Research Article

Amino Acid and Proximate Composition of Saccharomyces cerevisiae Biomass Cultivated in Cassava Mill Effluents  

Sylvester Chibueze Izah , Sunday Etim Bassey , Elijah Ige Ohimain
Department of Biological Sciences, Faculty of Science, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria
Author    Correspondence author
Molecular Microbiology Research, 2017, Vol. 7, No. 3   doi: 10.5376/mmr.2017.07.0003
Received: 16 Oct., 2017    Accepted: 24 Nov., 2017    Published: 08 Dec., 2017
© 2017 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Izah S.C., Bassey S.E., and Ohimain E.I., 2017, Amino acid and proximate composition of Saccharomyces cerevisiae biomass cultivated in Cassava mill effluents, Molecular Microbiology Research, 7(3): 20-29 (doi: 10.5376/mmr.2017.07.0003)

Abstract

Cassava mill effluent (CME) is generated during cassava processing into cassava flake or gari. The effluents affects the quality of the receiving environment such soil. It is could affect domestic animals such as goat and sheep when they ingest it, and retard normal growth and productivity of plants in the affected soil. This study evaluated amino acid and proximate composition of Saccharomyces cerevisiae cultivated in cassava mill effluents. S. cerevisiae used in this study was isolated from palm wine based on cultural, morphological, and physiological/biochemical characteristics. 10 ml of S. cerevisiae broth was inoculated into 100ml of sterile cassava mill effluents filtered with double muslin cloth. The medium was shaken intermittently between 7.00 to 19.00 hours’ time interval. The medium was decanted and subsequently filtered using Whatman filter paper. The resultant biomass was washed with distilled water and re-filtered. The sludge/biomass recovered were oven dried. Amino acid and proximate composition of the yeast biomass produced was analyzed using spectrophotometer. Proximate composition was also analyzed following standard procedure. Result showed that the total essential amino acid (40.88 g/100 g) was slightly higher than the Food and Agricultural Organization/World Health Organization (FAO/WHO) standard for feed use. While the total non-essential amino acid (21.56 g/100 g) was lower than FAO/WHO limits. Mean proximate composition was 17.01% (crude protein), 7.34% (ash), 56.40% (carbohydrate) and 407.13 kcal/100 g (calorific value/energy content). The findings of this study showed that during fermentation of cassava mill effluents with S. cerevisiae an appreciable amount protein is enriched considering the fact that the substrate is a waste.

Keywords
Amino acid; Biotechnology advances; Cassava mill effluents; Waste

Background

Cassava (Manihot esculenta) is one of the world most staple foods. Cassava belongs to the Euphorbiaceae family. Cassava is extensively cultivated in both tropical and subtropical regions probably due to its ability to grow in several soil conditions (Boonnop et al., 2009). Global cassava production is dominated by Nigeria (Ohimain, 2013; Ohimain et al., 2013; Okudoh et al., 2014; Izah and Ohimain, 2015), which accounts for over 20.3% of global output at the end of 2014 economic year. Nigeria cassava production have exceeded the combined production output of the world second (Thailand) and Third (Indonesia) largest producing nations. Furthermore, Democratic Republic of Congo is African second largest cassava producing country.

 

Like oil palm, cassava production is an agroenterprise and major source of livelihood to several families in Nigeria especially in the southern region. Cassava cultivation and processing is dominated by smallholder that accounts for about 80% of Nigeria cassava industry (Izah et al., 2017a-d). During cassava processing, large waste water often called cassava mill effluents is generated predominantly during bagging and pressing of grated cassava (Izah et al., 2017a-g). According to Ohimain et al. (2013), cassava mill effluent accounts for about 16% of total weight of cassava tuber in a smallholder cassava process mills.

