in microbial metabolism. The action of minerals on gas production and protozoa population density was complex and involved several experimental factors to an equal degree with a non-linear behaviour. The most striking result was the promotion of protozoa numbers by high Na/K ratios, which had not been documented previously, but was confirmed by subsequent experiments on ciliate maintenance (Broudiscou et al., 1997b). Methanogens and protozoa are prominent agents in the rumen ecosystem and difficult to maintain in continuous cultures of mixed rumen microbes. Thus, values for the methane production and protozoa population density are good biological criteria for determining the most suitable composition of a rumen content simulating buffer. The composition of the buffer resulting from the maximisation procedure was close to that of the artificial saliva proposed by Rufener et al. (1963), which has rarely been used in dual outflow continuous cultures. If we take into account the data on fermentation, the intermediate amounts of buffer salts should warrant that small variations in the composition of the saliva would not ÿ alter the values of fermentative parameters. Though the co-ordinates for HPO2ÿ 4 , HCO3 , ÿ and Cl concentrations were central, the high value taken by the Na /K ratio located the
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optimal point at the edge of the experimental domain. This feature was likely to be observed since a constrained optimisation was performed. In our case, the major constraint was to confine the factors within physiological values, in order to allow the proper comparison of our future results with published data. The optimised saliva contained 6.082 g lÿ1 Na2HPO412H2O, 5.293 g lÿ1 NaHCO3, 0.566 g lÿ1 KHCO3, 0.363 g lÿ1 NaCl and 1.333 g lÿ1 NaOH. It was tested in a verification experiment, where the fermenters were run identically for two 11-day experimental periods (12 data points). Protozoa numbers and average methane production ± 45.9 mlÿ1 (SD of 8.7 mlÿ1) and 1.39 mmol ÿ1 (SD of 0.135 mmol hÿ1), respectively, ± were slightly higher than predicted values. The development of this saliva has validated conditions suitable for investigation of a number of current factors influencing rumen functions, in particular the screening of methane inhibitors or the nutritional interest of rumen ciliates. Acknowledgements The authors would like to thank Dr. S. Komisarzuk-Bony for analysis of phosphorus in feeds, Mrs. B. Lassalas for laboratory analyses, and L. L'Hotelier for taking care of experimental animals. References Aafjes, J.H., Nijhof, J.K., 1967. A simple artificial rumen giving good production of volatile fatty acids. Br. Vet. J. 123, 436±445. Atlas, R.M., Bartha, R., 1993. Microbial Ecology, Fundamentals and Applications, Benjamin/Cummings, Redwood city. Bowie, W.C., 1962. In vitro studies of rumen microorganisms, using a continuous-flow system. Am. J. Vet. Res. 23, 858±868. Box, G.E.P., Hunter, W.G., Hunter, J.S., 1978. Statistics for Experimenters: An Introduction to Design, Data Analysis and Model Building, Wiley, New York. Box, G.E.P., Wilson, K.B., 1951. On the experimental attainment of optimum conditions. J. Royal Stat. Soc. Series B 13, 1±45. Broudiscou, L.P., Papon, Y., Broudiscou, A.F., 1997a. Effect of minerals on methane production and protozoa numbers in continuous culture of rumen microorganisms. Reprod. Nutr. Develop. (Suppl. 1), 70±71. Broudiscou, L.P., Papon, Y., Fabre, M., Broudiscou, A.F., 1997b. Maintenance of rumen protozoa populations in a dual outflow continuous fermenter. J. Sci. Food Agric. 75, 273±280. Bryant, M.P., Robinson, I.M., Chu, H., 1959. Observations on the nutrition of Bacteroides succinogenes ± a ruminal cellulolytic bacterium. J. Dairy Sci. 42, 1831±1847. Caldwell, D.R., Keeney, M., Barton, J.S., Kelley, J.F., 1973. Sodium and other inorganic growth requirements of Bacteroides amylophilus. J. Bacteriol. 114, 782±789. Clarke, R.T.J., 1977. Methods for studying gut microbes. In: Clarke, R.T.J., Bauchop, T. (Eds.), Microbial Ecology of the Gut. Academic Press, London, pp. 1±33. Cochran, W.G., Cox, G.M., 1957. Experimental Designs. Wiley, New York. Cote, R.J., Gherna, R.L., 1994. Nutrition and media. In: Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R. (Eds.), Methods for General and Molecular Bacteriology. ASM, Washington, pp. 155±177. Davies, A.W., Taylor, K., 1965. Application of the autoanalyser in a river authority laboratory. In: Symposium Technicon, Technicon, Tarrytown, pp. 294±300.
