Chemical Industry & Chemical Engineering Quarterly www.ache.org.rs/CICEQ
Chem. Ind. Chem. Eng. Q. 20 (4) 457−461 (2014) CI&CEQ
OGBONNAYA NWOKORO
Industrial Microbiology and Biotechnology Laboratory, Department of Microbiology, University of Nigeria, Nsukka, Nigeria
SCIENTIFIC PAPER
UDC 633.493:66.094.491:663 DOI 10.2298/CICEQ130325027N
PRODUCTION OF
L
-LACTIC ACID FROM
CASSAVA PEEL WASTES USING SINGLE
AND MIXED CULTURES OF
Rhizopus
oligosporus
AND
Lactobacillus plantarum
Article Highlights
• Production of reducing sugar from Cassava peels was highest when acid was used to hydrolyze the peels
• Lactic acid production was highest in acid hydrolyzed peels than in alkali hydrolysate • Mixed cultures produced the best lactic acid yield than single cultures in both acid and
alkali hydrolysate
Abstract
Production of L-lactic acid using cultures of Rhizopus oligosporus and Lacto-bacillus plantarum was investigated. Cassava peels were hydrolyzed by boiling for 1 h in either NaOH or HCl solutions followed by neutralization to a pH of 6.2. Reducing sugar produced from the hydrolysates increased with increasing concentrations of alkali or acid. Samples hydrolyzed with HCl produced a max-imum reducing sugar concentration of 402 mg/g substrate while alkali hydrol-yzed samples produced a maximum reducing sugar concentration of 213 mg/g substrate. Hydrolysates were amended with 0.5% ammonium sulphate solu-tion and inoculated with either single or mixed cultures of R. oligosporus and L. plantarum and incubated for 48 h for lactic acid production. The best lactic acid production of 50.2 g/100 g substrate was observed in a mixed culture ferment-ation of acid hydrolyzed peels. Mixed culture fermentferment-ation of alkali hydrolyzed peels produced a maximum lactic acid concentration of 36.4 g/100g substrate. Unhydrolyzed Cassava peels inoculated with a mixed culture of the microorg-anisms produced only 4.6 g/100g substrate. This work reports an efficient use of cassava peels for bio-product formation through microbial fermentation.
Keywords: Cassava peels, Rhizopus oligosporus, Lactobacillus plant-arum, hydrolysis methods.
Lactic acid (CH3CHOHCOOH) is among the most widely utilized organic acids in the food, pharm-aceutical, cosmetics and chemical industries. Its pro-duction is currently attracting a great deal of research and development. Microbial fermentation of starch and sugar is an important method for lactic acid pro-duction. The cost of raw materials such as sugar or starch may hinder commercial production of lactic acid [1]. Utilization of cheap agricultural substrates and wastes in bioprocess provide a cheaper alter-native for lactic acid production. Lactic acid has been produced from renewable cheap substrates in the
Correspondence: E-mail: ogb883@yahoo.com Paper received: 25 March, 2013
Paper revised: 4 June, 2013 Paper accepted: 22 July, 2013
The widely used substrates for lactic acid pro-duction are refined sugars, which are expensive [13]. Polysaccharides such as starch or cellulose can be utilized to reduce the cost of lactic acid production but it is necessary that they are pretreated to release fer-mentable carbohydrates. The use of mild acid or alk-ali hydrolysis of cellulose materials prior to microbial fermentation has been reported by many researchers [14,15].
Cassava tubers are normally peeled and the peels are discarded and often contribute to environ-mental pollution. Cassava peels make up to 10% of the wet weight of the roots and therefore constitute an important potential resource if properly processed in a bio-system [16]. Cassava peels and cassava powder have been used to produce lactic acid [17]. Cassava tubers are rich in starch while cassava peels contain high amounts of cellulose [18].
Nitrogen sources have been added into the fer-mentation media for lactic acid production from agri-cultural products [2]. In an economic analysis of the use of nitrogen sources for lactic acid production, the largest contributors were found to be yeast extract and peptone accounting to a very high cost of the pro-duction media [19]. There is need for investigation of the possibility of replacing yeast extract and peptone with cheaper nitrogen sources.
