Methane production in an in vitro fermentation of cassava pulp with urea was reduced by supplementation with leaves from bitter, as opposed to sweet, varieties of cassava

L T B Phuong, D N Khang and T R Preston¹

Nong Lam University, Viet Nam
binhphuonglt@gmail.com
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV),
Carrera 25 No 6-62 Cali, Colombia

Abstract

Leaves from a sweet variety of cassava (Gon) and from bitter varieties (Japan, KM 94 and KM 140-1) were included as sources of protein in an in vitro fermentation of cassava root pulp supplemented with urea.

Methane production was lower when leaves from bitter rather than sweet cassava were the protein source. There was a negative curvilinear relationship between the levels of HCN in the leaves and methane production. Condensed tannins were in the range (2-2.6% in DM) considered to favor escape of dietary protein from intestinal digestion and did not differ between sweet and bitter varieties. Ammonia concentration in the digesta after 24h fermentation was higher when leaves from sweet rather than bitter cassava were the protein source.

Key words: condensed tannins, escape protein, HCN

Introduction

According to the General Department of Customs, in 2013 Vietnam exported 3.1 million tonnes of cassava and cassava products. Cassava root yield in Vietnam currently is 17.6 tonnes/ha placing it in tenth place among countries with high yield ( http://www.moit.gov.vn/en/News/508/production-and-export-of-cassava-in-2013.aspx ).

A recent development has been the industrial development of cassava root processing for extraction of starch for export. This has generated a major increase in the market for cassava roots such that, in Vietnam, cassava is now the second most important crop after rice. As a result there are large quantities of by-products at farm level (the foliage and stems) and at the factories (cassava pulp), all of which have potential feeding value for livestock.

Cassava pulp represents approximately 10 to 15% of the original root weight (Khempaka et al 2007). On a dry matter (DM) basis, cassava pulp contained 70% starch, 1.70% ash, 1.55% crude protein (CP, 27.8% crude fiber (CF) and 0.12% ether extract (EE) (Sriroth et al 2000). The pulp is very low in protein; however, the foliage is high in CP with content of more than 20% in DM (Lukuyu et al 2014). It was reported by Ffoulkes and Preston (1978) that fresh cassava foliage could replace soybean meal as the only protein source in a fattening diet for cattle based on ad libitum molasses-urea. Preston and Leng (2009) postulated that part of the cassava leaf protein had “rumen-escape” characteristics which helped to balance the microbial protein produced from the rumen fermentation of molasses supplemented with urea .

Cassava products contain cyanogenic glucosides which liberate hydrocyanic acid (HCN) when emzymically degraded. Cyanogenic glucosides exist as linamarin and lotaustralin in unbruised leaf (Nartley 1968). When the cellular structure is broken, the glucoside is exposed to extracellular enzymes such as linamarase which gives rise to toxic hydrocyanic acid. In studies on biodigestion of cassava residues it was shown that the HCN liberated in the digestion process was toxic to methanogenic bacteria (Smith et al 1985; Rojas et al 1999). It is therefore postulated that a similar process could take place in the rumen of cattle fed cassava products, which could be an advantage as a strategy for reducing greenhouse gas emissions from ruminant animals.

Cassava varieties are generally categorized into “sweet” varieties suitable for human consumption, and “bitter” varieties more appropriately used for industrial production of starch. It is understood that the ‘bitter” varieties are so-called because they have higher concentrations of cyanogenic glucosides making them potentially toxic to humans and animals.

The hypothesis of this study was that methane production in an in vitro rumen fermentation would be reduced when urea-supplemented cassava root pulp was incubated with the leaves from bitter, rather than sweet, varieties of cassava.

Materials and methods

Location and duration

The in vitro experiments were conducted in the laboratory of Nong Lam University, Ho Chi Minh city, Viet Nam, in December, 2014.

Experimental design

The four treatments in a completely randomized design (CRD) were the leaves of a “sweet” variety of cassava (Gon) and leaves from three bitter varieties (Japan, KM94 and KM 140-1). The substrates were cassava pulp and urea (Table 1). The leaves were added to provide an overall level of 12.8% crude protein in substrate DM.

A simple in vitro system was used based on the procedure reported by Inthapanya et al (2011).