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Demeyer, D.I., Van Nevel, C.J., 1975. Methanogenesis, an integrated part of carbohydrate fermentation and its control. In: McDonald, I.W., Warner, A.C.I. (Eds.), Digestion and Metabolism in the Ruminant. University of New England Publishing, Armidale, pp. 366±382. Derringer, G., Suich, R., 1980. Simultaneous optimization of several response variables. J. Quality Technol. 12, 214±219. Durand, M., Kawashima, R., 1980. Influence of minerals in rumen microbial digestion. In: Ruckebush, Y., Thivend, P. (Eds.), Digestive Physiology and Metabolism in Ruminants. MTP Press, Lancaster, pp. 375±408. Durand, M., Komisarczuk, S., 1988. Influence of major minerals on rumen microbiota. J. Nutr. 118, 249±260. Franquart, P., 1992. Optimisations multicriteÁres et meÂthodologie de la recherche expeÂrimentale. TheÁse en Sciences, Universite d'Aix-Marseille. Gray, F.V., Weller, R.A., Pilgrim, A.F., Jones, G.B., 1962. A stringent test for the artificial rumen. Aust. J. Agric. Res. 13, 343±349. Halpern, Y.S., Barash, H., Dover, S., Druck, K., 1973. Sodium and potassium requirements for active transport of glutamate by Escherichia coli K-12. J. Bacteriol. 114, 53±58. Hino, T., Sugiyama, M., Okumura, K., 1993. Maintenance of protozoa and methanogens, and fiber digestion in rumen-simulating continuous culture. J. Gen. Appl. Microbiol. 39, 35±45. Hoover, W.H., Crooker, B.A., Sniffen, C.J., 1976. Effects of differential solid±liquid removal rates on protozoa numbers in continuous cultures of rumen contents. J. Anim. Sci. 43, 528±534. Hungate, R.E., 1966. The Rumen and its Microbes. Academic Press, New York. Iijima, T., Diesterhaft, M.D., Freese, E., 1977. Sodium effect of growth on aspartate and genetic analysis of a Bacillus subtilis mutant with high aspartase activity. J. Bacteriol. 129, 1440±1447. Ingram, L.O., Thurston, E.L., 1976. Potassium requirements for cell division in Anacystis nidulans. J. Bacteriol. 125, 369±371. Iwami, Y., Guha-Chowdhury, N., Yamada, T., 1997. Effect of sodium and potassium ions on intracellular pH and proton excretion in glycolysing cells of Streptococcus mutans NCTC 10449 under strictly anerobic conditions. Oral Microbiol. Immunol. 12, 77±81. Jouany, J.P., 1982. Volatile fatty acid and alcohol determination in digestive contents, silage juices, bacterial cultures and anaerobic fermentor contents. Sci. Aliments 2, 131±144. Kettenring, J.R., 1971. Canonical analysis of several sets of variables. Biometrika 58, 433±451. Khuri, A.I., 1988. A measure of rotatability for response-surface designs. Technometrics 30, 95±104. Mackie, R.I., Therion, J.J., 1984. Influence of mineral interactions on growth efficiency of rumen bacteria. In: Gilchrist, F.M.C., Mackie, R.I. (Eds.), Herbivore Nutrition, Craighall Science Press, London, pp. 455±477. McDougall, E.I., 1948. Studies on Ruminant nutrition 1. The composition and output of sheep's saliva. Biochem. J. 43, 99±109. NEMROD, 1995. Guide d'Utilisation. LPRAI Sarl., Marseille. O'Brien, R.W., Stern, J.R., 1969. Requirements for sodium in the anaerobic growth of Aerobacter aerogenes on citrate. J. Bacteriol. 98, 388±393. Peissik, A., 1995. MeÂthodologie de la Recherche ExpeÂrimentale: proprieÂteÂs et caracteÂristiques des matrices d'expeÂriences pour les modeÁles polynomiaux du second degreÂ. TheÁse en Sciences, Universite d'AixMarseille. Rufener, W.H., Nelson, W.O., Wolin, M.J., 1963. Maintenance of the rumen microbial population in continuous culture. Appl. Microbiol. 11, 196±201. Russell, J.B., Chow, J.M., 1993. Another theory for the action of ruminal buffer salts: decreased starch fermentation and propionate production. J. Dairy Sci. 76, 826±830. SAS/STAT1, 1990. User's Guide, Release 6.04. SAS Institute, Cary. Sistrom, W.R., 1960. A requirement for sodium in the growth of Rhodopseudomonas spheroides. J. Gen. Microbiol. 22, 778±785. Stewart, C.S., 1977. Factors affecting the cellulolytic activity of rumen contents. Appl. Environ. Microbiol. 33, 497±502. Weller, R.A., Pilgrim, A.F., 1974. Passage of protozoa and volatile fatty acids from the rumen of the sheep and from a continuous in vitro fermentation system. Br. J. Nutr. 32, 341±351. Williams, A.G., 1986. Rumen holotrich ciliate protozoa. Microbiol. Rev. 50, 25±49. Zak, J.H., 1984. Biostatistical Analysis, 2nd ed. Prentice-Hall, Englewood Cliffs, NJ, pp. 328±359.
