Effect of rice-wine distillers’ byproduct, biochar and sweet or bitter cassava leaves on gas production in an in vitro incubation using ensiled cassava root as substrate

Sengsouly Phongphanith and T R Preston¹

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

Abstract

This study was to evaluate the effect of sweet or bitter cassava foliage, rice distillers’ byproduct and biochar on methane production in an in vitro incubation system with ensiled cassava root as the energy source. The design was a 2*2*2 factorial with 4 replications: rice distillers’ byproduct (RDB) at 0 or 4% of substrate DM, biochar: 0 or 1% of substrate DM and leaves of sweet or bitter cassava at 30% of substrate DM. Urea (3% of ensiled root DM) and sulphur-rich minerals (2% of substrate DM) were included in the fermentation medium. 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 cattle immediately after being slaughtered”. The bottles were then filled with carbon dioxide and incubated at 38 ⁰C in a water bath for 24 hours.

Methane production was reduced when: leaf meal from bitter rather than sweet cassava was the protein source in an in vitro fermentation of ensiled cassava root; biochar was added to the fermentation medium.

Keywords: biofilm, cassava leaf, by-product, ensiling, in vitro, methane

Introduction

Greenhouse gases (GHG) will have to peak by 2020 and drop by 75-80 per cent in the period to 2050 to limit global warming to two degrees (The Climate Group 2008). The total GHG emissions in 2010 were estimated to have increased by more than 6 per cent, and for 2011 were estimated to have increased by 3.2 per cent (The Guardian 2011; IEA 2011). The methane emissions from enteric fermentation in herbivorous animals, especially ruminants, are considered a major source of greenhouse gases (Stavi and Lal 2013).

Cassava (Manihot esculenta Crantz) is grown in over 90 countries and is a most important food crop worldwide. It is the primary staple for more than 800 million people in the world (Lebot 2009). Of importance in a warming world is that it appears that cassava is potentially highly resilient to future climatic changes and according to Jarvis et al (2012) “could provide Africa with options for adaptation whilst other major food staples face challenges”.

Cassava foliage is considered to be a good source of bypass protein for ruminants (Ffoulkes and Preston 1978; Wanapat 2001; Keo Sath et al 2008). It has been fed as a major component of the diet for sheep (Hue et al 2008), goats (Ho Quang Do et al 2002; Dung et al 2005; Phengvichith and Ledin 2007; Seng Sokerya and Preston 2003) and cattle (Wanapat et al 2000; Thang et al 2010) in fresh, wilted or dried form.

Cassava leaves are known to contain variable levels of condensed tannins; about 3% in DM according to Netpana et al (2001) and Bui Phan Thu Hang and Ledin (2005). Condensed tannins at moderate levels are known to have positive effects on the nutritive value of the feed by forming insoluble complexes with dietary protein, resulting in "escape" of the protein from the rumen fermentation (Barry and McNabb 1999). Numerous studies have also shown the potential of the tannin content in cassava leaves to play an anthelminthic role for the control of nematode parasites in ruminants (Seng Sokerya and Preston 2003; Seng Sokerya et al 2009; Netpana et al 2001; Khoung and Khang 2005). Condensed tannins (CT) are also reported to decrease methane production and increase the efficiency of microbial protein synthesis (Makkar et al 1995; Grainger et al 2009). Reductions of CH₄ production due to presence of tannins were reported by Carulla et al (2005), Waghorn et al (2002), Grainger et al (2009) and Woodward et al (2004), apparently through a direct toxic effect on methanogens.

Previous research showed that methane production in a rumen in vitro fermentation system was reduced when the protein source was leaf meal derived from “bitter” as opposed to “sweet” varieties of cassava (Le Thuy Binh Phuong et al 2011).

