Effect of biochar and leaves from sweet or bitter cassava on gas and methane production in an in vitro rumen incubation using cassava root pulp as source of energy

Phanthavong Vongsamphanh, Viengsakoun Napasirth¹, Sangkhom Inthapanya² and T R Preston³

Department of Livestock and Fisheries, Ministry of Agriculture and Forestry
PO Box 6644 Vientiane, Lao PDR
ptvkivor@yahoo.com
¹ Faculty of Agriculture, National University of Laos,Nabong Campus, P.O.Box7322, Vientiane, Lao PDR
² Souphanouvong University, Luang Prabang, Lao PDR
³ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV),
Carrera 25 No 6-62 Cali, Colombia

Abstract

The aim of this study was to evaluate the effect of processing of cassava leaves and of supplementation with biochar on methane production in an in vitro rumen incubation with ensiled cassava pulp  as substrate. The design was a 2*2*2 factorial in CRD of 8 treatments with 4 replications. The factors were: Processing:  dried vs fresh cassava leaf; Variety:  bitter vs sweet; Biochar: with or without biochar. The quantity of substrate was 12 g with 65% as cassava root pulp and 31% as cassava leaf (DM basis).  To the 12 g of substrate DM were added 240 ml rumen fluid (from slaughtered cattle) and 960 ml of buffer solution. The incubation was for 24 h with measurements of total gas production, methane percentage and determination of residual unfermented substrate at the end.

 The percentage of methane in the gas was lower for: (i)  bitter compared with sweet cassava; (ii)  fresh versus dried leaves; and (iii) from substrates with biochar than for those without  biochar. The DM solubilized after 24h was not affected by the treatments. Methane produced per unit DM solubilized followed the same trends as for percent methane in the gas, with the exception of the effect of processing where there were no differences.

Key words: biochar, climate change, gas production, greenhouse gases

Introduction

Cassava planting in Lao PDR occurs in the rainy season starting in April for harvest some 8 months later. Almost all the production is processed into starch for export. There are five cassava starch factories with total planted area of 43,975 ha, giving an average yield of fresh roots of 24 tonnes/ha. Annual production is of the order to one million tonnes (Department of Agriculture 2012).

The cassava for the starch factory in Nashaw village, Pakngum District in Vientiane Capital is supplied by 235 households in 85 villages with total cassava harvested area of 5,207 ha producing 100.000 tonnes per year of cassava roots into starch for export. During the 6-7 month harvest season from October to March-April (the dry season in Lao PDR) this amounts to 480 tonnes of roots daily. The byproduct remaining after starch extraction, known as cassava pulp, represents from 10 to 15% of the original weight of fresh roots (Sriroth et al 2000). Over the past 4 years, very little of the cassava pulp was bought by farmers and almost all of it had been stored in a pit adjacent to the factory, which had not been covered or protected in any way. A recent study showed that the stored pulp had ensiled perfectly with a pH of 3.5 and that its potential feed value was only slightly less than that of the fresh whole cassava root (Phanthavong et al 2014).   

 For every tonne of roots that are harvested there are an additional 600 kg of stems and leaves which also have a high potential feeding value for cattle (Ffoulkes and Preston 1978; Wanapat et al 1997), and goats (Ho Quang Do et al 2002). The protein content in cassava leaves is around 22-25% in dry matter (DM), however, the famers in the Factory area have no experience in the utilization of cassava leaves as the protein supplement to feed to animals, especially cattle.

The varieties used for industrial starch production are known as “bitter” varieties due to the high content of cyanogenic glucosides that are converted into the highly toxic hydrocyanic acid when consumed by animals and people. The cassava varieties that are planted for human consumption are known as “sweet” varieties as they have a lower content of cyanogenic glucosides. Much research on evaluating the use of cassava for livestock feeding has thus concentrated on methods to reduce the HCN content, such as sun-drying and ensiling (Phuc et al 1995).

Recent research by Phuong et al (2012) showed that from the point of view of the environment, and especially the problem of global warming,  the presence of the cyanogenic glucosides in cassava could be an advantage. Methane emissions in an in vitro fermentation were found to be lower when the cassava leaves in the fermentation substrate were from “bitter” varieties rather than from “sweet “varieties”. 

