Open Access Original Research Article

Statistical Optimization of Process Variables for Biodiesel Production from Waste Cooking Oil Using Heterogeneous Base Catalyst

O. A. Aworanti, S. E. Agarry, A. O. Ajani

Biotechnology Journal International, Page 116-132
DOI: 10.9734/BBJ/2013/1381

In this study, the effects of methanol-to-oil molar ratio, catalyst amount and reaction time on the transesterification of waste cooking oil (WCO) to biodiesel were investigated. Methanol with calcium oxide as a heterogeneous catalyst was used for the transesterification process at a temperature of 60oC and 3000 rpm stirring speed. Response surface methodology (RSM) with central composite rotable design (CCRD) was used at five levels of oil-to-methanol molar ratio (9:1 – 14:1), catalyst (1- 5 %) and reaction time (30 – 90 min) as independent variables and WCO biodiesel yield as dependent variable (response). A statistically significant (P < 0.0001) second-order quadratic polynomial regression model with a coefficient of determination, R (= 0.9964) was obtained for biodiesel production (using Design-Expert Statistical program (v. 6.0.8)) and verification experiment confirmed the validity of the predicted model. Numerical optimization technique based on desirability function was carried out to optimize the WCO conversion to biodiesel. The optimum combinations for transesterification to achieve a predicted maximum biodiesel yield of 94.15 percent were found to be: oil-to-methanol molar ratio, 9.14:1; catalyst amount, 3.49 % and reaction time, 60.49 min. At this optimum condition, the observed biodiesel yield was found to be 94.10 percent. In addition, the fuel properties of the produced biodiesel were in the acceptable ranges according to international standards for biodiesel specifications. The statistical analyses and the closeness of the experimental results to model predictions show the reliability of the regression model and thus, the results will be helpful in selecting an efficient and economical method for biodiesel production from cheap raw materials with high free fatty acid.

Open Access Original Research Article

The Effect of Picloram on Somatic Embryogenesis of Different Explants of Strawberry (Fragaria ananassa Duch.)

Mohammad Gerdakaneh, Majid Zohori

Biotechnology Journal International, Page 133-142
DOI: 10.9734/BBJ/2013/2370

The present investigation was conducted to study the effects of different concentrations of picloram on somatic embryogenesis induction, development and maturation of three strawberry (Kurdistan, Paros and Camarosa) cultivars. For this purpose, leaf blade, nodal, petiole, stamen and flower bud calli were cultured on MS medium supplemented with picloram at 0.25, 0.5, 1 and 2 mg/L concentrations. The concentration of growth regulator, cultivar and explant type were found critical to somatic embryogenesis induction, development and maturation. Results obtained from the studies revealed that all explants with the exception of petiole and stamen incubated on medium formed embryonic calli. 2 mg/L picloram yielded the highest percentage of embryonic calli and number of globular-stage embryos and 1 mg/L picloram yielded the highest number of cotyledonary-stage embryos in all types of explants. The leaf explant calli and Paros cultivar were the most responsive to produce somatic embryogenesis induction, development and maturation

Open Access Original Research Article

Using Aedes aegypti larvae to Assess Pesticide Contamination of Soil, Groundwater and Vegetables

Claude Ahouangninou, Thibaud Martin, Françoise Assogba-Komlan, Serge Simon, Luc Djogbénou, Ibrahim Siddick, Cédric Pennetier, Vincent Corbel, Benjamin Fayomi

Biotechnology Journal International, Page 143-157
DOI: 10.9734/BBJ/2013/2161

In Benin, the use of synthetic pesticides in vegetable production poses a risk to the environment and human health. Vegetables, water and soil quality assessment is very important for monitoring and mitigation of these risks. The evaluation of pesticide contamination of vegetables and agricultural environment is often made using expensive methods. It is crucial for research in so-called developing countries to develop less expensive tools for pesticide risks assessment and monitoring.  The aim of this study was to assess the potential of using Aedes aegypti larvae as a bio-indicator to measure the pesticide contamination of soil, groundwater and vegetables. Vegetables just before harvest, groundwater and soils samples from three production sites and vegetables samples from markets were collected from March to August 2011. Ethanol extracts of these samples were tested on first stage larvae of Aedes aegypti. The method made it possible to detect residues of chlorpyrifos-ethyl and deltamethrin in cabbage until 4 and 8 days respectively after treatment with the recommended doses for crop protection. It proved inappropriate to measure pesticides residues in market-gardening soils, since these soils contain some amounts of nitrite, nitrate and phosphate, coming from the decomposition of fertilizers which are poisonous for the first stage larvae of Aedes aegypti. Overall, the results revealed the presence of small amounts of pesticides residues in 12.5% of the vegetables collected from markets. Pesticides residues were also detected in 30.0% of vegetables collected just before harvest. Residues of pesticide were not detected in groundwater samples collected from vegetable growing areas. First stage larvae of Aedes aegypti could be used as a bio-indicator to characterize and monitor risk of pesticide contamination of vegetables in southern Benin. It could also be used for a monitoring program before running a more thorough chemical analysis to identify and quantify the pesticide molecules present in samples.  