Rice distillers’ by-product is another potential source of high quality protein in rural areas of Lao PDR. Rice distillers’ by-product is the residue after distilling the alcohol derived by yeast fermentation of sticky rice (Taysayavong Lotchana and Preston 2010). The farmers traditionally use it as a mixture with other feeds such as rice bran and broken rice in diets for fattening pigs (Oosterwijk et al 2003). The farmers in Vietnam also use rice distillers’ by-product (known as “hem”) as a traditional feed for pigs (Luu Huu Manh 2000). The protein content of "hem" ranged from 17 to 33% (mean of 23%) in dry matter with a well-balanced array of amino acids (Luu Huu Manh et al 2003). These authors reported that this product could replace completely the fish meal in growing and fattening pig diets with no loss of performance.

Biochar derived from partial combustion of rice husks in an updraft gasifier stove (Olivier 2010) reduced methane production in an in vitro rumen incubation of cassava root meal and cassava leaf meal supplemented with urea or potassium nitrate as the major fermentable N source (Leng et al 2012).

The objectives of the present study were to evaluate the effect of sweet or bitter cassava foliage, rice distillers’ byproduct and biochar on methane production in an in vitro incubation system with ensiled cassava root as the energy source.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of the Faculty of Agriculture and Forest Resource, Souphanouvong University, LuangPrabang Province, Lao PDR, from January to February, 2016

Treatments and experimental design

The design was arranged as a 2*2*2 factorial with 4 replications. The factors were:

• RDB: Rice distillers’ byproduct at 0 or 4% of substrate DM
• BC: Biochar at 0 or 1% of substrate DM
• CLM: Leaf meal from sweet or bitter cassava varieties at 30% of substrate DM

The basal substrate was ensiled cassava root and urea at 3% of the DM of the ensiled root plus sulphur-rich minerals at 2% of substrate DM.

The in vitro system

The in vitro system was made from recycled “PEP” water bottles as described by Inthapanya et al (2011) (Photos 1, 2 and 3).

Photo 1. The in vitro system	Photo 2. Measurement of methane production in the gas	Photo 3. The substrate residue filtered through cloth

Experimental procedure

The cassava root was chopped into small pieces around 1-2 cm of length, then ensiled in sealed plastic bags for 7days. Cassava leaves (harvested from a sweet and a bitter variety) were chopped into small pieces around 1-2 cm of length, then dried in sunlight before grinding (1mm sieve).

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. The biochar was ground through a 1 mm sieve. Rice distillers’ byproduct was bought from farmers who produce “rice wine”.

Amounts of the substrates equivalent to 12g DM were put in the incubation bottle in the in vitro system followed by 0.96 liters of buffer solution (Table 1) and 240 ml of rumen fluid obtained from a steer immediately after being slaughtered. The bottles was then filled with carbon dioxide and incubated at 38 ⁰C in a water bath for 24 h.

Data collection and measurements

During the incubation the gas volume was recorded at 12 and 24h (Photo 1). After each time interval, samples of gas were taken for measurement of the methane concentration using an infra-red meter (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).

Chemical analyses

The samples of ensiled cassava root, rice distillers’ byproduct, and cassava leaf meals were analyzed for DM, ash and crude protein according to AOAC (1990) methods.

Statistical analysis

The data from the experiment were analyzed by the general linear model option of the ANOVA program in the Minitab software (version 16.0). The sources of variation were: replicates, RDB, biochar, sweet or bitter leaves of cassava, interaction RDB*biochar, RDB*cassava leaf, RDB*biochar*cassava leaf and error.

Results

Chemical composition

The composition of the substrate ingredients is in Table 2.

Gas production

Gas production was not affected by rice distillers’ byproduct nor by biochar but was less for bitter compared with sweet cassava leaf meal (Table 3).

Methane

The methane concentration in the gas increased with fermentation time (Table 3). Per unit of substrate methane production was not affected by RDB (Figure 1), was lower for bitter compared with sweet cassava leaves (Figures 2 and 6) and for addition of biochar (Figures 3 and 5). Substrate DM mineralized was increased by RDB, and decreased by bitter compared with sweet cassava leaves and by addition of biochar.