Optimal mineral composition of artificial saliva for fermentation and methanogenesis in continuous culture of rumen microorganisms Laurent-Philippe Broudiscou1,a,*, Yves Papona, Anne F. Broudiscoub a
DeÂpartement Elevage et Nutrition des Animaux, Institut National de la Recherche Agronomique, Paris, France b LPRAI, 31190, Miremont, France Received 13 July 1998; accepted 28 December 1998
Abstract In dual outflow continuous fermenters on a 75 : 25 hay : barley diet, fermentation and gas production ÿ ÿ by mixed rumen microbes were tested in relation to the concentrations of HPO2ÿ 4 , HCO3 and Cl and the Na /K ratio in artificial saliva, by applying a 17-run Franquart design, and by fitting second-order ÿ ÿ polynomial models. The HPO2ÿ 4 , HCO3 , Cl concentrations and Na /K ratio ranged from 0.1 to ÿ1 ÿ1 ÿ1 ÿ1 4 g l , from 0.5 to 7 g l , from 0.1 to 0.5 g l and from 0.5 to 15 g g , respectively. The major factor ÿ 2ÿ was the concentration of HCOÿ 3 , followed by HPO4 and Na /K , while the concentration of Cl had negligible effects. The volatile fatty acid (VFA) production rate was mostly influenced by changes in 2ÿ pH, from 6.7 to 7.2, mediated by HCOÿ 3 and HPO4 concentrations. The analysis of the fermentation ÿ pattern confirmed the predominant action of HCO3 . Butyrate (C4) and branched chain VFA production rates, as well as C4 molar proportion in the fermentation broth were also lowered by high or low values of the Na/K ratio. The action of minerals on gas production and protozoa population density was complex and involved to an equal degree several experimental factors with a non-linear behaviour. Protozoa numbers, which varied from 14 to 38 mlÿ1, were favoured by high Na/K ratios. Using response surface analysis, the composition of a mineral base was optimised to simultaneously promote protozoa numbers and methanogenesis in vitro, at 39 mlÿ1 and 1.20 mmol hÿ1, respectively. The ÿ1 ÿ1 HCOÿ Clÿ, and had a resulting artificial saliva contained 1.65 g lÿ1 HPO2ÿ 4 , 4.19 g l 3 , and 0.22 g l ÿ1 Na /K ratio of 14.2 g g . # 1999 Elsevier Science B.V. All rights reserved. Keywords: Rumen; Methanogenesis; DOE; Fermentation; Micro-organism; Fermenter * Corresponding author. Tel.: +33-(0)-144-081-756; fax: +33-(0)-144-081-853. 1 Present address: Laboratoire de Nutrition et Alimentation, I.N.A.P.G., 16 rue Claude Bernard 75231, Paris cedex 05, France. 0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 0 0 2 - 4
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1. Introduction In ruminants, the minerals supplied by the diet or salivary secretion directly act on rumen fermentation by providing micro-organisms with enzymatic cofactors and essential elements for biosynthesis, and by modifying rumen pH and osmolality. They may, also, affect the host digestive physiology, e.g. rumen contents dilution rate and absorption rates of metabolites, with indirect consequences on rumen microbes. Most studies on the action of salts on rumen metabolism have been performed in husbandry conditions, and did not differentiate between the ways in which minerals influenced the microbes or take account of their actual influx. In vitro continuous cultures offer another experimental technique allowing the quantitation of the direct response of rumen microbes to a change in mineral supply under controlled conditions, without the confounding effect of absorption and secretion in vivo. An immediate application of this procedure is in the compounding of a balanced mineral based buffer solutions. In the present study, we investigated the effects of mineral salts on fermentation and gas production rate by mixed rumen microbes maintained on a standard diet, by applying response surface methodology. The resulting polynomial models were used to optimise the chemical composition of an artificial saliva which favours the in vitro maintenance of two sensitive rumen microbial populations, protozoa and methanogens. Some preliminary results have been published in abstract form (Broudiscou et al., 1997a). 2. Materials and methods Dual outflow continuous fermenters were used to run these experiments (Broudiscou et al., 1997b). In these culture systems, the fermentation broth was characterised by a differential turnover of particulate and liquid phases owing to the presence of an overflow and an outlet equipped with a filter (in our system, a 100 mm pore size stainless steel gauze). The design and operation of our fermenters differed from the original apparatus (Hoover et al., 1976) in the daily collection and analysis of fermentation gases, the supply of solid substrates in separate meals rather than on a semi-continuous mode, and the use of a marine impeller and baffles rather than flat-blade turbine impellers for fermentation broth mixing. The working volumes of vessels were 1100 (10) ml. 2.1. Experimental strategy The major inorganic components of natural and artificial salivas are potassium or sodium salts of bicarbonate, of hydrogen- and dihydrogen phosphate, and of chloride. In vitro, the pH may be adjusted by the addition of KOH or NaOH. The main characteristics of any saliva can, thus, be defined by four variables: (1) the amount of phosphates, expressed as hydrogen phosphate equivalent (HPO2ÿ 4 ), (2) the amount of carbonates ), (3) the amount of chloride (Clÿ), and (4) expressed as bicarbonate equivalent (HCOÿ 3 the ratio of sodium to potassium amounts (Na /K ). These variables were taken as experimental factors in our study, and we aimed at determining their quantitative effect ÿ ÿ on rumen microbial metabolism. The values assumed by HPO2ÿ 4 , HCO3 and Cl in our
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45
Table 1 Limits of the experimental domain Independent variables
Symbols
Levels
Coded
Natural
Amount of hydrogen phosphate
