Effect of supplementation with urea or calcium nitrate and cassava leaf meal or fresh cassava leaf in an in vitro fermentation using a basal substrate of sugar cane stalk
Outhen Phommasack, T R Preston* and R A Leng**
Savannakhet University, Lao PDR
Outhen@hotmail.com
*Finca Ecologica, TOSOLY, UTA (Colombia)
AA#48, Socorro, Santander, Colombia
**University of New England, NSW, Australia
Abstract
A substrate of sugar cane stalk supplemented with cassava leaf meal or fresh cassava leaves was fermented in a simple in vitro system in which the sources of fermentable N were calcium nitrate or urea.
Replacing urea with calcium nitrate reduced methane production in an in vitro system in which the substrate was sugarcane stalk and dried or fresh cassava leaf. Increasing the fermentation time increased the production of methane, probably because of reduction of the VFA. After 5h of fermentation the methane percentage in the gas was lower with fresh than with dried cassava leaf, but there were no differences with longer fermentation times. The potential of HCN precursors in cassava leaf and roots to reduce methane production in ruminants merits further investigation.
Key words: Cassava, greenhouse gases, HCN, methane,
Introduction
Methane resulting from fermentative digestion of organic matter in the rumen
represents a loss of dietary energy to the animal (Johnson and Johnson 1995)
and ruminants are a significant contributor to world greenhouse gas
emmissions(Steinfeld et al 2006). These factors have led to a global search
for
strategies
to mitigate methane emissions from ruminants.
In ruminants, H2 produced in the pathways of conversion of organic matter to VFA with growth of cells, is normally removed by the reduction of CO2 to form methane. However, nitrate (present in some fresh pasture grasses) has a higher affinity for H2 than CO2 and, when it is present, H2 is effectively used in the reduction of NO3 to ammonia (NH3) thereby lowering the production of methane. The possibility of nitrate as an alternative hydrogen sink to carbon dioxide has been discouraged because of the possible toxic effects of nitrite, which is spilled into the medium under some feeding regimes (Lewis 1951).
After comprehensively reviewing the literature, Leng (2008) concluded that the inclusion of nitrate in feed supplements appeared to be entirely feasible as a means of reducing enteric methane emissions from ruminant livestock. While there is a risk of nitrite toxicity, nitrate supplementation also has potential advantages in addition to inhibiting methane emissions, viz. (i) the end product of nitrate reduction is ammonia, which for ruminants on low digestibility diets, is a major source of N for microbial growth; and (ii) nitrate reduction to ammonia is accompanied by formation of ATP from ADP and therefore theoretically microbial growth efficiency should also be improved.
The protein in cassava (Manihot esculenta, Crant) leaves is considered to be a good source of bypass protein (Ffoulkes and Preston 1978; Wanapat et al 1997, Keo Sath et al 2008). It is widely cultivated in all tropical counties and is thus a logical forage produced locally to provide the additional protein required in low protein high energy diets
Cassava contains cyanogenic glucosides, mainly linamarin (92-98%); which release hydrogen cyanide after hydrolysis in the rumen by an endogenous linamarase (Butler et al 1965; Com 1969). Anaerobic digestion can be inhibited by cyanide, because of the high sensitivity of methanogenic bacteria to this compound (4 mg 1itre-1 in pure culture) (Eikmanns and Thauer 1984; Smith et al 1985).
Cuzin and Labat (1992) showed that additions of 5, 10, and 25 mg 1itre-l cyanide (KCN or linamarin) temporarily inhibited methanogenesis but when the concentration of cyanide reached its initial value (before KCN or linamarin addition), methane production recovered. The biodigester methanogenic microflora were sensitive to cyanide addition, but tolerated the low concentrations (6 mg 1itre-1) normally present in the fermenter.
As production of HCN from cyanogenic glucosides is reduced by drying (from 190 mg/kg DM in the fresh leaf to 20 mg/kg after sun-drying according to Bui Huy Nhu Phuc et al 1995), it is possible that methane production in an in vitro fermentation would be less when the substrate contained fresh cassava leaves rather than dried leaves.
The purpose of the present study was to develop and use a simple in vitro method to determine methane production from a diet based on sugar cane stalk as substrate supplemented with protein from cassava leaf meal or fresh cassava leaf using calcium nitrate and urea as sources of non-protein nitrogen.
