Effect of water spinach (Ipomoea aquatica) and cassava leaf meal (Manihot esculenta Crantz) with or without biochar on methane production in an in vitro rumen incubation using ensiled or dried cassava root meal as source of carbohydrate

Sengsouly Phongphanith, T R Preston¹ and R A Leng²

Animal Science Department, Faculty of Agriculture and Forest Resource, Souphanouvong University Lao PDR
ssl.souphanouvong@gmail.com
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
² University of New England, Armidale NSW, Australia

Abstract

The objective of this study was to evaluate the effect of source of protein as cassava leaf meal or water spinach and supplementation with or without biochar on methane production in an in vitro rumen incubation system using ensiled or sun-dried cassava root as the source of energy. The experimental design was a 2*2*2 factorial in a completely randomized design with 8 treatments and 4 replications. The factors were: ensiled or sun-dried cassava root; water spinach meal or cassava leaf meal and with or without biochar. Urea was added as NPN source at 3% of substrate DM. The total substrates equivalent to 12g DM were put in the incubation bottle, followed by 960 ml of buffer solution and 240 ml of rumen fluid obtained from a cow immediately after being slaughtered. The bottles were then filled with carbon dioxide and incubated at 38 ⁰C in a water bath for 48 hours with measurements of total gas production and methane percentage at intervals of 6, 12, 18, 24 and 48 h and determination of residual unfermented substrate DM at the end.

The gas production was lower for ensiled than for dried cassava root; lower for cassava leaves than for water spinach; and lower for presence of biochar in the substrate.  The percent methane in the gas and the percent DM mineralized followed the same pattern . The proportion of methane in the gas increased with the length of the incubation. 

It is proposed that the lower net production  of methane per unit substrate DM digested, for cassava leaf meal  compared with water spinach meal, was because of the lower solubility of the protein in the cassava leaf meal (25.6%) compared with the water spinach (66.3%) and that this retarded the fermentation of the substrate and particularly of methane formation.   

Keywords: cyanide, cyanogenic precursors, fermentation, solubility, urea

Introduction

The agriculture sector is a major contributor to greenhouse gas emissions which cause global warming, producing 14-22% of global anthropogenic greenhouse gas (GHG) emissions (Ecofys 2013).   Ruminant livestock such as cattle, buffalo, sheep and goats are a substantial source of the methane (Lassey 2007; Chabra et al 2009; Hristov et al 2013). Reducing greenhouse gas (GHG) emissions from agriculture and especially from ruminant livestock should therefore be a top priority since it could help to curb global warming (Sejian et al 2010).

Cassava (Manihot esculenta, Crantz) is extensively cultivated throughout the tropics and subtropics regions due to its ability to grow in diverse soil conditions and with minimal management (Wanapat 2003; Wanapat et al 2006; Wanapat and Khampa 2007). The root is composed almost entirely of carbohydrate which can be used as an important food source. However, it contains variable amounts of cyanogenic glucosides depending on the variety (Stupak et al 2006; Cumbana et al 2007) which could limit cassava root utilization for human consumption and for livestock feeding.

Cassava leaves are considered to be a good source of bypass protein (Ffoulkes and Preston 1978; Wanapat et al 2001; Sath et al 2008) and are thus a logical source of forage to provide the additional bypass protein required in diets high in non-protein nitrogen (Preston and Leng 2009).

Water spinach (Ipomoea aquatica) plays an important role for farmers in rural areas. It is easy to grow and has a very high yield of biomass with a short growth period (Sophea and Preston 2001). The crude protein content in the leaves and stems can be as high as 32 and 18 % on a dry basis, respectively (Le Thi Luyen 2003). Water spinach is widely used for human food, but at the same time this vegetable is nutritious for animals such as rabbits, pigs, poultry and small ruminants.  

A recent development arising from studies on promoting renewable sources of energy has been the finding that biochar - the residue from the gasification of rice husks - appeared to act as a biofilm in rumen fermentation studies with beneficial effects manifested by reduced production of enteric methane and higher growth rates when added at low levels to cattle (Leng et al 2012). In vitro rumen fermentation studies have shown that methane production increases linearly over 48 hours independently of the substrate (Inthapanya et al 2011, Binh Phuong et al 2011, and Thanh et al 2011). Leng (2014) has suggested that when the fermentation in an in vitro incubation exceeds 24h it will more resemble what occurs in a biodigesters than in the rumen.  In ruminants fed well balanced diets, the rumen retention time rarely exceeds 24h.

The objective of this study was to evaluate the effect of source of protein as cassava leaf meal or water spinach and supplementation with or without biochar on methane production in an in vitro rumen incubation system using ensiled or sun-dried cassava root as the source of energy.  