Therefore, the objective of this research was to compare the methane production from cassava leaves derived from both bitter and sweet varieties.  As drying is known to reduce the HCN produced from cassava leaves (Phuc et al 1995), a comparison was made of leaves in both the fresh and dry state.

Biochar is the residue when fibrous biomass is carbonized at high temperatures. It has been shown to play an active role in systems involving microbial fermentation (Lehmann and Joseph 2009). It is believed that the biochar, which is highly porous,   acts a support for biofilms that facilitate the functioning of consortia of micro-organisms and their nutrients (Leng et al 2012).

Hypothesis

• It was hypothesized that in an in vitro rumen fermentation of cassava root pulp, the methane production would be reduced: (i) when the protein source was the leaf of bitter cassava compared with leaf of sweet cassava; (ii) when the leaves were in the fresh rather than the dry state; and (iii) when biochar was included in the fermentation medium.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of the Faculty of Agriculture, National University of Laos from September to October 2014

Treatments and experimental design

The experimental design was arranged as a 2*2*2 factorial in a completely randomized design of 8 treatments with 4 replications.

The factors were:

Processing:

• Dried (DCL) or fresh (FCL) cassava leaves

Source:

• Bitter (B) or sweet (SW) cassava

Biochar:

• Presence (BC) or absence of biochar (NBC)

The basic substrate was cassava root pulp (CRP) to which was added urea (3% of cassava pulp DM) and a mineral mixture (supplying phosphorus and sulphur). The leaves in the fresh or dry state were ground and added to the substrate at levels of 30% of the total DM. Biochar was added at 1% of the total substrate DM.

The in vitro system

The equipment and procedure were as described by Inthapanya et al (2011) (Photos 1 and 2).

Photo 1. The in vitro system	Photo 2. Gas production after fermentation

Experimental procedure

Cassava leaves from sweet and bitter varieties were collected in the morning from plots in the  Faculty of Agriculture, National University of Laos, Nabong Campus. The  leaves and petioles were separated from the rest of the foliage and immediately chopped into small pieces (0.5-1.0 cm) and then ground (1mm sieve). Ensiled cassava pulp was collected from the storage pit at the Cassava Starch Factory at Nashaw village (Phanthavong et al 2014).  Biochar was produced locally by combusting rice husks in a top lit updraft (TLUD) gasifier stove (Olivier 2010). Urea and minerals, and biochar (in the BC  treatment)  (Table 1), were mixed with the cassava pulp and cassava leaves, and put in the fermentation bottle prior to adding 0.96 liters of buffer solution (Table 2) and 240 ml of rumen fluid (obtained from a newly slaughtered animal of the local “Yellow” breed in the Saythany District abattoir). The residual air in the fermentation bottle was flushed out with carbon dioxide. The bottles were incubated at 38ºC in a water bath for 24 hours.  

Data collection and measurements

The gas volume and the percentage of methane in the gas (Crowcon infra-red analyser; Crowcon Instruments Ltd, UK) were recorded for the separate incubations after 12h, 18 and 24h. At the end of each incubation the residual DM in the incubation bottle was determined by filtering through cloth and drying (100°C for 24h) the residue. The filtrate was analysed for ammonia nitrogen.

Chemical analysis

Dry matter (DM), nitrogen (N), ether extract, crude fiber and ash were determined by standard methods (AOAC 1900). Protein solubility was determined by extraction with M NaCl solution (Whitelaw and Preston 1963).

Statistical analysis

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

Results and discussion

Chemical composition

On all processing treatments were analyzed as of % DM (Table 3).

Gas production

Gas production did not differ among the treatments (Table 4).

Methane and DM solubilized

The percentage of methane in the gas was lower for: (i) bitter compared with sweet cassava; (ii) fresh versus dried leaves; and (iii)  substrates with biochar than for those without biochar (Table 4; Figures 1a,b). The DM solubilized after 24h was not affected by the treatments. Methane produced per unit DM solubilized followed the same trends as for percent methane in the gas, with the exception of the effect of processing where there were no differences (Figure 2).