HPo
HPO2ÿ 4
Amount of bicarbonate
HCo
ÿ1 HCOÿ 3 (g l )
Amount of Chloride
Cl
Clÿ (g lÿ1)
Ratio of sodium : potassium
NaK
Na/K (g gÿ1)
ÿ1
(g l )
Coded
Natural
ÿ1 1 ÿ1 1 ÿ1 1 ÿ1 1
0.1 4 0.5 7 0.1 0.5 0.5 15
trial (Table 1) reproduced physiological variations around the average values reported by Clarke (1977) and Durand and Kawashima (1980). A distinctive feature of factor Na/K was the location of the values of greatest interest at the periphery of the experimental domain rather than at its centre, since the Na/K ratios in most salivas were close to either 14 or 2 (McDougall, 1948; Bowie, 1962; Gray et al., 1962; Rufener et al., 1963; Aafjes and Nijhof, 1967; Weller and Pilgrim, 1974; Hino et al., 1993). We thus dimensioned the interval of variation of this factor to include both sets of values. Published data have clearly demonstrated linear or quadratic responses of bacterial growth (Sistrom, 1960; O'Brien and Stern, 1969; Caldwell et al., 1973; Ingram and Thurston, 1976; Iijima et al., 1977; Atlas and Bartha, 1993; Cote and Gherna, 1994) or enzymatic activity (Halpern et al., 1973; Atlas and Bartha, 1993) to physiological changes in mineral supply, in accordance with the law of ecological tolerance formulated by V.E. Shelford in 1913. We thus modelled the relationships between experimental factors and microbial metabolism parameters by second-order polynomial equations. Response surface methodology, initiated by Box and Wilson (1951) and extensively reviewed in the statistics literature (Cochran and Cox, 1957; Box et al., 1978), appeared to be suited to our study by providing efficient experimental strategy and analysis to determine the relationships between the four independent variables and the responses relative to microbial metabolism. We explored the spherical domain comprised within the values given in Table 1, using an experimental design published by Franquart (1992). This design has been found using simulated annealing with D-optimality for criterion. Rather than selecting a central composite design for this trial, we opted in favour of Franquart design, which also led to a valid estimate of the response surface model, though with a much smaller number of experiments. As the Franquart design has not been published in the agronomic or statistical literatures yet, we have compared the main characteristics of both designs in Table 2, as summarised by Peissik (1995). Maximal inflation factors are equally satisfactory (below 4), and the Franquart design is close to orthogonality. Using this design leads to an adequate prediction of dependent variables (or responses), with a maximal variance function dMax of 1. The Franquart design is almost rotatable, as shown by the Khuri index which is above 98% (Khuri, 1988). Our experimental worksheet is developed in Table 3. All the runs but No. 16 are evenly distributed at the edge of the four-dimensional experimental domain. Run No. 16, at the
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Table 2 Characteristics of Franquart and central composite designs
Number of runs Factor levels Trace (X0 X)ÿ1 Fi Fii Fij Khuri Index (%) G-efficiency (%)
Franquart
Central composite
16 7; 7; 7; 7 18.34 1 1.62 1.01 99.6 93.75
25 5; 5; 5; 5 15.22 1 1 1 89.2 33.74
X: design matrix. Fi, Fii, Fij: maximal inflation factors for the terms Xi, Xi2 and Xij. Table 3 Experimental worksheet Run No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Experimental factors ÿ1 HPO2ÿ 4 (g l )
ÿ1 HCOÿ 3 (g l )
Clÿ (g lÿ1)
Na/K (g gÿ1)
1.3597 1.3597 2.7403 2.7403 0.5875 0.5875 3.5125 3.5125 0.6714 3.4287 2.0500 2.0500 2.0500 2.0500 2.0500 2.0500
1.7610 5.7390 1.7610 5.7390 2.3428 5.1573 2.3428 5.1573 3.7500 3.7500 0.9355 6.5645 3.7500 3.7500 3.7500 3.7500
0.4224 0.1776 0.1776 0.4224 0.2134 0.3866 0.3866 0.2134 0.3000 0.3000 0.3000 0.3000 0.1268 0.4732 0.3000 0.3000
10.3165 10.3165 10.3165 10.3165 9.5625 9.5625 9.5625 9.5625 2.6243 2.6243 4.1250 4.1250 4.1250 4.1250 15.0000 7.7500
See Table 1 for symbols.
centre, was applied three times to give an estimation of experimental error. The 18 runs were randomly assigned to six independent fermenters, identically assembled, each operated for three 7-day contiguous experimental periods. This randomisation was justified by a preliminary trial, where the fermenters were run identically for two 11-day experimental periods with no statistically significant differences between periods or between fermenters for most measurements (Broudiscou, unpublished data). 2.2. Experimental procedure Two wethers, fed on 1000 g per day chopped hay and 200 g per day ground and pelleted barley, were used as donors of rumen contents. 4 l of rumen contents were
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Table 4 Composition of feeds
ÿ1
Dry matter (DM g kg ) Composition of DM (g kgÿ1 DM) Organic matter Crude protein (N 6.25) Neutral detergent fibre Acid detergent fibre
Hay
Barley
921
911
913 148 634 342
924 129 157 60