Materials and methods

Location and duration

The experiment was conducted in the laboratory of the Research and Technology Transfer Center, in Nong Lam University, Vietnam, from May to June 2015.

Treatments and experimental design 

The experimental design was arranged as a 2*2*2 factorial in a completely randomized design with 8 treatments and 4 replications of each treatment. The factors were:

Source of carbohydrate:

• ECR: ensiled cassava root

• DCR: dried cassava root

Source of protein:

• WSM: water spinach meal

• CLM: cassava leaf meal

Source of biochar

• With or without biochar

The in vitro system

The in vitro system was made from recycled “PEP” water bottles as described by Inthapanya et al (2011).

Diets

Ensiled and dried cassava root

The cassava root was chopped into small pieces (1-2 cm of length), which were either: (i) ground in a liquidizer and then ensiled in a sealed plastic bag for 5 days; or (ii) dried in the open air for 24h before being ground in a coffee grinder.

Cassava leaves and water spinach

Leaves and petioles from a bitter variety of cassava and leaves and stems of water spinach were collected in the farm of the Research and Technology Transfer Center, Nong Lam University, chopped into small pieces around 1-2 cm of length, then dried in the open air for 24h before grinding in a coffee grinder.  

Biochar

The biochar was produced by burning rice husks in a top lit updraft (TLUD) gasifier stove (Olivier 2010) at a temperature of 900-1000^oC. Urea as NPN source was added in all the substrates at 3% of DM.

Buffer medium and rumen fluid

Amounts of the substrates equivalent to 12g DM were put in the incubation bottle, followed by 0.96 liters of buffer solution (Table 1) and 240 ml of rumen fluid obtained from a cattle immediately after being slaughtered. The bottles were then filled with carbon dioxide and incubated at 38 ⁰C in a water bath for 48h.

Data collection and measurements

During the incubation the gas volume was recorded at 6, 12, 24 and 48h. After each time interval, the methane concentration in the gas was measured with a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK) (Photo 2).  At the end of the incubation, the residual DM in the incubation bottle was measured to determine mineralization of the DM (Photo 3).

Photo 2. Measurement of methane production in the gas	Photo 3. The substrate residue filtered through cloth

Chemical analyses

Samples of ensiled cassava root, dried cassava root meal, cassava leaf meal and water spinach meal were analyzed for DM and crude protein by AOAC (1990) methods. Soluble nitrogen was determined as the nitrogen remaining in the liquid fraction after extraction of the protein source in M NaCl (Whitelaw and Preston 1963). 

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab Software (Minitab 2000). Sources of variation in the model were:  source of protein, source of carbohydrate, biochar, interaction carbohydrate*protein*biochar and error.

Results

Chemical composition

Crude protein values were similar within the sources of carbohydrate and protein (Table 2). N solubility was lower in the ensiled than in the dried cassava root, and much lower in  cassava leaf meal than in water spinach meal.

Gas production, percent methane in the gas  and percent DM mineralized
 

The gas production was lower for ensiled than for dried cassava root; lower for cassava leaves than for water spinach; and lower for presence of biochar in the substrate (Table 3; Figure 1).  The percent methane in the gas and the percent DM mineralized followed the same pattern (Table 3; Figure 4). The proportion of methane in the gas increased with the length of the incubation (Figures 2 and 3).

Methane production per unit substrate mineralized

The methane per unit of DM mineralized was: similar for ensiled and dried cassava root;  lower (by 12%) for cassava leaf compared with water spinach leaf; and lower (by 10%) for supplementation with biochar than for no biochar (Table 3; Figure 4). The combined effect of combining cassava leaf meal with biochar led to a 25% reduction in methane production per unit substrate DM mineralized compared with the diet having no biochar and with water spinach as the protein source (Figure 5).

Figure 2: Effect of fermentation time on methane content of the gas from dried cassava root

Figure 3: Effect of fermentation time on methane in the gas from ensiled cassava root

Figure 4: Effect of treatments on methane produced per unit substrate mineralized

Figure 5: Effect of treatments on methane production per unit DM mineralized

Discussion

The increased methane production with duration of incubation is indicative of the transition to a secondary or aceticlastic fermentation of acetate to methane, which is supported by the findings of Sangkhom Inthapanya et al (2011), Le Thi Binh Phuong et al (2011), Thanh et al (2011) and Outhen et al (2011).