 

Raw cassava mill effluents is acidic (Olorunfemi and Lolodi, 2011; Patrick et al., 2011; Rim-Rukeh, 2012; Orhue et al., 2014; Izah and Ohimain, 2015; Izah et al., 2017g) and could lead to acidification (Izonfuo et al., 2013). Cassava mill effluents contain heavy metals (Adejumo and Ola, Undated; Olorunfemi and Lolodi, 2011; Patrick et al., 2011; Orhue et al., 2014; Omomowo et al., 2015; Izah et al., 2017a) and general physicochemical characteristics such as total dissolved solid (Orhue et al., 2014), low dissolved oxygen, high chemical oxygen demand (Rim-Rukeh, 2012), and other nutrient oriented parameters such as anions, cations among other (Adejumo and Ola, Undated; Olorunfemi and Lolodi, 2011; Patrick et al., 2011; Rim-Rukeh, 2012; Orhue et al., 2014; Omomowo et al., 2015). The characteristics of the effluents often exceed the limit specified by FEPA (1991) for all categories of industrial effluents to be discharge into the environment.

 

The impact of cassava mill effluents is severe to the environmental components such as soil and surface water. Authors have variously reported that cassava mill affects microbial, heavy metal, and general physicochemical parameters of the receiving soil (Nwaugo et al., 2007, 2008; Ehiagbonare et al., 2009; Eneje and Ifenkwe, 2012; Nwakaudu et al., 2012; Okechi et al., 2012; Osakwe, 2012; Chinyere et al., 2013; Izonfuo et al., 2013; Omotiama et al., 2013; Okunade and Adekalu, 2013; Ezeigbo et al., 2014; Ibe et al., 2014; Eze and Onyilide, 2015; Igbinosa, 2015; Igbinosa and Igiehon, 2015; Omomowo et al., 2015; Izah et al., 2017b).

 

On surface water it induces toxicity on fisheries with regard to behavioral response, mortality, enzymatic, haematological and histopathological parameters (Adeyemo, 2005; Asogwa et al., 2015). Furthermore, it has also been reported to induce toxicity that could lead to death in domestic animals such as goat and sheep (Ero and Okponmwense, Undated; Ehiagbonare et al., 2009). It is also known to inhibit vegetation growth (Otunne and Kinako, Undated) and its productivity.

 

Some biotechnological advances have been demonstrated toward sustainable management of cassava mill effluents. Some of the technologies focus in the field of bioenergy viz: bioethanol (Izah and Ohimain, 2015), biogas (Eze, 2010; James et al., 2013; Kullavanijaya and Thongduang, 2012; Jijai et al., 2014) and bioelectricity using microbial fuel cells (Kaewkannetra et al., 2011) and enzyme production viz: cellulose, protease and amylase (Arotupin, 2007; Oshoma et al., 2010; Santhi, 2014).

 

Previous studied have attempted the suitability of S.cerevisiae biomass cultured in cassava mill effluents for animals feed. Some of these studies were carried out with regard to heavy metal concentration (Izah et al., 2017c), cyanide and macronutrients (Izah et al., 2017d). Therefore, this study aimed at providing another outlook toward sustainable management of cassava mill effluents for potential animal feed production. The study utilizes cassava mill effluents for the cultivation of Saccharomyces cerevisiae biomass. The resultant biomass was assessed with regard to amino acid and proximate composition.

 

1 Materials and Methods

1.1 Sample collection

Raw cassava mill effluents containing palm oil were collected from small-scale cassava processor at Ndemili in Ndokwa west Local Government Area of Delta state, Nigeria. 4-litres clean containers were used to collect the sample. The samples were transported to the laboratory using ice pack. The samples were used immediately at the laboratory.