withdrawn after a 24 h fasting period. They were immediately strained through a 1 mm stainless steel screen and kept at 398C under a continuous flow of N2. Within minutes, the vessels, previously filled with 700 ml of artificial saliva (pH 8.0) and 20 g of pelleted feed, were inoculated with 400 ml of strained rumen fluid, flushed with N2, then closed and started up. Each fermenter, maintained at 398C, was continuously infused with one of the 16 tested salivas. All the salivas were supplemented with 0.4 g lÿ1 HCl±Cysteine as a reducing agent. The fermentation broths were separately supplemented with 31.7 mg per day CaCl2, 47.5 mg per day MgCl2 and 0.755 g per day (NH4)2SO4. Ten g of a pelleted diet made of 750 g kgÿ1 orchard-grass hay and 250 g kgÿ1 ground barley was supplied to the fermenters at 10:00 hours and 20 g at 18:00 hours. The composition of feeds is given in Table 4. The saliva and the filtered effluent were pumped so as to obtain dilution rates of 0.03 hÿ1 and 0.06 ( 0.002) hÿ1, respectively for particle and liquid phases. All effluents were separately collected in a container kept at 48C. The fermentation gas volumes and the amount of effluents collected and saliva delivered were measured every day. After a 5-day adaptation period, the displaced and filtered effluents were collected on days 6 and 7, pooled and stored at ÿ208C, prior to subsampling for volatile fatty acids (VFA) and ammonia nitrogen (NH3-N) analysis. On day 6, the fermentation broths were sampled at 11:00 hours for pH, redox potential, osmotic pressure, VFA and NH3-N determinations. The samples for VFA and NH3-N analysis were mixed with 0.1 volume of H3PO4 8.2% (w/w) and stored at ÿ208C until analysis. On days 6 and 7 the fermentation gas composition was determined. At the end of the experimental period on day 7, a sample of filtered fermenter contents was fixed by addition of 3 volumes of a 13.3 ml lÿ1 glutaraldehyde, 666 ml 1ÿ1 glycerol solution and the protozoa population density determined. The osmotic pressure was measured by freezing point depression, VFA were determined as described by Jouany (1982), and NH3-N was determined as described by Davies and Taylor (1965). Fermentation gases were analysed by gas chromatography as described by Broudiscou et al. (1997b). The hydrogen balance was calculated as described by Demeyer and Van Nevel (1975). Phosphorus in feeds was determined by AFNOR standard method NF V 18±106. 2.3. Statistical analysis The results were submitted to response surface polynomial regression by the SAS procedure REG (SAS/STAT, 1990). The following second-order polynomial model was
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fitted to data: Y b0
4 X
bi X i
i1
4 X i1
bii Xi2
3 X X
bij Xi Xj
(1)
i1 j>i
where Xi were the four coded variables presented in Table 1, and b0, bi, bii and bij the 15 coefficients to be estimated. The coded variables Xi, used in multiple linear regression, varied from ÿ1 to 1. They were related to the experimental factors Ni, also called natural variables, by the equation: Xi
2 Ni ÿ Ni0 Ni
(2)
in which Ni0 is the value at the centre of the domain, and Ni is the interval of variation of Ni. For instance, Ni0 and Ni equalled 2.05 and 3.9 g lÿ1, respectively, for variable HPO2ÿ 4 . We assessed the goodness or the lack of fit of the models by visual analysis of the response residuals. Using the resulting polynomials, the mineral composition of the saliva which maximised simultaneously methane production and the protozoa population density was calculated by maximising a desirability function (Derringer and Suich, 1980), using a NEMROD procedure (DESIR/NEMROD, 1995). 3. Results All the regression analyses were performed on 17 runs, as one repetition of experiment No. 16 was stopped due to technical failure. These analyses were still valid because the number of data points was greater than the number of terms in the model by three (Zak, 1984). The estimates of coefficients for daily VFA production are given in Table 5. The VFA production rate ranged from 85 to 121 mmol per day and was poorly described by ÿ the model, with only quadratic effects of HPO2ÿ 4 and HCO3 , and an interaction between ÿ ÿ HCO3 and Cl . It was highest at the centre of the domain. For daily productions of individual VFA, the models provided unequal fits to data. Butyrate (C4) production varied from 10.3 to 17.8 mmol per day. It demonstrated a curvilinear negative effect of HCOÿ 3 , and was lowered by extreme values of Na /K . Acetate (C2) and propionate (C3) productions ranged from 56.9 to 77.1 mmol per day and from 15.1 to 22.2 mmol per day, respectively. C2 production was influenced in a non-linear way, mainly by HCOÿ 3 , then by Clÿ, with an interaction between both factors, and it was decreased by extreme values 2ÿ of HPO2ÿ 4 . The model for C3 production showed a curvilinear negative effect of HPO4 2ÿ inducing a sharp decrease in the response for high values of HPO4 , and a linear negative effect of HCOÿ 3. The fermentation broth variates (Table 6) were satisfactorily modelled except for VFA concentration, only influenced by HPO2ÿ 4 in a quadratic way. The pH ranged from 6.64 to 7.26 and regression analysis showed a linear increase with respect to HCOÿ 3 and a . The redox potential values ranged from ÿ364 to ÿ403 mV, quadratic effect of HPO2ÿ 4 2ÿ and HPO . The VFA molar proportions ranged and were mainly influenced by HCOÿ 3 4 from 59.7% to 68.6% for C2, from 15.8% to 22.3% for C3, and from 12.0% to 15.7% for
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49
Table 5 Linear regression applied to the production (mmol per day) of VFA, acetate (C2), propionate (C3), isobutyrate (IC4), butyrate (C4), isovalerate (IC5), valerate (C5) VFA R-square Adjusted R2 RSD Terms Intercept HPoa HCo Cl NaK HPo2 HCo2 Cl2 NaK2 HPo HCo HPo Cl HPo NaK HCo Cl HCo NaK Cl NaK
0.92 0.33 6.84
C2
C3
IC4
0.93 0.46 3.23
0.93 0.42 1.60
0.95 0.61 0.079
75.9 ÿ1.4 4.7 4.8 ÿ0.9 ÿ11.2 ÿ15.4* ÿ5.5 ÿ7.3 0.8 ÿ2.5 3.9 10.5 ÿ5.6 4.3
20.9 ÿ1.9 ÿ3.1* 0.3 ÿ0.1 ÿ3.4 ÿ1.5 ÿ2.3 ÿ0.2 1.6 ÿ0.5 1.0 3.1 ÿ1.6 0.2
0.79 0.02 0.03 0.14* 0.03 ÿ0.22* ÿ0.39* ÿ0.06 ÿ0.20* ÿ0.10 0.07 0.06 0.13 ÿ0.12 0.17
C4
IC5
C5
0.97 0.73 1.01
0.96 0.69 0.103
0.97 0.74 0.102
17.7 ÿ0.6 ÿ2.5 ** 0.8 ÿ0.7 ÿ2.7* ÿ4.1* ÿ1.1 ÿ3.1* ÿ1.3 ÿ2.8 2.0 2.4 ÿ2.1 1.6
1.21 ÿ0.01 0.09 0.15* 0.00 ÿ0.41* ÿ0.55** ÿ0.13 ÿ0.36* ÿ0.24 ÿ0.01 0.18 0.24 ÿ0.08 0.18
2.00 ÿ0.10 ÿ0.30 ** 0.10 0.01 ÿ0.39* ÿ0.24 ÿ0.04 ÿ0.23 ÿ0.02 ÿ0.10 0.08 0.24 ÿ0.32 * 0.19
Coefficients 119.3 ÿ4.2 ÿ1.5 6.3 ÿ1.6 ÿ18.6* ÿ22.4* ÿ9.0 ÿ11.6 0.6 ÿ5.7 7.4 16.9 ÿ10.6 6.8
a See Table 1 for symbols. Levels of significance for the null hypothesis: *p < 0.15,
**
p < 0.05.