Figure 1. Effect of rice distiller’s by product with bitter or sweet cassava leaf meal on methane per unit substrate	Figure 2. Effect of bitter or sweet cassava leaf meal with or without rice distiller’s by product on methane per unit substrate
Figure 3. Effect of biochar with or without rice distiller’s by product on methane per unit substrate	Figure 4. Effect of rice distiller’s by product with or without biochar on methane per unit substrate
Figure 5. Effect of biochar with bitter or sweet cassava leaf meal on methane per unit substrate	Figure 6. Effect of bitter or sweet cassava leaf meal with or without biochar on methane per unit substrate

Discussion

The reduction in methane when leaf meal from bitter rather than sweet cassava was the protein source is in agreement with the findings of Binh Phuong et al (2011) in an in vitro rumen system. A similar result was reported by Binh Phuong et al (2016, personal communication) in cattle fed foliage from a bitter compared with a sweet cassava variety as a supplement to ensiled cassava root pulp.

In research reported by Phuong et al (2012), there were only minor differences in the solubility of the protein between bitter (31.9% soluble protein) and sweet (28.8 – 30.4% soluble protein) cassava varieties. The higher concentrations of cyanogenic glucosides (precursors of HCN) in leaves from bitter compared with sweet cassava is a more likely explanation of the reduced production of methane from leaves of bitter cassava. The research of Eikmanns and Thauer (1984) and Smith et al (1985) supports the concept that cyanide is somewhat toxic to methanogens or reduces their potential growth by lowering the availability of sulphur by formation of thiocyanates (Majak and Cheng 1984). Additions of 5, 10, and 25 mg 1itre^-l cyanide (from KCN or linamarin) temporarily inhibited methanogenesis in biodigesters charged with cassava root waste (Cuzin and Labat 1992). The biodigester methanogenic microflora were sensitive to the added cyanide.

The observed reduction in methane by addition of biochar to the fermentation medium is supported by many reports that incorporation of biochar in in vitro rumen systems (Leng et al 2012a,b; Leng et al 2013; Phanthavong et al 2015; Silivong et al 2015; Vongkhamchanh et al 2015) or fed directly to cattle (Leng et al 2012c; Sengsouly and Preston 2016) reduces methane production.

Conclusions

• Methane production was reduced when: leaf meal from bitter rather than sweet cassava was the protein source in an in vitro fermentation of ensiled cassava root; biochar was added to the fermentation medium

Acknowledgements

The authors acknowledge support for this research from the MEKARN II project financed by Sida. Special thanks are given to Mr Sangkhom Inthapaya who provided valuable help in the laboratory and I also acknowledge Souphanouvong University, Faculty of Agriculture and Forest Resources, Department of Animal Science, providing the facilities to carry out this research.

References

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

Belibasakis N and Tsirgogianni D 1996 Effects of wet brewers grains on milk yield, milk composition and blood components of dairy cows in hot weather. Animal Feed Science and Technology, 57: 175–181

Binh Phuong L T, 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 June 1, 2016, from http://www.lrrd.org/lrrd23/2/phuo23022.htm

Bui Phan Thu Hang and Ledin Inger 2005 Utilization of Melastoma (Melastoma affine, D. Don) foliage as a forage for growing goats with cassava (Manihot Esculenta, Crantz) hay supplementation. Proceedings International Workshop on Small Ruminant Production and Development in South East Asia (Editor: Inger Ledin), Hanoi, Vietnam, 2-4 March 2005.http://www.mekarn.org/procsr/hangctu.pdf

Carulla J E, Kreuzer M, Machmüller A and Hess H D 2005 Supplementation of Acacia mearnsii tannins decreases methanogenesis and urinary nitrogen in forage-fed sheep. Australian Journal of Agricultural Research 56, 961-970.

