• International Journal of Technology (IJTech)
  • Vol 10, No 3 (2019)

Optimization of Solid State Fermentation Conditions for Cyanide Content Reduction in Cassava Leaves using Response Surface Methodology

Optimization of Solid State Fermentation Conditions for Cyanide Content Reduction in Cassava Leaves using Response Surface Methodology

Title: Optimization of Solid State Fermentation Conditions for Cyanide Content Reduction in Cassava Leaves using Response Surface Methodology
Mohamed Hawashi, Hakun Aparamarta, Tri Widjaja, Setiyo Gunawan

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Cite this article as:
Hawashi, M., Aparamarta, H., Widjaja, T., Gunawan, S., 2019. Optimization of Solid State Fermentation Conditions for Cyanide Content Reduction in Cassava Leaves using Response Surface Methodology. International Journal of Technology. Volume 10(3), pp. 624-633

Mohamed Hawashi -Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember (ITS) -Central Scientific Research Laboratory, Sebha University, Sebha
Hakun Aparamarta Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember (ITS), Surabaya, 60111, Indonesia
Tri Widjaja Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember (ITS), Surabaya 60111, Indonesia
Setiyo Gunawan Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember (ITS), Surabaya 60111, Indonesia
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Optimization of Solid State Fermentation Conditions for Cyanide Content Reduction in Cassava Leaves using Response Surface Methodology

Cassava leaves are a good source of protein. However, their use is limited because of the presence of cyanogenic glucosides. These require a further detoxification process in order to reduce the cyanide to a safe level prior to human consumption. The main objectives of this work are: (i) to demonstrate the effectiveness of solid-state fermentation using Saccharomyces cerevisiae on the cyanide content degradation of cassava leaves; and (ii) to optimize the independent variables for the minimum cyanide content level of cassava leaves by the application of response surface methodology (RSM). The various process parameters investigated for these purposes were sucrose concentration, urea concentration, moisture content, and fermentation time. The degradation of cyanide content was described by the quadratic model, which resulted in an excellent fit of the experimental data (p < 0.01). The statistical tests show that linear terms for sucrose concentration, urea concentration, moisture content and fermentation time had a significant effect on cyanide content (p < 0.01). Moreover, the interaction coefficients between sucrose concentration and fermentation time; urea concentration and moisture content; and nitrogen concentration and fermentation time were significant model terms (p < 0.05). A minimum cyanide content of 0.81 ppm was obtained at 1% (w/w) sucrose concentration, 0.5% (w/w) urea concentration, 60% (v/w) moisture content and with a fermentation time of 78 hours. The optimal level made a significant reduction in cyanide content of 97.96%, which is lower than the toxicity level suggested by the World Health Organization of 10 ppm.

Cassava leaves; Cyanide content; Response surface methodology; Solid state fermentation


With the growth in food consumption, the majority of people rely heavily on food crops as their primary food sources. Root crops, such as cassava, are grown in developing countries as a primary source of carbohydrates (Hawashi et al., 2018). This crop represents one of the primary sources of food for Indonesian people, along with other staples such as rice, sago and corn.  Reports indicate a production rate of nearly 20 million tons per year, harvested from 1.93 million hectares (Agustian, 2016). Cultivation of cassava plants can take place even in marginal environmental conditions, due to their high drought tolerance, with an optimal yield of approximately 50% for leaves and 6% for roots upon plant maturity (Tewe & Lutaladio, 2004). Cassava leaves contain valuable protein and nutrients and are consumed in many countries, including Indonesia, Malaysia, the Congo, Madagascar and Nigeria (Latif & Muller, 2015). However, they contain both nutritive and non-nutritive compounds. Among the anti-nutrients, particular concern is paid to cyanide acid (HCN), whose concentration in fresh cassava leaves is much higher than the safe limit recommended by the World Health Organization (WHO) for human consumption (10 ppm). As an effect of consuming a high concentration of cyanide, HCN poses health problems to the human body. Such a condition is known as Konzo disease, an irreversible neurological disorder associated with cyanide consumption (Bradbury, 2006). Other long-term exposure to cyanogenic glycosides from eating cassava includes tropical ataxic neuropathy, neurological effects, and damages to goiter, and thyroid functions (WHO, 2008). In addition, HCN is an inhibitor of the oxidation processes occurring in the mitochondria, which can lead to chronic toxicity. Therefore, it is essential that the content of cyanide be reduced below 10 ppm to allow people to safely consume cassava (WHO, 1995).