Rhizopus species are important microorganisms that metabolize carbohydrate substrates to L-lactic acid [20]. Lactobacilli have been used for L-lactic acid production from cellulose materials [6,21]. This work reports the fermentative production of L-lactic acid from hydrolyzed cassava peels using microorganisms.
MATERIALS AND METHODS
Collection of Cassava tuber samples
Cassava tubers (TMS 0581) were collected from the International Institute of Tropical Agriculture (IITA) Ibadan, Nigeria. About 2 kg of the peels were dried in an oven at 105 °C for 24 h, after which it was ground with a grater (Corona Mill, Mendellin, Columbia) to a size of approximately 0.5 mm. Samples (100 g each) contained in conical flasks were hydrolyzed with either 400 ml of 0.5% HCl or 400 ml of 0.5% NaOH by boiling in a thermostatic water bath (Kotterman, Bre-men, Germany). The pH of the slurry was adjusted to 6.2 with either sterile lactic acid for alkali hydrolysate or NaOH for acid hydrolysate. Hydrolyzed samples were loaded into sterile conical flasks and amended with 20 ml of 0.5% ammonium sulphate solution. Unhydrolyzed samples were separately prepared.
Isolation of microorganisms
About 10 g of decomposing cassava peels were collected from a refuse dump near a cassava proces-sing mill. The sample was ground with a pestle and mortar containing 5 ml of sterile distilled water. Seri-ally diluted samples were plated in three replicates on MRS agar (Oxoid, UK) for the isolation of lactic acid bacteria or on potato dextrose agar (Oxoid, UK) con-taining 0.1% chloramphenicol for the isolation of fungi. Plating was done in triplicates. Lactobacillus plantarum and Rhizopus oligosporus gave the highest counts and were selected and identified. Identification of L. plantarum was done using the taxonomic sche-mes given in Bergey’s Manual of Determinative Bact-eriology [22]. The carbohydrate fermentation patterns of the bacterium were determined by using API 50 CHL test kit. Bio Merieux online software (www.api-web.biomerieux.com) was used to identify the isolate. R. oligosporus was identified based on the taxonomic schemes described by Pitt and Hocking [23].
Each conical flask containing the hydrolysates and the control was either inoculated with 20 ml each of 107 colony forming units (CFU)/ml of L. plantarum or 20 ml each of 107 spores/mL of R. oligosporus. Mixed culture media contained 10 ml of 107 spores/ml of R. oligosporus and 10 ml of 107 cfu/ml of L. plantarum. A control treatment containing unhydrol-yzed Cassava peels was separately inoculated. The peels were incubated under static condition at room temperature (28±2 °C). After 48 h incubation, the con-tents in each flask was filtered with a Muslin cloth and re-filtered with Whatman No. 1 filter. The filtrate was tested for lactic acid.
Analyses
The pH was determined using a glass electrode pH meter (PYE Unicam, England). Reducing sugar concentrations were determined by the dinitrosalicylic acid (DNS) method of Miller [24] using 50–200 µg glu-cose as the standard. Lactic acid was converted to acetaldehyde by heating with 0.8 M H2SO4 and colour was developed by treatment in the acid solution with p-hydroxydiphenyl in the presence of 20% CuSO4⋅5H2O. Lactic acid concentration was estimated according to the colorimetric method of Barker and Summerson [25]. Stereospecificity of lactic acid was determined using D- and L-lactate assay kit from Megazyme Inter-national, Ireland.
RESULTS AND DISCUSSION
by microorganisms for lactic acid production. Maxi-mum reducing sugar yields increased with acid or alkali strengths. Results in Table 1 shows reducing sugar yields in the range of 89 to 402 mg/g substrate were obtained with acid strengths in the range of 0.1– –1%, whereas a concentration range of 42–242 mg/g substrate was produced with 0.1–1% NaOH. Acid hyd-rolysates generally produced more reducing sugars from cassava peels than alkali hydrolysates.