The decrease in methane per unit substrate mineralized when cassava leaf meal replaced water spinach as the protein source agrees with the findings of Inthapanya and et al (2015).  This effect could be due to the presence of HCN precursors in cassava leaves as cyanide inhibits the metabolism of acetate to methane as happens inprolonged incubations such as occurs in biodigesters  or sludge fermentation in general (Smith et al 1985). A possible furtherexplanation is that the lower solubility of the protein in the cassava leaves, relative to that in the water spinach leaves, retarded the process of fermentative digestion (as was observed in the lower rate of gas production and reduced mineralization of the DM for the cassava leaf compared with water spinach leaf treatment) and that the formation of methane was affected to an even greater extent, as observed by the lower percent of methane in the gas at all stages of the fermentation. It appeared that the addition of biochar to the substrate had a similar effect in reducing the rate of fermentation and hence of the formation of methane. We have no explanation for the mechanism by which biochar could have this effect.

Conclusions

• Methane production in an in vitro rumen fermentation of cassava root was reduced when cassava leaf meal replaced water spinach leaf meal as the major protein source, and when biochar was added to the substrate.

Acknowledgements

This research was done by the senior author as part of the requirements for the MSc degree in Animal Production "Specialized in Response to Climate Change and Depletion of Non-renewable Resources" of Cantho University, Vietnam. The authors acknowledge support for this research from the MEKARN II project financed by Sida. They also acknowledge the Research and Technology Transfer Center, Nong Lam University, Vietnam for providing infrastructure support.

References

AOAC 1990 Official methods of anaimilarlysis 15th ed. AOAC, Washington, D.C

Chabra A, Manjunath K R, Panigrahy S and Parihar J S 2009 Spatial pattern of methane emissions from Indian livestock. Current Science 96(5): 683-689.

Cumbana A, Mirione E, Cliff J and Bradbury J H 2007 Reduction of cyanide content of cassava flour in Mozambique by the wetting method. Food Chemistry v.101, p.894-897      

Ecofys 2013 World GHG emissions flow chart 2010. Accessed Oct. 31, 2013. http://www.ecofys.com/files/files/asn-ecofys-2013-world-ghg-emissions-flow-chart-2010.pdf

Ffoulkes D and Preston T R 1978 Cassava or sweet potato forage as combined sources of protein and roughage in molasses based diets: effect of supplementation with soybean meal. Tropical Animal Production. http://www.utafoundation.org/TAP/TAP33/ 331.pdf

Hristov A N, Oh J, Firkins JL, Dijkstra J, Kebreab E, Waghorn G, Makkar HPS, Adesogan AT, Yang W, Lee C, Gerber PJ, Henderson B and Tricarico J M 2013 Mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options. Journal of Animal Science 91, 5045–5069. doi:10.2527/jas.2013-6583

Inthapanya S and Preston T R 2014 Methane production from urea-treated rice straw is reduced when the protein supplement is cassava leaf meal or fish meal compared with water spinach meal in a rumen in vitro fermentation. Livestock Research for Rural Development. Volume 26, Article #159. http://www.lrrd.org/lrrd26/9/sang26159.html

Inthapanya S, Preston T R and Khang D N 2015 Methane production was reduced when cassava root (Manihot esculenta, Crant) was ensiled rather than dried, and when cassava leaves replaced water spinach (Ipomoea aquatic) as the protein source, in an in vitro rumen fermentation. Livestock Research for Rural Development. Volume 27, Article #183. Retrieved March 2, 2016, from http://www.lrrd.org/lrrd27/9/sang27183.htm

Gijzen H J, Bernal E and Ferrer H 2000 Cyanide toxicity and cyanide degradation in anaerobic wastewater treatment. Water Research. Volume. 34, No. 9, pp. 2447-2454

Kean Sophea and Preston T R 2001 Comparison of bio digester effluent and urea as fertilizer for water spinach vegetable. Livestock Research for Rural Development, Volume 13, Number 6, December 2001. http://www.lrrd.org/lrrd13/6/kean136.htm

Keo Sath, Borin K and Preston T R 2008 Effect of levels of sun-dried cassava foliage on growth performance of cattle fed rice straw. Livestock Research for Rural Development. Volume 20, supplement. http://www.lrrd.org./lrrd20/supplement/sath2.htm

Lassey K R 2007 Livestock methane emission: From the individual grazing animal through national inventories to the global methane cycle. Agriculture Meteorology, 142: 120-132.