C4. The molar proportion of C2 was linearly increased with respect to HCOÿ 3 concentration. This factor also acted on C3 in a curvilinear way, with a minimum reached ÿ1 for an HCOÿ 3 amount of approximately 5.7 g l . The effect on C4 was more complex. A canonical analysis, which is a multivariate statistical technique used to analyse secondorder response surfaces (Kettenring, 1971), showed that the proportion of C4 was lowest ÿ 2ÿ at extreme values of HCOÿ 3 and Na /K , central values of HPO4 and low values of Cl , ÿ and was maximised by central values of HCO3 and Na /K and by high values of and Clÿ. NH3-N concentration was always greater than 135 mg lÿ1. It was HPO2ÿ 4 linearly increased with respect to Na/K, but was lowered by the extreme values of 2ÿ HCOÿ and Clÿ and a negative interaction 3 . A positive interaction between HPO4 ÿ between Cl and Na /K was also observed. The osmotic pressure of the fermentation broth ranged from 107 to 296 milli-osmol lÿ1 and was correlated with the osmotic pressure of the saliva. The volume of gases ranged from 2.6 to 3.6 l per day, and the amount of methane produced from 0.85 to 1.58 M hÿ1, respectively. Both responses were satisfactorily modelled (Table 7). The daily volume of collected gases was mainly influenced by ÿ 2ÿ 2ÿ HCOÿ 3 and HPO4 , and to a lesser extent by Cl . It reached a maximum for HPO4 , ÿ ÿ1 ÿ1 ÿ1 ÿ HCO3 and Cl concentrations of 2.4 g l , 4.6 g l and 0.3 g l , respectively. The effects of experimental factors on methane production were more complex, with a ÿ quadratic effect of HPO2ÿ 4 , curvilinear effects of Na /K and HCO3 , and with significant
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Table 6 Results of the stepwise regression applied to pH, redox potential (Eh), VFA concentration, acetate (C2), propionate (C3) and butyrate (C4) molar proportions and ammoniacal nitrogen (NH3-N) concentration in the vessels at 16:00 hours pH R-square Adjusted R2 RSD
0.99 0.99 0.014
Eh (mv)
VFA (mM) C2 (%)
C3 (%)
C4 (%)
NH3-N (mg lÿ1)
0.98 0.87 4.09
0.91 0.30 3.40
0.99 0.99 0.070
0.98 0.84 0.693
0.99 0.95 0.255
0.96 0.71 7.88
Terms
Coefficients
Intercept HPoa HCo Cl NaK HPo2 HCo2 Cl2 NaK2 HPo HCo HPo Cl HPo NaK HCo Cl HCo NaK Cl NaK
7.00 ÿ380.2 0.04** ÿ4.6 0.32** ÿ22.1** 0.06** 1.9 0.02 ÿ1.1 0.14** ÿ14.2* ÿ0.06* ÿ0.2 0.05* ÿ6.7 0.03 ÿ7.7 ÿ0.04 4.4 ÿ0.10** 5.9 ÿ0.06* 6.7 ÿ0.03 8.5 ÿ0.09** ÿ3.8 0.08** ÿ2.0
72.5 ÿ2.8 2.1 ÿ1.8 ÿ0.2 ÿ12.4* ÿ6.1 ÿ6.9 ÿ1.9 ÿ8.9 2.7 3.9 1.9 2.2 ÿ3.0
63.55 0.57** 5.06** 0.48** 0.33** 0.05 0.32* 0.95** 0.13 0.95** 0.83** ÿ1.02** ÿ0.46** 1.09** 2.44**
17.76 ÿ0.97* ÿ2.73** ÿ0.92* 0.30 0.05 2.35* ÿ1.38 1.30 1.04 0.42 0.14 0.20 0.47 ÿ2.07*
14.36 0.34* ÿ1.78** 0.28 ÿ0.78** 0.13 ÿ1.74** 0.32 ÿ0.87* ÿ1.76** ÿ1.42** 0.69 0.35 ÿ0.75 ÿ0.35
175.5 4.2 5.2 9.0 16.3* ÿ16.4 ÿ30.4* ÿ10.7 ÿ4.2 ÿ10.4 27.9* 11.9 9.8 10.3 ÿ19.0
a See Table 1 for symbols. Levels of significance for the null hypothesis: *p < 0.15,
**
p < 0.05.
ÿ 2ÿ interactions between HCOÿ 3 , HPO4 , and Cl . A canonical analysis showed that 2ÿ ÿ methanogenesis was highest for HPO4 , HCO3 , Clÿ and Na/K, at 2.6 g lÿ1, 4.7 g lÿ1, 0.48 g lÿ1 and 5.8 g gÿ1, respectively. The protozoa population density comprised between 14 and 38 mlÿ1 and was clearly linked to Na/K by a positive curvilinear relationship. It was lowered by high or low values of HCOÿ 3 , and by an interaction 2ÿ between HCOÿ and HPO . The hydrogen balance was satisfactory in all experimental 3 4 runs, with hydrogen recovery rates higher than 0.9. The simultaneous maximisation of methanogenesis and protozoa numbers maintenance led to the mineral composition of artificial saliva given in Table 8. Fig. 1(a) and (b) show the contour plots of protozoa ÿ ÿ population density and methane production for HCOÿ 3 and Na /K , with HCO3 and Cl held at the values determined for the optimised saliva. Its composition was characterised by a high Na/K ratio, a low Clÿ content and intermediate values for HCOÿ 3 and . The variance function at this point was 0.862, the standard variations of both HPO2ÿ 4 responses are given in Table 8.