Dung N T, Mui N T and Ledin I 2005 Effect of replacing a commercial concentrate with cassava hay (Manihot esculenta Crantz) on the performance of growing goats. Animal Feed Science and Technology, 119, 271–281

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

Grainger C, Clarke T, Auldist M J, Beauchemin K A, McGinn S M and Waghorn G C 2009 Potential use of Acacia mearnsii condensed tannins to reduce methane emissions and nitrogen excretion from grazing dairy cows, Canadian Journal of Animal Science, 89, 241–251

Ho Quang Do, Vo Van Son, Bui Phan Thu Hang, Vuong Chan Tri and Preston T R 2002 Effect of upplementation of ammoniated rice straw with cassava leaves or grass on intake, digestibility and N retention by goats.Livestock Research for Rural Development. Volume 14, Article #143b. http://www.lrrd.org/lrrd14/3/do143b.htm

Hue K T, Van D T T and Ledin I 2008 Effect of supplementing urea treated rice straw and molasses with different forage species on the performance of lambs. Small Ruminant Research, 78, 134–143

IEA 2011 CO₂ emission from fuel combustion: 2011 Edit. Paris Available at: www.iea.org/co2highlights.

Jarvis A, Ramirez-Villegas J, Herrera Campo B V and Navarro-Racines C 2012 Is Cassava the Answer to African Climate Change Adaptation? Tropical Plant Biology.5 (1) 9-29.

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

Khuong L H and Khang D N 2005 Effect of fresh cassava foliage on growth and feacal nematoda egg counts in Sindhi x yellow cattle fed urea-treated rice straw basal diet. Workshop-seminar "Making better use of local feed resources" (Editors: T. R. Preston and B. Ogle) MEKARN-CTU, Cantho, 23-25 May, 2005.

Lebot V 2009 Cassava tropical root and tuber crops: cassava, sweet potato, yams and aroids. CAB International, Wallingford

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. Retrieved August 4, 2015, from http://www.lrrd.org/lrrd24/6/sang24103.htm

Leng R A, Inthapanya S and Preston T R 2012 Methane production is reduced in an in vitro incubation when the rumen fluid is taken from cattle that previously received biochar in their diet. Livestock Research for Rural Development. Volume 24, Article #211. http://www.lrrd.org/lrrd24/11/sang24211.htm

Leng R A, Inthapanya S and Preston T R 2013 All biochars are not equal in lowering methane production in in vitro rumen incubations. Livestock Research for Rural Development. Volume 25, Article #106. http://www.lrrd.org/lrrd25/6/leng25106.htm

Leng R A, Preston T R and Inthapanya S 2012 Biochar reduces enteric methane and improves growth and feed conversion in local “Yellow” cattle fed cassava root chips and fresh cassava foliage. Livestock Research for Rural Development. Volume 24, Article #199. ttp://www.lrrd.org/lrrd24/11/leng24199.htm

Luu Huu Manh 2000 Composition and nutritive value of rice distillers’ by-product (hem) for small-holder pig production, from: http://www.mekarn.org/sarpro/manh.htm

Luu Huu Manh, Nguyen Nhut Xuan Dung and Lindberg J E 2003 Effects of replacement of fish meal with rice distiller’s waste (hem) on performance and carcass quality of growing pigs In: Proceedings of Final National Seminar-Workshop on Sustainable Livestock Production on Local Feed Resources (Editors: Reg Preston and Brian Ogle). HUAF-SAREC, Hue City, 25 – 28 March, 2003. Retrieved May 18, 110, from: http://www.mekarn.org/sarec03/manh3.htm

Makkar H P S, Blu¨mmel M and Becker K 1995 In vitro effects of and interactions between tannins and saponins and fate of saponins tannins in the rumen, Journal of the Science of Food and Agriculture. pp. 481–493.