Le Thuy Binh Phuong, Preston T R and Leng R A 2011 Mitigating methane production from ruminants; effect of supplementary sulphate and nitrate on methane production in an in vitro incubation using sugar cane stalk and cassava leaf meal as substrate. Livestock Research for Rural Development. Volume 23, Article #22. Retrieved, from http://www.lrrd.org/lrrd23/2/phuo23022.htm

Leng R A 2014 Interactions between microbial consortia in biofilms: a paradigm shift in rumen microbial ecology and enteric methane mitigation. Animal Production Science 54 (5) 519-543 . http://dx.doi.org/10.1071/AN13381

Leng R A, Inthapanya S and Preston T R 2012 Biochar lowers net methane production from rumen fluid in vitro. Livestock Research for Rural Development. Volume 24, Article #103. http://www.lrrd.org/lrrd24/6/sang24103.htm

Ly Thi Luyen 2003 Effect of the urea level on biomass production of water spinach (Ipomoea aquatica) grown in soil and in water.  Retrieved, from MEKARN Mini-projects. http://www.mekarn.org/MSc2003 05/minipro jects/luye.htm

Minitab 2000 Minitab Software Release 16.0

Outhen P, Preston T R and Leng R A 2011 Effect of supplementation with urea or calcium nitrate and cassava leaf meal or fresh cassava leaf in an in vitro incubation using a basal substrate of sugar cane stalk. Livestock Research for Rural Development. Volume 23, Article #23. http://www.lrrd.org/lrrd23/2/outh23023.htm

Phonethep P, Preston T R and Leng R A 2016: Effect on feed intake, digestibility, N retention and methane emissions in goats of supplementing foliages of cassava (Manihot esculenta Crantz) and Tithonia diversifolia with water spinach (Ipomoea aquatica). Livestock Research for Rural Development. Volume 28, Article #72. Retrieved May 1, 2016, from http://www.lrrd.org/lrrd28/5/phon28072.html

Rojas Ch O, Alazard D, Aponte R L and Hidrobo L F 1999 Influence of flow regime on the concentration of cyanide producing anaerobic process inhibition. Water Science Technology. Volume.40.No.8.pp. 177-185

Sangkhom I, Preston T R, Khang D N and Leng R A 2012 Effect of method of processing of cassava leaves on protein solubility and methane production in an in vitro incubation using cassava root as source of energy. Livestock Research for Rural Development. Volume 24, Article #36.  http://www.lrrd.org/lrrd24/2/sang24036.htm

Sangkhom Inthapanya, Preston T R and Leng R A 2011 Mitigating methane production from ruminants; effect of calcium nitrate as modifier of the fermentation in an in vitro incubation using cassava root as the energy source and leaves of cassava or Mimosa pigra as source of protein.  Livestock Research for Rural Development. Volume 23, Article #21. http://www.lrrd.org/lrrd23/2/sang23021.htm

Sejian V, R Lal, Lakritz J and Ezeji T 2010 Measurement and prediction of enteric methane emission. International Journal. Biometeorology DOI: 10.1007/s00484-010-0356-7.

Smith M R, Lequerica J L and Hart M R 1985 Inhibition of methanogenesis and carbon metabolism in Methanosarcina sp. by cyanide, Journal of Bacteriology, 162, 67-71.

Stupak M, Vanderschuren H, Gruissem W, Zhang P 2006 Biotechnological Approaches To Cassava Protein Improvement. Trends in Food Science & Technology, V.17, P.634-641, 2006.    

Tilley J M A and Terry R A 1963 A two stage technique for the in vitro digestion of forage crops. Journal of the British Grassland Society 18: 104     

Thanh V D, Preston T R and Leng R A 2011 Effect on methane production of supplementing a basal substrate of molasses and cassava leaf meal with mangosteen peel (Garcinia mangostana) and urea or nitrate in an in vitro incubation. Livestock Research for Rural Development. Volume 23, Article #98. http://www.lrrd.org/lrrd23/4/than23098.htm

Wanapat M 2001 Role of cassava hay as animal feed in the tropics. In: International Workshop Current Research and Development on Use of Cassava as Animal Feed, Khon Kaen University, Thailand July 23-24, 2001. http://www.mekarn.org/procKK/wana3.htm

Wanapat M 2003 Manipulation of cassava cultivation and utilization to improve protein to energy biomass for livestock feeding in the tropics. Asian-Australasian Journal of Animal Science, 16, 463-472.

Wanapat M 1999 Feeding of Ruminants in the tropics based on Local Feed Resources. Khon Kaen Publ. Comp. Ltd., Khon Kaen, Thailand. pp 236

Wanapat M, Khampa S 2007 Effect of Levels of Supplementation of Concentrate Containing High Levels of Cassava Chip On Rumen Ecology, Microbial N Supply And Digestibility Of Nutrients In Beef Cattle. Asian-Australasian Journal of Animal Science, V.20, P.75-82, 2007.   

Wanapat M, Promkot C, Wanapat S 2006 Effect Of Cassoy-Urea Pellet As A Protein Source In Concentrate On Ruminal Fermentation And Digestibility In Cattle. Asian-Australasian Journal of Animal Science, V.19, P.1004-1009, 2006.

Received 2 March 2016; Accepted 27 May 2016; Published 2 June 2016

Go to top