Table 1. Release of reducing sugar from hydrolyzed cassava peels (mg carbohydrate/g substrate); unhydrolyzed control sample released 38 mg carbohydrate/g substrate
Concentration, % HCl NaOH
0.1 89 42
0.2 98 48
0.3 107 69
0.4 214 92
0.5 312 142
0.6 324 176
0.7 349 198
0.8 358 206
0.9 396 213
1.0 402 242
Rhizopus oligosporus and Lactobacillus plant-arum were tested for their ability to produce L – lactic acid from cassava peels. Appropriate culture condi-tions for the growth of the microorganisms in the peels and their levels of lactic acid production were investigated. The peels were ground to approximately 0.5 mm size and hydrolyzed for 1 h either in HCl or in NaOH solutions after which the pH of the hydrolysate was adjusted to 6.2 and amended with 0.5% ammo-nium sulphate solution.
High yields of lactic acid are reported from the fermentation of cellulose substrates; however, these processes required additional nutrient supplement-ation [26,27]. Ammonium sulphate is the most widely used nitrogen source for lactic acid production [28,29]. Ammonium sulphate was found more suitable than ammonium nitrate, urea, yeast extract, peptone and corn steep liquor for lactic acid production [30]. Yin et al. [2] compared the impact of various nitrogen sources on the production of lactic acid by Rhizopus arrhizus NRRL 395. Corn steep liquor, yeast extract, polypeptone, and ammonium sulphate, were found to be the most suitable nitrogen source for lactic acid production by the microorganism. In contrast, Zhang et al. [31] used three organic nitrogen sources, namely CO(NH2)2, yeast extract and peptone and two inorganic nitrogen sources (NH4)2SO4 and NH4NO3 for lactic acid production from waste potato starch by
R. arrhizus. Among the tested nitrogen sources, NH4NO3 resulted in the highest increase in lactic acid production, which corresponded to a 90.6% yield.
R. oligosporus was used as a single culture for lactic acid production and a maximum L-lactic acid concentration of 32 g/100 g substrate was produced with 1% HCl hydrolysate (Table 2). When the fungus was grown in NaOH hydrolysate, the lactic acid con-centration produced was 22.5 g/100 g substrate (Table 2).
Table 2. Production of L-lactic acid from alkali and acid hyd-rolyzed cassava peels using a culture of Rhizopus oligosporus
Concentration, % Lactic acid concentration g/100 g
Yield, g lactic acid/g substrate
NaOH
0.1 89 42
0.2 98 48
0.3 107 69
0.4 214 92
0.5 312 142
0.6 324 176
0.7 349 198
0.8 358 206
0.9 396 213
1.0 402 242
HCl
0.1 17.2 0.172
0.2 17.5 0.175
0.3 19.6 0.196
0.4 20.1 0.201
0.5 22.2 0.222
0.6 22.1 0.221
0.7 28.6 0.286
0.8 28.6 0.286
0.9 30.1 0.301
1.0 32.0 0.320
Control 3.5 0.035
Maximum lactic concentration of 28.5 g/100 g substrate was produced in HCl hydrolysate when L. plantarum was used in single culture fermentation (Table 3), whereas a maximum concentration of 19.8 g/100 g substrate was produced when the bacterium was grown in NaOH hydrolysate (Table 3).
Table 3. Production of L-lactic acid from alkali and acid hyd-rolyzed Cassava peels using a culture of Lactobacillus plant-arum
Concentration, % Lactic acid concentration g/100 g
Yield, g lactic acid/g substrate
NaOH
0.1 13.6 0.136
0.2 13.3 0.133
0.3 14.2 0.142
0.4 14.8 0.148
0.5 15.6 0.156
0.6 16.3 0.163
0.7 16.1 0.161
0.8 17.0 0.170
0.9 17.7 0.177
1.0 19.8 0.198
HCl
0.1 16.2 0.162
0.2 16.9 0.169
0.3 18.2 0.182
0.4 18.2 0.182
0.5 20.9 0.209
0.6 21.2 0.212
0.7 21.6 0.216
0.8 25.7 0.257
0.9 26.2 0.262
1.0 28.5 0.285
Control 2.3 0.023
The production of lactic acid from this study is lower than the amount reported by Wang et al. [32] who obtained a yield of 0.71 g/g on cassava powder supplemented with yeast extract using Lactobacillus rhamnosus strain CASL. Linko and Javanainen [8] produced lactic acid yields of 0.87g/g on barley starch supplemented with yeast extract and peptone using Lactobacillus casei NRRL B-441. John et al. [33] rep-orted a lactic acid yield of 0.96 g/g through the fer-mentation of cassava bagasse by L. casei NCIMB 3254 using ammonium chloride and yeast extract as nitrogen sources. Lower lactic acid productivity was reported by Ray et al. [34] who obtained a maximum lactic acid yield of 0.2985 g/g from cassava fibrous residue using L. plantarum MTCC 1407. The use org-anic nitrogen sources such as yeast extract and pep-tone for lactic acid production through microbial fer-mentation makes the processes less economical and costly. The results presented in this work highlight the potential of using R. oligosporus and L. plantarum to produce high concentrations of L-lactic acid from Cas-sava peels taking into account that no optimization of culture conditions was performed in this study.
