Biochar improves the protein-enrichment of cassava pulp by yeast fermentation

Somphanh Philavong, T R Preston¹ and R A Leng²

Living Aquatic Resource Research Centre (LARReC, Lao PDR
Sph.philavong@yahoo.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 2351, Australia.

Abstract

The treatments, in a completely randomized design with 5 replicates, were five levels of biochar (0, 0.25, 0.5, 0.75 and 1% in DM) in a solid-state fermentation of cassava pulp with yeast, urea and diammonium phosphate (DAP). The substrates were 5kg of fresh cassava pulp (30% DM) to which were added (DM basis) urea 3%, DAP 1% and yeast 4%. The substrates were mixed and stored in closed polyethylene bags for fermentation over 7 days. Crude and true protein and pH of the substrates were determined at the beginning and after 7 days of fermentation.

The level of crude protein in the substrate DM was not affected by addition of biochar, either before (14.0% in DM) or after 7 days of fermentation (14.8%). In the absence of biochar the true protein in the fermentation medium increased from 2.9 to 5.2% (in DM), after 7 days of fermentation. With 1% of added biochar the corresponding increase was from 2.9 to 7.2%. There was a linear increase (R² =0.98) in the pH of the fermentation medium (from 3.6 to 4.4) as the biochar concentration was increased from 0 to 1%, and this was linearly related (R²=0.90) with the rate of conversion of non-protein-nitrogen (urea and DAP) to true protein. It is concluded that biochar acts as a support mechanism for consortia of microorganisms acting synergistically to enhance the rate of fermentation of starch and therefore the efficiency of microbial growth.

Keywords: biofilms, DAP, solid-state, urea

Introduction

Cassava has become a major crop in Lao PDR mainly because of the export of starch that is extracted from the cassava root. There are five cassava starch factories with total planted area of 60,475 ha of cassava, giving an average yield of fresh roots of 27 tonnes/ha. Annual root production is of the order of 1.6 million tonnes (Department of Agriculture 2014).

It is estimated that the five cassava starch factories in Lao PDR have a yearly production of 200,000 tonnes of pulp, which is the residue after the extraction of starch from the roots (Phanthavong et al 2014). Cassava pulp represents approximately 10 to 15% of the original root weight (Khempaka et al 2007). It is composed almost completely of non-structural carbohydrate, 65% of which is starch according to Sriroth et al ( 2000). It ensiles naturally (pH 3.0-3.5) and can be preserved over long periods (Phanthavong et al 2014). Its limitation as livestock feed especially for monogastric animals is the low protein content (<3% in DM; www.feedipedia.org).

Protein-enrichment of cassava pulp by solid-state fermentation with yeast and urea has been studied by several researchers but the results have been variable. Sengxayalth and Preston (2017) showed that the proportion of true to crude protein increased from 30% at the start of the fermentation to 70% after 7 days, equivalent to final percentages of 12.8 and 18.4% of true and crude protein in the pulp DM. Comparable data from the experiment of Hong et al (2017) were: true protein 34% of the crude protein at the start and 46% after 7 days fermentation, equivalent to final levels of true and crude protein in the pulp DM of 12 and 27%. In both of these studies it was apparent that at the end of the fermentation a considerable proportion of the urea and diammonium phosphate (DAP), added at the beginning, remained in some form of “non-protein-nitrogen” after 7 days fermentation.

Biochar is the residue from the carbonization of fibrous biomass at high temperatures (500-1000 °C) in either downdraft gasifiers used for generation of synthesis gas for heating or use in internal combustion engines (Rodriguez and Preston 2010); or in updraft gasifier stoves used for household cooking (Southavong and Preston 2011). Addition of biochar to soils has been shown to improve their capacity to retain water, nitrogen and organic matter (Lehman and Joseph 2009) and thereby to increase plant growth especially when combined with organic fertilizers such as biodigester effluent (Rodriguez et al 2009; Bouaravong et al 2017). Biochar has also been shown to enhance production of methane in biodigesters (Inthapanya et al 2012); to reduce methane in rumen in vitro incubations (Leng et al 2012a); and to enhance growth rate of cattle when included at low levels (1% of diet DM) in cassava-based feeding systems (Leng et al 2012b; Sengsouly et al 2017).

It is believed that the role of biochar in this wide range of biological systems is as a support mechanism for biofilms that host a diverse array of micro-organisms and nutrients acting synergistically with major benefits for the host system (Lehman et al 2011; Leng 2014).

Against this background it was hypothesized that inclusion of biochar in the process of protein-enrichment of cassava pulp enhances the efficiency of the growth of yeast and other micro-organisms thus improving the rate of conversion of non-protein nitrogen into true protein.

Materials and methods

Location and duration

The experiment was carried out from February to March 2016, in Thamouang Villagemajor benefits Hatsayfong District, Vientiane, Lao PDR location: 17°54'3.55"N 102°43'24.67"E

Treatments and design

In a completely randomized design with 5 replicates, the treatments were five levels of biochar (0, 0.25, 0.5, 0.75 and 1% in DM) in a solid-state fermentation of cassava pulp with yeast, urea and diammonium phosphate (DAP). The substrates were 5kg of fresh cassava pulp (22% DM; Table 2)) to which were added (DM basis) urea 3%, DAP 1% and yeast 4%. The substrates were mixed and stored in closed polyethylene bags for fermentation over 7 days.