Material preparation

The cassava leaves were from plants five months old growing in different locations in Cam My, Dong Nai province. Leaves (without petioles) were selected at a point approximately one third of the height of the plant measured from the top. They were stored in plastic bags to avoid loss of moisture. In the laboratory, the fresh leaves were chopped into small pieces and then ground (1mm sieve). Dry cassava pulp was taken from the Wuson starch factory, Binh Phuoc Province.

Urea, cassava pulp and the fresh cassava leaves were mixed with 0.24 liters of rumen fluid (taken from a Holstein male animal immediately after it was slaughtered at the local abattoir) and 0.96 liters of buffer solution (adapted from Tilly and Terry 1963). This mixture was put in the fermentation bottle, gassed with carbon dioxide, and incubated in a water bath at 39^°C for 24h.

Measurements

The gas volume was measured by water displacement from the receiving bottle suspended in water. The bottle was calibrated at intervals of 50ml. The methane percentage in the gas was measured with a Crowcom meter (Crowcom Instruments Ltd, UK). The DM and crude protein contents of the substrates were determined according to AOAC (1990) methods. Ammonia was analysed in the filtrate after separating the solids using a cloth filter. HCN was determined by titration with AgNO₃ after boiling the sample in KOH to concentrate the HCN. Tannin was analyzed by the Lowenthal method consisting of boiling the leaves in 0.1N H₂SO₄, adding indigo dye and titrating with potassium permanganate.

Results and discussion

Chemical composition of the substrate

The cassava leaves contained a high level of crude protein (27.5-31.8% CP in DM); the cassava pulp had less than 3% CP in DM (Table 2).

Gas production, ammonia concentration and DM mineralized

Gas production after 24 hours fermentation did not differ among the treatments (Table 3). The percent age of DM mineralized was lower in the sweet cassava variety than in the three bitter varieties among which there were no differences. Ammonia concentration in the fermentation medium at the end of the incubation was higher for the sweet cassava than for the bitter varieties. A similar effect of cassava variety on ammonia concentration was reported by Phuong et al (2012). It could be that the higher content of HCN precursors in the bitter varieties had an inhibitory effect on microbial breakdown of the cassava leaf protein.

Methane, HCN and condensed tannin

Methane production, expressed as percent of the gas production or per unit DM mineralized, was lower when leaves from the bitter varieties were the protein source compared with the sweet variety (Table 4; Figure 1. This is in agreement with the report by Phanthavong et al (2015) that methane production was lower when leaves from a bitter cassava variety, rather than a sweet variety, were the source of protein in an in vitro fermentation with cassava pulp and urea. The negative relationship between HCN and methane production indicates that it is the HCN precursors in the leaves of the bitter varieties that are responsible for the decline in methane production (Table 4; Figure 2). This is in agreement with reports of cyanide inhibiting methanogenesis in microbial systems (Smith et al 1985; Rojas et al 1999).

The content of condensed tannin (2 to 2.6% in DM) was in the range considered to favor escape of dietary protein from the rumen for subsequent hydrolysis in the abomasum and intestines (Barry and McNabb 1999). The negative relationship between tannin content and ammonia concentration after fermentation (Figure 3) supports this hypothesis. It is also in line with reports that impairment of proteolysis of dietary protein leads to lower concentration of ammonia in rumen fluid (Frutos et al 2004; Jayanegara 2011).

Figure 1. Mean values for percent methane in the gas in an in vitro rumen fermentation with sweet and bitter varieties of cassava leaves	Figure 2. Relationship between methane in the gas and the concentration of HCN in the cassava leaves	Figure 3. Effect of condensed tannin on ammonia production during the in vitro fermentation of cassava root pulp with urea and leaves of bitter or sweet cassava varieties

Conclusions

• In an in vitro fermentation of cassava root pulp supplemented with urea, methane production was lower when leaves from bitter rather than sweet cassava were the protein source.
  
  
• There was a negative curvilinear relationship between the levels of HCN in the leaves and methane production.
  
  
• Ammonia concentration in the digesta after 24h fermentation was higher when leaves from sweet rather than bitter cassava were the protein source.

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

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Received 10 July 2015; Accepted 20 July 2015; Published 1 August 2015

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