Minitab 2000 Minitab Software Release 16.0

Netpana N, Wanapat M, Poungchompu O and Toburan W 2001 Effect of condensed tannins in cassava hay on fecal parasitic egg counts in swamp buffaloes and cattle. In: Proceedings International Workshop on Current Research and Development on Use of Cassava as Animal Feed. T R Preston, B Ogle and M Wanapat (Ed) http://www.mekarn.org/procKK/netp.htm

Oosterwijk, G, Van Aken D and Vongthilath 2003 A manual on Improved Rural Pig Production (1st Edition, English Language). Department of Livestock and Fisheries, Ministry of Agriculture and Forestry, Vientiane, Lao PDR VIII + 113 pp. Page 21, from: http://www.smallstock.info/reference/FAO/APHCA/Pig_Eng_ebook.pdf

Phanthavong V, Viengsakoun N, Sangkhom I and Preston T R 2015 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. Livestock Research for Rural Development. Volume 27, Article #72. http://www.lrrd.org/lrrd27/4/phan27072.html

Phengvichith V and Ledin I 2007 Effect of feeding different levels of wilted cassava foliage (Manihot esculenta, Crantz) on the performance of growing goats. Small Ruminant Research, 71, 109–116

Phongphanith S, Preston T R and Leng R A 2016 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. Livestock Research for Rural Development. Volume 28, Article #112. http://www.lrrd.org/lrrd28/6/seng28112.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

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

Seng Sokerya and Preston T R 2003 Effect of grass or cassava foliage on growth and nematode parasite infestation in goats fed low or high protein diets in confinement. MSc. Thesis, MEKARN-SLU

Seng Sokerya, Try P, Waller P J and Hapglund J 2009 The effect of long-term feeding of fresh or ensiled cassava (Manihot esculenta) foliage on gastrointestinal nematode infections in goats. Tropical Animal Health and Production 41(2), 251-258.

Silivong P and Preston T R 2015 Effect of water spinach and biochar on methane production in an in vitro system with substrate of Bauhinia acuminata or Bitter Neem (Azadirachta indica) leaves. Livestock Research for Rural Development. Volume 27, Article #57. http://www.lrrd.org/lrrd27/3/sili27057.html

Stavi I and Lal R 2013 Agriculture and greenhouse gases, a common tragedy. A review. Agronomy for Sustainable Development. 33(2):275-289.

Taysayavong Lotchana and Preston T R 2010 Effect of rice distillers’ by-product on growth performance and digestibility of Moo Laat and Mong Cai pigs fed rice bran and water spinach http://www.mekarn.org/msc2008-10/theses/lotc_1.htm

Thang C M, Ledin I and Bertilsson J 2010 Effect of feeding cassava and/or Stylosanthes foliage on the performance of crossbred growing cattle. Tropical Animal Health Production. 42, 1–11

The Climate Group 2008 Breaking the climate deadlock. A global deal for our low-carbon future. Report submitted to the G-8 Hokkaido Toyako Summit. London. Available at www. http://www.theclimategroup.org/what-we-do/programs/Breaking-the-Climate-Deadlock/ .

The Guardian 2011 London, 30 May. Available at: www.guardian.co.uk/.

Vongkhamchanh B, Inthapanya S and Preston T R 2015 Methane production in an in vitro rumen fermentation is reduced when the carbohydrate substrate is fresh rather than ensiled or dried cassava root, and when biochar is added to the substrate. Livestock Research for Rural Development. Volume 27, Article #208. http://www.lrrd.org/lrrd27/10/bobb27208.html

Waghorn, G.C, Tavendale, M.H and Woodfield, D.R 2002 Methanogenesis from forages fed to sheep. Proceedings of the New Zealand Grassland Association 64, 167–171.

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, Puramongkon T and Siphuak W 2000 Feeding of cassava hay for lactating dairy cows. AsianAustralasian Journal of Animal Sciences, 13, 478–482

Woodward S L, Waghorn G C and Laboyrie P 2004 Condensed tannins in birdsfoot trefoil (Lotus corniculatus) reduced methane emissions from dairy cows. Proceedings of the New Zealand Society of Animal Production 64, 160–164.

Received 13 September 2016; Accepted 15 September 2016; Published 1 October 2016

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