Biochar production

A gasifier stove was constructed to process 2 kg of rice husk (Photo 1). Production of biochar after 35-40 minutes of combustion was 700g.

Photo 1. Rice huskstove

Protein enrichment of cassava pulp

The ‘ensiled’ cassava pulp (Phanthavong et al 2014) was obtained from the starch factory in Nashaw village, Pakngum District in Vientiane Capital, Lao PDR. Urea, DAP, minerals and yeast (Table 1) were added to 5kg of the ensiled pulp according to the proportions shown in Table 2. The yeast was a commercial product purchased in the market and traditionally used in household production of “rice wine”.

Measurements

Samples were taken from each treatment/replicate, before and after fermentation, and dried in an oven at 100°C for 24 h to determine the DM content. Other fresh samples were analyzed for crude and true protein (AOAC 1990). For estimation of true protein (see Krishnamoorthy et al 1982), 0.5 g of the fresh sample was put in a 125ml Erlenmeyer flask with 50 ml of distilled water, allowed to stand for 30 min, after which 10ml of 10% TCA (trichlor-acetic acid) were added and allowed to stand for a further 20-30 min. The suspension was then filtered through Whatman #54 paper by gravity. The filtrate was discarded, and the remaining filter paper and suspended substrate were transferred to a kjeldahl flask for standard estimation of total N by acid digestion, distillation and titration of the distillate with 0.1M HCl (AOAC 1990).

Statistical analysis

The data were analyzed by using the general linear option in the ANOVA program of the Minitab software (Minitab 2000). Linear and polynomial regressions were fitted to the data using the appropriate models in the Microsoft “Excel” program.

Results

Crude and true protein enrichment

There were curvilinear responses in percentage of crude and of true protein in substrate DM as the level of biochar was increased with optimum levels of both being reached with 0.75% biochar in the substrate DM (Table 3; Figure 1). The response was more pronounced for true protein (with a 50% increase from 5.2 to 7.8% in DM) compared with crude protein (a 16% response from 14.1 to 16.4%). The ratio of true to crude protein (Figure 2) increased from 0.37 to 0.49 (an increase of 32%).

Figure 1. Effect of biochar on protein enrichment of cassava pulp

Figure 2. The ratio of true protein to crude protein was increased by incorporation of biochar in the fermentation medium

Biochar and pH of the fermentation medium

There was a linear increase in the pH of the fermentation medium as the biochar concentration was increased from 0 to 1% (Figure 3). In turn, the pH was linearly related with the rate of conversion of non-protein-nitrogen (from urea and DAP) to true protein (Figure 4).

Figure 3. Effect of concentration of biochar on pH in the fermentation medium at the end of the fermentation	Figure 4. Relationship between pH in the fermentation medium and the ratio of true/crude protein

Discussion

The improvement in the conversion of NPN to true protein, as a result of including biochar in the fermentation substrate, implies that the biochar has a synergistic effect on the capacity of the yeast (and or other microorganisms) to utilize ammonia N (from urea and DAP) for growth. Leng (2014) advanced the concept that the mode of action of biochar in increasing methane production in biodigesters (Inthapanya et et al 2012), reducing methane in in vitro rumen incubations (Leng et al 2012a), and improving growth rates in cattle (Leng et al 2012b), could be due to the biochar acting as a support mechanism (providing increased surface areas for biofilms to form) for consortia of microorganisms acting synergistically to enhance the rate, and therefore the efficiency, of microbial growth. This idea is supported by recent research showing that “conductive carbon materials, such as biochar, facilitate inter-species electron transfer among micro-organisms by direct electron transport” (Lovley 2017).

An important finding is the linear increase in the pH of the fermentation medium as the biochar concentration was increased from 0 to 1% (Figure 3), and the fact that the increase in pH was linearly related with the rate of conversion of non-protein-nitrogen (urea and DAP) to true protein (Figure 4). These effects of biochar on pH of the fermentation medium are similar to those recorded when biochar is used as soil amendment (Lehmann et al 2011). Supporting evidence for pH being a determining factor for growth of yeast is in Figure 5. Yeast growth rate on a sugar-rich medium was increased by almost 50% when the pH of the medium was raised from 2.1 to 4.6.

Figure 5. Growth rate of yeast was increased with increase of pH in the fermentation medium (Arroyo-Lopez et al 2008)

Conclusions

• The true protein content of cassava pulp, in a solid-state fermentation with urea, diammonium phosphate and yeast, was increased from 5.2 to 7.4% in DM when biochar was included at 1% of the DM of the fermentation medium. The crude protein content of the fermentation medium was not affected by the presence of biochar (14.0 -14.8% in DM).
• There was a linear increase in the pH of the fermentation medium (from 3.6 to 4.4) as the biochar concentration was increased from 0 to 1%, and this was linearly related with the rate of conversion of non-protein-nitrogen (urea and DAP) to true protein.

Acknowledgements

The authors would like to thank the Swedish International Development Agency (Sida) and the Norwegian Programme for Development, Research and Higher Education (NUFU) for the financial support for this research. The research formed part of the requirement by the senior author for the MSc degree from Cantho University. The Living Aquatic Resource Research Center (LARReC) belonging to National Agricuture and forestry Research Institute (NAFRI) was the supporting institution for the experiment. Special thanks are given to Mr Siphao Thammavong in Thamouang village Hatxayfong district, Vientiane Capital for providing facilities for the research location and his valuable help in the farm.

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Received 8 November 2017; Accepted 28 November 2017; Published 1 December 2017

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