4. Discussion Our experimental domain complied with two constraints. It was within physiological limits (Clarke, 1977; Durand and Kawashima, 1980). Furthermore, it contained both
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Table 7 Results of the stepwise regression applied to the daily production of fermentation gases (volume), the production of methane (CH4) and the protozoa population density (Protozoa) CH4 (mmol hÿ1)
Volume (l per day) R-square Adjusted R2 RSD
0.99 0.92 0.089
Terms
Coefficients
Intercept HPoa HCo Cl NaK HPo2 HCo2 Cl2 NaK2 HPo HCo HPo Cl HPo NaK HCo Cl HCo NaK Cl NaK
3.63 ÿ0.30** 0.36** 0.03 ÿ0.03 ÿ0.71** ÿ0.71** ÿ0.40* ÿ0.17 0.05 0.00 0.15 0.28* ÿ0.08 ÿ0.12
a See Table 1 for symbols. Levels of significance for the null hypothesis: *p < 0.15,
Protozoa (mlÿ1)
0.99 0.90 0.0627
0.97 0.76 2.75
1.535 0.001 0.145** 0.078* ÿ0.137* ÿ0.485** ÿ0.379** ÿ0.152 ÿ0.280* ÿ0.314* 0.410** ÿ0.049 0.231* 0.135 ÿ0.043
25.7 0.9 0.6 ÿ3.9* 4.0* ÿ1.9 ÿ11.8* 0.2 7.0* ÿ9.8* ÿ4.6 ÿ5.2 5.5 3.9 ÿ5.3
**
p < 0.05.
Table 8 Characteristics of the saliva optimised by the procedure DESIR Response
SD
Methane production (mmol h ) Protozoa density (mlÿ1)
1.20 38.7
0.058 2.55
Coordinatesa ÿ1 HPO2ÿ 4 (g l ) ÿ1 HCOÿ (g l ) 3 Clÿ (g lÿ1) Na/K (g gÿ1)
1.65 4.19 0.22 14.2
ÿ1
a
See Table 1 for symbols.
artificial salivas concurrently used in continuous cultures of rumen microbes. The first saliva was proposed by Rufener et al. (1963), and adapted from the study of sheep salivary secretion by McDougall (1948). The second one was adapted by Weller and Pilgrim (1974) from the determination of the rumen fluid mineral composition in two cows by Aafjes and Nijhof (1967). The Rufener saliva is characterised by a high Na/K ratio, 13.6 versus 2.1, and provided slightly greater amounts of Clÿ and HPO2ÿ 4 . The rationale for both mineral compositions was essentially the accurate imitation of a natural
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Fig. 1. Contour plots of (a) protozoa population density and (b) methane bicarbonate concentration (HCOÿ 3 and ratio Na/K, at hydrogenophosphate and chloride concentrations of 1.65 and 0.22 g lÿ1.
state. From this standpoint, both artificial salivas based on either salivary secretion or rumen fluid, were equally justified. The determinations, however, were conducted on one or two animals, which is questionable. Moreover, assessing the sensitivity of rumen microbial metabolism to variations in the mineral content of an artificial saliva is important. A robust saliva would be able to sustain a stable rumen microbial population and activity in vitro, in spite of small changes in its mineral composition. This assessment justified the choice of an intermediate diet composed of ordinary feeds, while the use of synthetic substrates would have allowed a precise control of mineral influxes but would have been poorly representative of livestock feeds. In our experiment, the ions associated with each experimental factor had well defined metabolic roles. Bicarbonates are efficient buffers at pH values of approximately 6.5, and carbon dioxide is used by methanogens as an electron acceptor. Phosphates contribute to the buffering capacity of the medium, and phosphorus is a constituent of nucleic acids, phospholipids and coenzymes. Supplying potassium or sodium chloride merely changes the osmotic pressure. Potassium is a major cation in microbial cells, especially as a cofactor for such enzymes as phosphohexokinases. Sodium is required by a number of slightly halophilic rumen bacterial species (Caldwell et al., 1973; Durand and 2ÿ Kawashima, 1980). In general, the major factor was HCOÿ 3 , followed by HPO4 and ÿ Na /K , while Cl only had noticeable effects on VFA production. Unfortunately, the information available on quantitative effects of minerals on rumen microbe metabolism, with possible interactions, is scarce. The fermentation rate appeared to be mostly influenced by changes in pH mediated by amounts of bicarbonate, with a maximum reached at pH 7.0, and by the supply of phosphates. This could partly be explained by changes in the extent of carbohydrate hydrolysis. In fact, Stewart (1977) reported a similar relationship between pH and cotton degradation by mixed rumen microbes.