Figure 1a. Percentage methane in the gas from substrates containing: (i) leaves from bitter vs sweet cassava varieties; (ii) leaves that were fresh rather than dry; and (iii) biochar or no biochar

Figure 1b. Percentage methane in the gas from substrates containing: (i) leaves from bitter vs sweet cassava varieties; (ii) leaves that were fresh rather than dry; and (iii) biochar or no biochar

Figure 2. Methane iper unit DM solubilized from substrates containing: (i) leaves from bitter vs sweet cassava varieties; (ii) leaves that were fresh rather than dry; and (iii) biochar or no biochar

The lower methane production in substrates with fresh cassava leaves compared with dried leaves, and for the bitter compared with the sweet variety, indicates that cassava leaves with higher levels of cyanide precursors decrease the amount of methane produced. This may be explained if cyanide inhibits the metabolism of acetate to methane and carbon dioxide as happens in sludge type fermentations (Smith et al 1985; Gijzen et al 2000).

Conclusions

•  The percentage of methane in the gas was lower for: (i)  bitter compared with sweet cassava; (ii)  fresh versus dried leaves; and (iii) substrates with biochar than for those without  biochar.
  
  
• The DM solubilized after 24h was not affected by the treatments.
  
  
• Methane produced per unit DM solubilized followed the same trends as for percent methane in the gas, with the exception of the effect of processing where there were no differences.

Acknowledgements

This research is part of the requirement for the PhD of the senior author in the doctoral  program of Nong Lam University, Vietnam. Financial support from the Sida-financed project, MEKARN II, is gratefully acknowledged.

References

AOAC 1990 Official methods of analysis. 15th ed. AOAC, Washington, DC

Department of Agriculture 2012 Crop statistic year book, Lao PDR

Ho Quang Do, Vo Van Son, Bui Phan Thu Hang, Vuong Chan Tri and Preston T R 2002 Effect of supplementation of ammoniated rice straw with cassava leaves or grass on intake, digestibility and N retention by goatsLivestock Research for Rural Development. Volume 14, Article #29. http://www.lrrd.org/lrrd14/3/do143b.htm

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 Volume 3, Number 3 http://www.utafoundation.org/UTAINFO1/TAP/TAP33/3_3_1.pdf

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

Inthapanya S, 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

Lehmann J and Joseph S (Editors) 2009 Biochar for Environmental Management; Science and Technology. Earthscan, London, EC1N 8XA, UK, Sterling,VA 20166-2012, USA

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

Minitab 2000 Minitab Software Release 13

Olivier P 2010 The Small-Scale Production of Food, Fuel, Feed and Fertilizer; a Strategy for the Sustainable Management of Biodegradable Waste. http://www.mekarn.org/workshops/pakse/html/olivier.docx

Phanthavong V, Viengsakoun N, Sangkhom I and Preston T R 2014 Cassava pulp as livestock feed; effects of storage in an open pit. Livestock Research for Rural Development. Volume 26, Article #169. http://www.lrrd.org/lrrd26/9/phan26169.htm

Phuc B H N, Lai N V, Preston T R, Ogle B and Lindberg J E 1995 Replacing soya bean meal with cassava leaf meal in cassava root diets for growing pigs. Livestock Research for Rural Development. Volume 7, Article #21. http://www.lrrd.org/lrrd7/3/9.htm

Phuong L T B, Preston T R and Leng R A 2012 Effect of foliage from “sweet” and “bitter” cassava varieties on methane production in in vitro incubation with molasses supplemented with potassium nitrate or urea. Livestock Research for Rural Development. Volume 24, Article #189. http://www.lrrd.org/lrrd24/10/phuo24189.htm

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. http://jb.asm.org/content/162/1/67.full.pdf+html

Sriroth K, Chollakup R, Chotineeranat S, Piyachomkwan K and Oates C G 2000 Processing of cassava waste for improved biomass utilization. Bioresource Technology, Volume 71, pp 63-69

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

Wanapat M, Pimpa O, Petlum A and Boontao U 1997 Cassava hay: A new strategic feed for ruminants during the dry season. Livestock Research for Rural Development. Volume 9, Number 2, Article 18. http://www.lrrd.org/lrrd9/2/metha92.htm

Whitelaw F G and Preston T R 1963 The nutrition of the early-weaned calf. III. Protein solubility and amino acid composition as factor affecting protein utilization. Animal Production 5, 131-145

Received 14 February 2015; Accepted 15 March 2015; Published 1 April 2015

Go to top