In vitro fermentation and methane production of fava and soy beans

A. Guglielmelli, S. Calabr, M. Cutrignelli, O. Gonzalez, F. Infascelli, R. Tudisco, V. Piccolo Dipartimento di Scienze Zootecniche e Ispezione degli alimenti, Universit di Napoli Federico II, Via F. Delpino 1, 80137 Napoli, Italy Introduction Cultivation of legumes (fava bean, pea, lupine) can improve soil fertility, reduce supply of nitrogen (N) fertiliser and, due to the tannins presence (Woodward et al., 2001), restrict CH4 production. In addition, legume grain, in some agricultural condition, such as Mediterranean area, can replace partially or totally soybean in ruminant diets (Cutrignelli et al., 2008). In this study the in vitro fermentation characteristics of six diets for ruminants with fava bean (Vicia faba minor) or solvent extracted (s.e.) soybean as protein sources were studied. Materials and methods For each protein source (fava bean: FD and soybean s.e. SD), three different diets [CP: 10, 13 and 15% DM; NEl (MJ)/CP: 0.50, 0.46 and 0.41] were formulated with wheat straw and corn meal. The estimated protein digestible at intestinal level (PDI) of all the diets ranged between 8.3 and 16.2% DM. Fermentation characteristics were studied using the in vitro gas production technique, by incubating two series of bottles for each diet (1.015 g 0.012) in triplicate under anaerobic condition with buffered rumen fluid collected at the slaughterhouse from six cows fed a standard diet (Calabr et al., 2006). The gas produced during the incubation was recorded using a manual system and the cumulative gas production was related to the incubated organic matter (OMCV). For the 1st series, the fermentation was stopped at 72 h and the fermentation liquor was analyzed for NH3 by spectrophotometer and for volatile fatty acids (VFA) by gas chromatography; the organic matter disappearance (dOM) was measured by filtering and successively burning at 550C the bottles residues. CH4 was determined by a chemical method (Pell and Schofield, 1993) using NaOH (10%) at 39C to separate CH4 from CO2. CH4 production was also estimated from VFA using stoichiometric equations (Van Soest et al., 1994). In order to study the fermentation kinetics of the diets, the 2nd series was stopped at 120 h and the gas production profiles were fitted to a sigmoid model. Data were statistically processed with SAS (2000). Results and discussion The trend of fermentation rate was similar for all the diets and the process was almost completed at 72 h (Fig. 1). However, FD showed always higher gas production compared to SD; FD15 had the highest gas production and fermentation rate. Protein source and level significantly (P<0.01) affected most of fermentation characteristics recorded at 72 h (Tab. 1). In particular, FD showed higher dOM and OMCV, CH4, NH3, and total VFA production compared to SD. The higher acetic and butyric acid production and higher AB/P ratio in FD explains the higher CH4 production. As expected, dOM and VFA increased with the decrease of NEl/CP in the diets. CH4 showed an opposite trend (P<0.01); probably the shift in protein to carbohydrates changed the rumen H balance. Significant correlations (P<0.05) were found between CH4 (ml/g of degraded OM) and NEl/CP or PDI (0.838 and -0.877, respectively) confirming that the optimisation of rumen conditions for microbial growth is related to a decrease of CH4 production (Moss, 1994). The mean value of the chemically determined CH4 registered for all the diets (41.8 ml/g of incubated DM) was consistent with the data reported by Getachew et al. (2005) and similar to that estimated stoichiometrically (42.3 ml/g). In addition, the percentage of CH4 on the total gas (Fig. 2) was similar to the average value (18%) reported by Blmmel and rskov (1993). According to Fievez et al. (2005), the results concerning CH4 confirm the suitability of the chemical method which has the advantage to be very simple and inexpensive and compatible in those laboratories where GC equipments are

not available. However, some error can affect the data: not all CO2 may be dissolved in NaOH, and CH4 is the main, but not the only, gas of fermentation which remain insoluble in alkaline solution. FD had higher CH4 production than SD, which is negative from the environmental point of view. The more intensive fermentation process with FD led to a higher dOM and VFA production than SD, which may be beneficial to the energy supply of ruminants.
Fig. 1. Gas production and rate over time
360 320 280 240
gas (ml/g)
FD_10 FD_13 FD_15 SD_10 SD_13 SD_15

Fig. 2. Methane production at 72 h

14.0 12.0 10.0 8.0 6.0 4.0 2.0 0.0 120
rate (ml/h)

100 90 80 70 60 50 40 30 20 10 0 FD_10 FD_13

ml/g iDM % of total gas

ml/g dOM ml/g iDM estimated

200 160 120 80 40 0 0 12 24 36 48 60 72 time (h) 84 96 108

FD_15 SD_10

SD_13

SD_15

Table 1. In vitro fermentation parameters at 72 h

Diets FD SD CP 10 CP 13 CP 15 dOM % 73.2A 69.8B 66.8C 71.4B 76.3A OMCV ml/g OM 310A 284B 294 295 302 NH3 mg/g DM 44.5 39.3 42.8B 36.9C 46.1A CH4 ml/g DM 46.3A 37.2B 46.6A 41.4AB 37.3B CH4 ml/g dOM 75.7A 63.6B 82.9A 68.3Ba 57.8Bb Ace Prop But VFA Mm /g litre of incubated DM 45.9 27.3 10.4A 96.2 40.7 27.5 9.59B 84.2 B 41.2 26.8 8.93 84.1 41.9 25.7 9.13AB 91.3 46.8 29.7 10.4A 95.2 79.5 NS AB/P 2.11 1.79 1.87 2.05 1.94 0.089 NS

MSE 1.86 41.3 2.17 21.7 61.6 31.5 9.14 0.654 D*CP NS NS *** *** NS NS NS NS VFA: ace + prop + but + isobut + valeric + isovaleric; AB/P: (acetate + butyrate)/propionate. NS: not significant. a, b P<0.05; A, B, C and *** P<0.01; MSE: mean square error.

References
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