 

1.2 Isolation and identification of saccharomyces cerevisae

The S. cerevisiae used in this study was isolated from palm wine bought from Rumuomasi, Port Harcourt, Rivers state, Nigeria. Pure culture of the isolate was obtained following the pour plate method previously described by Benson (2002), Pepper and Gerba (2005) using potato dextrose agar supplemented with chloramphenicol. The resultant isolates were streaked in another potato dextrose agar plate. The S. cerevisiae used was identified based on conventional microbiological techniques (cultural, morphological, and physiological/biochemical characteristics) using carbon fermentation and assimilation, glucose-peptone-yeast extract broth, lacto-phenol cotton blue stain and growth based on temperature as previously described by Kurtzman and Fell (1998), APHA (2006), Benson (2002) and have been applied by Iwuagwu and Ugwuanyi (2014), Abioye et al. (2015), Okoduwa et al. (2017), Izah et al. (2017a,c,d). The characteristics of the isolates were compared with the guide provided by Ellis et al. (2007).

 

1.3 Growth of yeast biomass

The S. cerevisiae was cultured based on the methodology previously described by Abioye et al. (2015), Okoduwa et al. (2017) with slight modifications. 100 ml of the sterile cassava mill effluents were measured into 250 ml Erlenmeyer’s flask under aseptic condition and 10 ml of S. cerevisiae inoculum was added into the flask (Izah et al., 2017c; d). The flask was capped with cotton wool wrapped with aluminum foil paper. The flasks were shaked intermittently between 7.00-19.00 hours interval. At the end of 15 days experimental period, 60 ml of the medium were decanted into another flask and the remaining 40 ml was filtered using Whatman Number 42 filter paper (Izah et al., 2017c; d). The resultant sludge/biomass was washed with distilled water, filtered and oven dried (Izah et al., 2017c; d). The biomass was packaged in Ziploc bag prior to analysis.

 

1.4 Analysis of proximate composition and amino acid

Proximate composition: Moisture, total ash, crude fat (ether extraction), crude protein, carbohydrate and crude fibre of was determined by standard methods (AOAC, 1984, 1990, 1998). While the energy content was calculated based on the formula:

 

Energy value (kcal/100 g) = 9 x % fat + 4(%protein + %carbohydrates) (Hassan et al., 2008; Galla et al., 2012).

 

Amino acid determination: Amino acids was analyzed using Spectrophotometric method based on ninhydrin chemical reaction following the method previously described by Schroeder et al. (1990), Spies and Chambers (1950), Gaitonde (1967). Typically, ninhydrin combines with amino acids to form coloured complexes, the intensity of the colours depend on the amount of amino acid present.

 

1.5 Statistical analysis

SPSS software version 20 was used to carry out the statistical analysis. The results were expressed as mean ± standard deviation.

 

2 Results and Discussion

The proximate composition of S. cerevisiae biomass cultured in cassava mill effluents is presented in Table 1. The protein content increases as fermentation proceeds using microbes; as such it was compared with previous works on non-microbial biomass as well. Basically protein in carbohydrate crop is very low. As fermentation duration increases using microbes, the protein content of carbohydrate crops such as cassava tuber increases (Gunawan et al., 2015). This could be due to the fact that most microbes that play essential role in fermentation of carbohydrate crop are able to convert carbon and nitrogenous compound into protein (Hu et al., 2012; Gunawan et al., 2015). This may be associated to the fact that some microbes that play essential role in fermentation of cassava products. Some of these microbes include Lactobacillus plantarum, S. cereviseae, and Rhizopus oryzae and they could secrete some extracellular enzymes (proteins) into the cassava during fermentation (Gunawan et al., 2015). Specifically the increase in protein content using S. cerevisiae in the fermentation medium could be due to their ability to secrete some extracellular enzymes such as amylases, linamarase and cellulase into the cassava during metabolic processes (Oboh and Akindahunsi, 2003; Gunawan et al., 2015). This could eventually lead to yeast growth (Oboh and Akindahunsi, 2003; Gunawan et al., 2015). The amount of protein enriched during fermentation in influenced by duration of fermentation (Gunawan et al., 2015) and culture conditions such as temperature and pH.