Table 4. Production of L-lactic acid from alkali and acid hyd-rolyzed Cassava peels using mixed cultures of Rhizopus oligo-sporus and Lactobacillus plantarum
Concentration, % Lactic acid concentration g/100 g
Yield, g lactic acid/g substrate
NaOH
0.1 15.2 0.152
0.2 18.6 0.186
0.3 22.4 0.224
0.4 28.9 0.289
0.5 30.4 0.304
0.6 30.6 0.306
0.7 32.8 0.328
0.8 32.7 0.327
0.9 34.2 0.342
1.0 36.4 0.364
HCl
0.1 17.9 0.179
0.2 19.2 0.192
0.3 22.6 0.226
0.4 29.3 0.293
0.5 33.5 0.335
0.6 38.4 0.384
0.7 40.2 0.402
0.8 41.0 0.410
0.9 42.5 0.425
1.0 50.2 0.502
Control 4.6 0.046
CONCLUSION
Mixed cultures of Rhizopus oligosporus and Lactobacillus plantarum produced the highest yield of
L-lactic acid from acid hydrolyzed Cassava peels. Single cultures of the microorganisms produced lower yields of L-lactic acid from alkali and acid hydrolyzed peels. Lowest yield of lactic acid was observed in unhydrolyzed samples inoculated with the microorg-anisms. This work proposes an economic method of lactic acid production from cassava waste material.
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OGBONNAYA NWOKORO
Industrial Microbiology and Biotechnology Laboratory, Department of Microbiology, University of Nigeria, Nsukka, Nigeria
NAUČNI RAD
PRODUKCIJA
L
-MLE
Č
NE KISELINE IZ OTPADAKA
KORE MANIOKE POMO
Ć
U POJEDINA
Č
NIH I
MEŠOVITIH KULTURA
Rhizopus oligosporus
I
Lactobacillus plantarum
U radu je proučavana produkcija L-mlečne kiseline pomoću pojedinačnih i mešovitih kultura Rhizopus oligosporus i Lactobacillus plantarum. Otpaci kore manioke su hidro-lizovani u toku jednog sata na temperaturi ključanja pomoću rastvora NaOH ili HCl, a zatim je hidrolizat neutralizovan na pH 6,2. Koncentracija redukujućih šećera dobijenih hidrolizom se povećava sa povećanjem koncentracije alkalije i kiseline. Uzorci kiselin-skih, odnosno baznih hidrolizata sadrže maksimalnu koncentraciju šećera od 402, odnosno 213 mg/g supstrata. Hidrolizatima je dodat 0,5% rastvor amonijum-sulfata, a zatim su zasejani pojedinačnim ili mešovitim kulturama R. oligosporus i L. plantarum i držani 48 sati radi produkcije mlečne kiseline. Najveći prinos mlečne kiseline od 50,2 g/100 g supstrata je zapažen kod fermentacije kiselinskog hidrolizata sa mešovitom kul-turom. Fermentacijom alkalnog hidrolizata mešvotom kulturom ostvaren je maksimalni prinos mlečne kiseline od 36,4 g/100 g supstrata. Na nehidrolizovanim otpacima kora manioke zasejanih mešovitom kulturom ostvaren je prinos od samo 4,6 g/100 g sup-strata. Rad pokazuje efikasnu upotrebu kore manioke za dobijanje bioproizvoda mikrob-nom fermentacijom.