Hypothesis
-
Methane production in an in vitro fermentation (artificial rumen) of sugar cane stalk would be reduced when calcium nitrate replaced urea as NPN source, and fresh cassava leaf was the protein source rather than cassava leaf meal.
Materials and Methods
Location
The experiment was conducted in the laboratory of An Giang University, Vietnam from August to October 2010.
Treatments and experimental design
A 2 x 2 factorial design was used to evaluate the effect of (i) supplementation of cassava leaf meal or fresh cassava leaf; and (ii) supplementation of urea or calcium nitrate using sugar cane stalk as basal substrate. There were four replications of the following treatments.
- Cassava leaf meal plus urea (CLM-U)
- Cassava leaf meal plus calcium nitrate (CLM -CaN)
- Fresh cassava leaf plus urea (FCL-U)
- Fresh cassava leaf plus calcium nitrate (FCL- CaN)
Preparation of substrate and the in vitro system
The sugar cane stalk and cassava leaf meal (or fresh cassava leaf) were milled in a coffee grinder and mixed with the sources of N (urea or calcium nitrate). A representative sample (12 g DM) of the mixtures (Table 1) were put in a 1500 ml flask (a recycled water bottle) to which was added 0.96 litres of buffer solution (Table 2) and 240ml of rumen fluid (obtained immediately after slaughter of a buffalo in the municipal abattoir), prior to eliminating the remaining air in the flask with carbon dioxide. The incubation flask was connected by a plastic tube (id 3mm) to a second flask (a calibrated recycled water bottle with the bottom removed) suspended in water so as to measure the gas production by water displacement. The incubation flasks were then incubated at 38 0 C in a water bath for 48h. Full details of the in vitro system (Photo 1) are given in Sangkhom et al (2011).
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Table 1. Ingredients (g) in the substrate |
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|
CLM-U |
FCL-U |
CLM-CaN |
FCL-CaN |
|
|
Sugar cane stalk |
8.76 |
8.76 |
8.76 |
8.76 |
|
Cassava leaf meal |
3.00 |
3.00 |
|
|
|
Fresh cassava leaf |
3.00 |
|
3.00 |
|
|
Urea |
0.24 |
0.24 |
|
|
|
Ca(NO3)2.4H2O |
0.46 |
0.46 |
||
|
Table 2. Ingredients of the buffer solution (adapted from Tilly and Terry 1964) |
|||||||
|
Ingredients |
CaCl2 |
NaHPO4.12H2O |
NaCl |
KCl |
MgSO4.7H2O |
NaHCO3 |
Cysteine |
|
(g/liter) |
0.04 |
9.30 |
0.47 |
0.57 |
0.12 |
9.80 |
0.25 |
|
|
|
|
Photo 1. The in vitro fermentation system using recycled water bottles and water displacement to measure gas production |
Photo 2. Measurement of percentage of methane in the gas |
Data collection and measurements
Gas production was measured after 5, 15 and 43h and samples analyzed for methane percentage using an infra-red analyser (Crowcon Instruments Ltd, UK) (Photo 2). Each time, after the gas volume was measured and analyzed for methane, the gas was released to empty the bottles for the next measurement. Residual undigested substrate in the fermentation flask was determined by filtration through cloth (Photo 3) and drying of the residue in the oven at 105°C for 24h.
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|
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Photo 3. The residual substrate filtered through cloth
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Statistical analysis
The data were analyzed by
the General Linear Model (GLM) option in the ANOVA program of the Minitab
Software (version13.2) (Minitab 2000). Sources of variation in the model
were: N source, cassava leaf, interaction N*cassava leaf and error.
Results and Discussion
Gas production was higher for fresh than for dried cassava at 5h and for urea versus nitrate at 15h; however, there were no treatment differences at other sampling times and only a tendency (P=0.15) for total gas production at 43h to be higher on urea than on nitrate (Table 3). After 5h (Figure 1) and at all subsequent sampling times the per cent methane in the gas, and the quantity of methane produced, were reduced in presence of nitrate compared with urea. Comparing fresh with dried cassava leaves, after 5h the per cent methane in the gas was lower for the former both before and after correcting the data for differences in gas volume (Figure 2). However, there were no differences at 15 and 43 h, and no differences at all times for volume of methane produced. The proportion of substrate fermented, and the gas production per unit substrate fermented, were not affected by the treatments. However, the production of methane as per cent of total gas production (Figure 3) , and as total volume per unit substrate fermented (Figure 4), were lower for nitrate than for urea. There were no interactions between N source and cassava leaf processing for any of the measurements.