 

 

Table 1 Proximate composition of the S.cerevisiae biomass produced from cassava mill effluent in comparison with previous work on yeast biomass and plants

Note: Data was expressed as mean ± standard deviation

 

The protein content (17.01%) is lower than the values previously reported in S. cerevisiae  biomass cultured in POME (Iwuagwu and Ugwuanyi, 2014), Spirulina sp biomass grown at pH of 8.5-10.0 and temperature of 30ºC (Ogbonda et al., 2007), and vegetation such as seed of Sterculia urens (Galla et al., 2012), Parkia biglobosa (Hassan and Umar, 2015) and Citrullus ecirrhosus (Umar et al., 2013), but higher than the value in seed of Annonas squamosa (Hasssan et al., 2008) (Table 1). Variation in protein content could be due to the difference in the biochemical constituents of the various feedstocks compared.

 

Furthermore, Boonnop et al. (2009) reported that cassava fermented with S. cerevisiae in solid-liquid media for 132 hours and dried at 30ºC had protein content of 30.4%. Also, Polyorach et al. (2013) reported protein content of 47.5% during fermentation of cassava chip with S. cerevisae. Bekatorou et al. (2006) reported that Saccharomyces cerevisiae contain 40.6–58.0 % of protein depending on the yeast and growth conditions.

 

The crude fat content (12.57%) was higher in this study compare the values previously reported in S. cerevisiae biomass cultured in POME (Iwuagwu and Ugwuanyi, 2014), Spirulina sp biomass (Ogbonda et al., 2007), fermented cassava tuber (Irtwange and Achimba, 2009), but lower than the value previously reported in seed of Sterculia urens (Galla et al., 2012), Citrullus ecirrhosus (Umar et al., 2013), Annonas squamosa (Hasssan et al., 2008) (Table 1). Bekatorou et al. (2006) reported that Saccharomyces cerevisiae contain 4.0-6.0% of lipids depending on the yeast and growth conditions.

 

Crude fiber (2.71%) in this study was comparable to the values reported in fermented cassava (Irtwange and Achimba, 2009), seed of Citrullus ecirrhosus (Umar et al., 2013), Sterculia urens (Galla et al., 2012), and lower than the values S. cerevisiae biomass cultured in POME (Iwuagwu and Ugwuanyi, 2014), seed of Annonas squamosa (Hasssan et al., 2008) and Spirulina sp biomass (Ogbonda et al., 2007).

 

The moisture content (7.12%) in this study was lower than the values previously reported in different feedstocks by authors (Ogbonda et al., 2007; Hasssan et al., 2008; Irtwange and Achimba, 2009; Galla et al., 2012) and lower than the values reported in seed of Citrullus ecirrhosus by Umar et al. (2013) (Table 1).

 

The ash content (7.34%) in this study is higher than the values variously reported by authors in different substrate (Hasssan et al., 2008; Galla et al., 2012; Umar et al., 2013; Iwuagwu and Ugwuanyi, 2014), and lower than the value reported in Spirulina sp biomass by Ogbonda et al. (2007).

 

The carbohydrate content (56.40%) was higher than the values previously reported by author in S. cerevisiae  biomass cultured in POME (Iwuagwu and Ugwuanyi, 2014), Spirulina sp biomass (Ogbonda et al., 2007) seed of Sterculia urens (Galla et al., 2012), Citrullus ecirrhosus (Umar et al., 2013), and Annonas squamosa (Hasssan et al., 2008). Typically carbohydrate is a major source of energy. Furthermore, Bekatorou et al. (2006) reported that Saccharomyces cerevisiae contain 35.0-45.0 % of carbohydrates depending on the type of yeast and growth conditions.

 

The energy content (407.13 kcal/100 g) was lower than the value reported in seed of Sterculia urens (Galla et al., 2012), Citrullus ecirrhosus (Umar et al., 2013), and Annonas squamosa (Hasssan et al., 2008) (Table 1). The variation in energy content and proximate composition of the various substrate/feedstock compared could be due to differences in their biochemical constituents.