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However, in our experiment, the pH was always above 6.3, which is the generally recognised lower limit for optimal fibre degradation (Durand and Kawashima, 1980) and varied within a narrow range. This observation stresses the need for a precise control of pH in continuous cultures, with a tolerance of less than 0.1 pH unit. The analysis of the fermentation pattern confirmed the predominant action of bicarbonates on VFA production rates. A comparison of the present VFA data with in vivo data is of limited value due to the differential absorption of endproducts from the rumen. Phosphates probably also influenced VFA production by means of a change in pH. Phosphorus was supplied in a readily available form which met microbial requirements in all experimental runs, with at least 20 mg gÿ1 OMF (Durand and Komisarczuk, 1988). Moreover, both buffers only modified the redox potential within a narrow range below ÿ350 mV. The production of propionate followed a particular pattern which was characterised by the negative linear effect of bicarbonate. The pH increase may have directly penalised the C3-producing microorganisms, and it may be speculated that, in animals, bicarbonates increase the rumen fluid dilution rate and favour the escape of readily fermentable carbohydrates (Russell and Chow, 1993). The nature of cations was a relatively minor factor. However, it significantly affected C4 and branched chain VFA production rate and C4 molar proportion, probably because of specific requirements for the growth of some of the bacterial strains releasing these endproducts. Bryant et al. (1959) reported that Na and K were essential for the growth of Bacteroides succinogenes in pure culture. The effect of Na/K ratio on C4 production was probably related to bacterial metabolism, rather than to protozoa, which are active butyrate producers (Hungate, 1966; Williams, 1986) but whose numbers in our essay were influenced by the Na/K ratio in a different way. Mackie and Therion (1984) have stressed the greater sensitivity of bacteria to higher concentrations of potassium than sodium. Iwami et al. (1997) reported that Na and K ions had opposite effects on proton excretion in Streptococcus mutans grown in an anaerobic continuous culture system. The pattern of NH3-N concentration tended to mirror the VFA production rate, probably as a consequence of the effect of pH on fermentation, but it was never limiting and nor could it explain many of the changes in microbial metabolism. The action of minerals on gas production and protozoa population density was complex and involved several experimental factors to an equal degree with a non-linear behaviour. The most striking result was the promotion of protozoa numbers by high Na/K ratios, which had not been documented previously, but was confirmed by subsequent experiments on ciliate maintenance (Broudiscou et al., 1997b). Methanogens and protozoa are prominent agents in the rumen ecosystem and difficult to maintain in continuous cultures of mixed rumen microbes. Thus, values for the methane production and protozoa population density are good biological criteria for determining the most suitable composition of a rumen content simulating buffer. The composition of the buffer resulting from the maximisation procedure was close to that of the artificial saliva proposed by Rufener et al. (1963), which has rarely been used in dual outflow continuous cultures. If we take into account the data on fermentation, the intermediate amounts of buffer salts should warrant that small variations in the composition of the saliva would not ÿ alter the values of fermentative parameters. Though the co-ordinates for HPO2ÿ 4 , HCO3 , ÿ and Cl concentrations were central, the high value taken by the Na /K ratio located the
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optimal point at the edge of the experimental domain. This feature was likely to be observed since a constrained optimisation was performed. In our case, the major constraint was to confine the factors within physiological values, in order to allow the proper comparison of our future results with published data. The optimised saliva contained 6.082 g lÿ1 Na2HPO412H2O, 5.293 g lÿ1 NaHCO3, 0.566 g lÿ1 KHCO3, 0.363 g lÿ1 NaCl and 1.333 g lÿ1 NaOH. It was tested in a verification experiment, where the fermenters were run identically for two 11-day experimental periods (12 data points). Protozoa numbers and average methane production ± 45.9 mlÿ1 (SD of 8.7 mlÿ1) and 1.39 mmol ÿ1 (SD of 0.135 mmol hÿ1), respectively, ± were slightly higher than predicted values. The development of this saliva has validated conditions suitable for investigation of a number of current factors influencing rumen functions, in particular the screening of methane inhibitors or the nutritional interest of rumen ciliates. Acknowledgements The authors would like to thank Dr. S. Komisarzuk-Bony for analysis of phosphorus in feeds, Mrs. B. Lassalas for laboratory analyses, and L. L'Hotelier for taking care of experimental animals. References Aafjes, J.H., Nijhof, J.K., 1967. A simple artificial rumen giving good production of volatile fatty acids. Br. Vet. J. 123, 436±445. Atlas, R.M., Bartha, R., 1993. Microbial Ecology, Fundamentals and Applications, Benjamin/Cummings, Redwood city. Bowie, W.C., 1962. In vitro studies of rumen microorganisms, using a continuous-flow system. Am. J. Vet. Res. 23, 858±868. Box, G.E.P., Hunter, W.G., Hunter, J.S., 1978. Statistics for Experimenters: An Introduction to Design, Data Analysis and Model Building, Wiley, New York. Box, G.E.P., Wilson, K.B., 1951. On the experimental attainment of optimum conditions. J. Royal Stat. Soc. Series B 13, 1±45. Broudiscou, L.P., Papon, Y., Broudiscou, A.F., 1997a. Effect of minerals on methane production and protozoa numbers in continuous culture of rumen microorganisms. Reprod. Nutr. Develop. (Suppl. 1), 70±71. Broudiscou, L.P., Papon, Y., Fabre, M., Broudiscou, A.F., 1997b. Maintenance of rumen protozoa populations in a dual outflow continuous fermenter. J. Sci. Food Agric. 75, 273±280. Bryant, M.P., Robinson, I.M., Chu, H., 1959. Observations on the nutrition of Bacteroides succinogenes ± a ruminal cellulolytic bacterium. J. Dairy Sci. 42, 1831±1847. Caldwell, D.R., Keeney, M., Barton, J.S., Kelley, J.F., 1973. Sodium and other inorganic growth requirements of Bacteroides amylophilus. J. Bacteriol. 114, 782±789. Clarke, R.T.J., 1977. Methods for studying gut microbes. In: Clarke, R.T.J., Bauchop, T. (Eds.), Microbial Ecology of the Gut. Academic Press, London, pp. 1±33. Cochran, W.G., Cox, G.M., 1957. Experimental Designs. Wiley, New York. Cote, R.J., Gherna, R.L., 1994. Nutrition and media. In: Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R. (Eds.), Methods for General and Molecular Bacteriology. ASM, Washington, pp. 155±177. Davies, A.W., Taylor, K., 1965. Application of the autoanalyser in a river authority laboratory. In: Symposium Technicon, Technicon, Tarrytown, pp. 294±300.
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