Reduction of cyanide levels can also be made in cyanide-rich raw food sources such as cassava.

Worldwide, the most common methods of cassava leaf processing include boiling and soaking in water, steaming, sun drying, and oven drying. These approaches aim to reduce the toxic compounds in the leaves for human consumption (Fasuyi, 2005). Cassava leaf processing is mainly based on the endogenous cassava enzyme (linamarase), which catalyzes the conversion of cyanide-containing compounds (linamarin) into acetone cyanohydrin, which either enzymatically or spontaneously decomposes into HCN and acetone (Montagnac et al., 2009). However, some methods (such as steaming and oven drying) have been proven to be ineffective for lowering the cyanide content in cassava leaves to the safe limit. Studies have shown that the fermentation of cassava leaves is a promising method for reducing cyanide content (Kobawila et al., 2005; Morales et al., 2018). These reports show respective reductions of at least 70% and 94.18% in cyanide content during fermentation. They further validate the preference for the fermentation technique over conventional methods. The SSF technique has several advantages, including high productivity and reduced processing time (Febrianti et al., 2017). However, reports indicating the efficiency of cassava leaf fermentation are quite scarce compared to those which investigate tubers.

Various process conditions such as moisture content, pH, inoculum size, fermentation time, concentration of nutrient supplementation and temperature can affect the microbial growth, enzyme production, and formation of the product during the fermentation process (Ezekiel & Aworh, 2013). The optimization processes using the “One Variable at One Time (OVAT)” technique (changing one single variable, while keeping others at constant levels) is an inefficient way of determining the interaction between the process variables as it involves high cost and requires various experiments to obtain the optimum levels (Braga et al., 2011; Hadiyat & Wahyudi, 2013). Recently, the application of RSM has attracted the attention of researchers working with fermentation to optimize process conditions and evaluate the correlation between independent variables and their responses (Istianah et al., 2018).

Yeast and lactic acid bacteria (LAB) are the most investigated microorganisms for the production of linamarase during cassava fermentation and the development of flavor. Yeast, such as Saccharomyces cerevisiae, has several advantages, including its availability, low cost, ability to secrete extracellular enzymes, non-pathogenic character, and widespread use in traditional fermentation, particularly in fermented foods (Oboh & Akindahunsi, 2003). Furthermore, Saccharomyces cerevisiae is able to use cyanogenic glucosides and their metabolites during food processing, making it one of the micro-organisms which is most involved in the cassava fermentation process (Lambri et al., 2013). Therefore, the objective of this work is to demonstrate the effectiveness of solid-state fermentation using Saccharomyces cerevisiae in the reduction of cyanide in cassava leaves. Furthermore, the optimization of the independent variables (moisture content, incubation time and nutrient supplementation) to achieve a minimum cyanide content level in cassava leaves by employing response surface methodology (RSM), is also studied in detail.


The research has investigated the effect of solid state fermentation using Saccharomyces cerevisiae on the removal of cyanide content from cassava leaves.  The study has shown that response surface methodology (RSM) was a high-performance technique for optimization of the process conditions for minimizing cyanide content in fermented cassava leaves through solid-state fermentation. The optimal process condition was obtained at 1% (w/w) sucrose concentration, 0.5% (w/w) urea concentration and 60% (v/w) moisture content, with a fermentation time of 78 hours. It was observed that an exponential decrease in cyanide content over time can lead to satisfactory detoxification in cassava leaves, with cyanide concentration falling to levels lower than 10 ppm after 60 hours of fermentation, and thus providing a safe and healthy food source.


This work was supported by grant no. 849/PKS/ITS/2018 provided by the Ministry of Research, Technology and Higher Education of the Republic of Indonesia.

Supplementary Material
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