 

Table 2 presents the composition of amino acid from S. cerevisiae cultured in cassava mill effluents substrate in comparison to FAO/WHO limits for single cell protein and other studies on plant amino acids. The values in this study suggest that isoleucine, leucine, tryptophan, histidine and combination of phenylalanine and tyrosine are superior and/ or comparable to FAO/WHO standard for single cell protein. While lysine, methionine, valine and threonine were lower in this study compared to the standard for single cell protein standard for feeds as recommended by FAO/WHO. Based on the total number of essential amino acid, the findings of this study were apparently higher compared to FAO/WHO limits. Nearly all the non-essential amino acid in this study has lower values compared to FAO/WHO standard for single cell protein meant for feed. The trend in this study had some similarity with the findings of Iwuagwu and Ugwuanyi (2014) that reported amino acid level in biomass of S. cerevisiae cultured in palm oil mill effluents (POME) to be comparable and/ or superior to the recommended standard with respect to a number of amino acids, and inferior to few others, especially non-essential amino acids. The authors also reported higher value of essential amino acid compared to the non-essential amino acid in their study. The lysine content in this study is lower than the values 8.5 g/100 g reported cassava fermented chip using S. cerevisiae by Boonnop et al. (2009).

 

 

Table 2 Amino acid profile of S. cerevisiae cultured in Cassava mill effluents expressed as g/100 g of protein in comparison to FAO/WHO limits and other plant amino acids

Note: Data in this study is expressed as Mean± Standard deviation

*six tropical seaweeds (Sargassum wightii, Ulva lactuca, Kappaphycus alvarezii, Hypnea musciformis, Acanthophora spicifera and Gracilaria corticata), data also expressed in percentage

**Data is expressed as g/16 gN

 

Furthermore, the findings of this study are higher than the values reported in six tropical seaweeds (Sargassum wightii, Ulva lactuca, Kappaphycus alvarezii, Hypnea musciformis, Acanthophora spicifera and Gracilaria corticata) by Vinoj and Kaladharan (2007). Galla et al. (2012) reported some superior amino acids especially the non-essential ones compared to the findings of this study. This trend has also been reported in seed of Citrullus ecirrhosus (wild melon) by Umar et al. (2013) and Parkia biglobosa (African locust bean) by Hassan and Umar (2015). The work of Ogbonda et al. (2007) on Spirulina sp biomass grown at pH of 9.0 and temperature of 30ºC has some of the amino acids superior to the findings of this study. The variation in the findings of this study compare to previous works could be associated to the difference in substrate as well as environmental conditions. Ugwuanyi (2008) also reported that culture conditions affect the amino acid profiles of microbial biomass. Hence culture condition can be optimized to enhance the amino acids content. Ogbonda et al. (2007) have reported that pH and temperature affect the concentration of individual amino acids and were manipulated and optimum yield was produced at pH of 9.0 and temperature of 30ºC.

 

Based on the amino acid concentration of this study, it showed that cassava waste water has the tendency to produce essential amino acid using S. cerevisiae. Therefore, considering the status of the substrate (culture medium - a waste), it’s remarkable. This is because of the adverse attendant environmental impacts associated with the waste water especially in high cassava producing communities in developing country like Nigeria that treatment method of the effluents prior to discharge into the environment is unavailable.

 

3 Conclusions

During microbial fermentation of cassava mill effluents, the physical and chemical characteristics of the effluents are altered, which varies according to microbial isolates and culture condition. This study demonstrated the possible potentials to use S. cerevisiae biomass cultured in cassava mill effluents for animal feed. The biomass produced showed an appreciable amount of protein bearing in mind that cassava contains low amount of protein. Amino acids were considerably high in some of the essential amino acids compared to FAO/WHO standard for feed.

 

Acknowledgements

This paper is based on part of PhD project work of S.C. Izah supervised by Dr S.E. Bassey and Prof. E.I. Ohimain at the Niger Delta University, Wilberforce Island, Nigeria. The abstract of this study was presented in 5th Annual conference of Environmental Health Sciences held at University of Ibadan, Nigeria between 20-24th of November 2017.

 

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