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Table 3. Mean values for gas production and concentration of methane after 5, 15 and 43h of in vitro fermentation of fresh sugarcane in presence of urea or calcium nitrate and fresh or dried cassava leaves |
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|
|
Effect of cassava |
Effect of N source |
P values |
SEM |
|||
|
|
CLM |
FCL |
CaN |
Urea |
Cassava |
N source |
|
|
0- 5h |
|
|
|
|
|
|
|
|
Gas production, ml |
875 |
975 |
950 |
900 |
0.030 |
0.24 |
28.9 |
|
Methane, % |
14.4 |
12.9 |
11.8 |
15.5 |
0.040 |
<0.001 |
0.46 |
|
Methane, %# |
14.3 |
12.9 |
12.1 |
15.2 |
0.046 |
<0.001 |
|
|
Methane, ml |
125 |
125 |
111 |
138 |
0.92 |
<0.001 |
4.46 |
|
5-15h |
|
|
|
|
|
|
|
|
Gas production, ml |
925 |
900 |
831 |
994 |
0.71 |
0.03 |
47.2 |
|
Methane, % |
19.6 |
19.3 |
16.5 |
22.4 |
0.68 |
<0.001 |
0.62 |
|
Methane, ml |
182 |
174 |
136 |
220 |
0.35 |
<0.001 |
5.99 |
|
15-43h |
|
|
|
|
|
|
|
|
Gas production, ml |
450 |
444 |
425 |
469 |
0.88 |
0.30 |
28.9 |
|
Methane, % |
37.4 |
37.0 |
34.9 |
39.5 |
0.74 |
<0.001 |
0.79 |
|
Methane, ml |
167 |
164 |
147 |
184 |
0.81 |
0.01 |
8.41 |
|
At 43h |
|
|
|
|
|
|
|
|
Total gas production, ml |
2250 |
2319 |
2206 |
2363 |
0.51 |
0.15 |
72.3 |
|
Total methane, ml |
474 |
464 |
395 |
543 |
0.04 |
<0.001 |
13.9 |
|
Overall methane, % |
21.0 |
19.9 |
17.9 |
23.0 |
0.60 |
<0.001 |
0.33 |
|
DM substrate fermented, g |
6.56 |
6.89 |
6.67 |
6.78 |
0.21 |
0.68 |
0.18 |
|
DM substrate fermented, % |
52.4 |
57.2 |
54.8 |
54.9 |
0.19 |
0.97 |
2.39 |
|
Gas production, ml/g substrate DM fermented |
343 |
337 |
331 |
348 |
0.79 |
0.56 |
16.9 |
|
Methane, ml/g DM substrate fermented |
72.6 |
67.6 |
59.8 |
80.4 |
0.27 |
<0.001 |
3.08 |
|
# Corrected for differences in gas production |
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|
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Figure 1. Effect of CaN vs urea on methane content of gas after 5h of incubation of sugar cane and cassava leaf |
Figure 2. Effect of fresh vs dry cassava leaf on methane content of gas after 5h incubation (corrected for differences in gas volume) |
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|
|
|
Figure 3. Effect of CaN vs urea on methane content of gas after 43h incubation of sugar cane and cassava leaf |
Figure 4. Effect of CaN vs urea on methane per unit substrate fermented after 43h incubation |
The lowering in methane production when nitrate, compared with urea, was the NPN source with an energy substrate of sugar cane stalk, mirrored the findings of Sangkhom et al (2011) who used cassava root as the substrate. Comparable comparisons for cassava root versus sugar cane showed the proportion of substrate fermented to be higher, while the production of methane per unit substrate fermented was lower, for the former.
The values recorded in this study for total gas production, percentage of methane at the end of the incubation period, and amount of methane production per DM substrate fermented, were higher than those reported by Le Thi Ngoc Huyen et al (2010). The differences can be attributed to the nature of the substrate: NaOH-treated rice straw in the experiment of Le Thi Ngoc Huyen et al (2010) compared with the more digestible sugar cane stalk in the present study.
The linear increase in the methane content of the gas with fermentation time (Figures 5 and 6) showed the same tendency as was reported by Sangkhom et al (2011). As discussed by those authors, these changes almost certainly reflected changes in the nature of the fermentation as carbohydrate was fermented first to VFA, methane and carbon dioxide followed by secondary fermentation of the VFA to methane and carbon diooxide.