Open Access Original Research Article

Changes in Heavy Metal Contents of a Waste Engine Oil Polluted Soil Exposed to Soil pH Adjustments

B. Ikhajiagbe, G. O. Anoliefo, E. O. Oshomoh, Nosakhare Airhienbuwa

Biotechnology Journal International, Page 158-168
DOI: 10.9734/BBJ/2013/2374

The present study investigated the role of soil pH adjustments in heavy metals concentrations in waste engine oil (WEO) - polluted soils. Sun-dried top soil (0-10cm) was measured into buckets. WEO was added to soil and mixed thoroughly to obtain similar concentrations of 2.5% w/w oil in soil. The polluted soil was thereafter amended with NPK (15:15:15) fertilizer to enhance microbial activity. The buckets were transferred into a well ventilated screen house with inherent constant room temperature (27ºC). The entire setup was divided into 5 sets. Each set was wetted daily with 200ml of different pH solutions (pH 3, 5, 7, 9, and 11) for a period of 3 months. There were significant reductions in heavy metal concentrations particularly at pH of 5. There were significant reductions in total hydrocarbons contents (THC) of polluted soils at 2 months after pollution from 1882.32 mg/kg at pH 3 to 325 mg/kg at pH 5, compared to THC of soil at 1 week after pollution at pH 7 (3425.63mg/kg).

Open Access Original Research Article

Optimization of Sweet Potato Starch Hydrolyzate Production and Its Potential Utilization as Substrate for Citric Acid Production

E. Betiku, O. A. Adesina

Biotechnology Journal International, Page 169-182
DOI: 10.9734/BBJ/2013/2768

Aims: The aims of this work was optimization of two-step enzymatic hydrolysis of sweet potato starch using statistical approach and subsequent utilization of the hydrolyzate obtained for citric acid production.

Methodology: Box Behnken design was used in this study to generate a total of 17 individual experiments for each step of the hydrolysis (liquefaction and saccharification steps). These were designed to study the effect of temperature, time and pH on the sweet potato starch hydrolyzate (SPSH) concentration. The optimization was carried out using response surface methodology (RSM). The SPSH obtained was used to culture Aspergillus niger for citric acid production.

Results: A statistically significant quadratic regression model (P<0.05) was obtained for the liquefaction step. Statistical model predicted the highest sweet potato starch hydrolyzate (SPSH) concentration to be 172.23 g/L at optimal condition of temperature 61.05ºC, time 55.02 min and pH 6.5. A statistically significant quadratic regression model was also obtained for the saccharification step. Statistical model predicted the highest SPSH concentration to be 241.92 g/L, established at the optimal condition of temperature 52ºC, time 44 min and pH 4.5. The optimal liquefaction and saccharification conditions were validated with the actual SPSH concentration of 172.00 and 241.01g/L, respectively. The maximum citric acid production of 86g/L was achieved on the 8th day of cultivation when the SPSH was used for the cultivation of A. niger.

Conclusion: RSM was successfully applied the two-step enzymatic hydrolysis of sweet potato starch. This work showed that the sweet potato starch hydrolyzate could serve as sole carbon source for citric acid production.

Open Access Original Research Article

Preparation of Protein Extraction from Flower Buds of Solanum lycopersicum for Two-Dimensional Gel Electrophoresis

Xiaocui Zhao, Jingqi Ren, Na Cui, Haiyan Fan, Guangchao Yu, Tianlai Li

Biotechnology Journal International, Page 183-190
DOI: 10.9734/BBJ/2013/2957

Aims: Find a suitable method for the protein extraction from flower buds of Solanum lycopersicum.

Study Design: Compare some kinds of protein extraction methods and find the best one among them suitable to tomato flower buds.

Place and Duration of Study: Biological Science and Technology College, between June 2010 and July 2011.

Methodology: The proteins for electrophoresis were extracted using different methods, such as trichloroacetic acid /acetone (TCA/acetone), Sodium dodecyl sulfate (SDS), Tris-saturated phenol (Tris-Phen), Phenol/SDS and Direct lysis method. After silver staining, different patterns of protein spots were observed in the gels.

Results: Few spots were found by SDS and Phenol/SDS extractions, more spots by immediate dissolution but the most impurities, less protein productivity though more spots by Tris-Phen extractions, and more protein productivity and better apart effect by TCA/acetone. The 2-DE image background was the clear and the protein spots were the most by TCA/acetone method.

Conclusion: TCA/acetone method is much more suitable as extraction method for protein two-dimensional electrophoresis of tomato flower buds.

Open Access Original Research Article

Anti-Inflammatory and Analgesic Activities of Soft Drink Leaf Extract of Phyllanthus amarus in Some Laboratory Animals

Adeolu A. Adedapo, Sunday O. Ofuegbe

Biotechnology Journal International, Page 191-204
DOI: 10.9734/BBJ/2013/2953

Aim: Phyllanthus amarus Schum (Euphorbiaceae) is an annual herbal shrub which has been used in traditional medicine in Nigeria to treat some disease conditions. The aim of this study is to evaluate the anti-inflammatory and analgesic activities of the aqueous extract of Phyllanthus amarus in experimental animal models hence confirming its folkloric use.

Study Design: Forty healthy white Wister strain albino rats (100–200g) and forty mice (15–30g) of either sex bred in the experimental animal house of the Faculty of Veterinary Medicine, University of Ibadan, Nigeria were used for the study. Forty rats were used for anti-inflammatory study while forty mice used for the analgesic study. In anti-inflammatory study, carrageenan and histamine-induced paw oedema were used while acetic acid-induced writhing test and formalin-induced paw lick test were deployed for analgesic test. 

Place and Duration of Study: Faculty of Veterinary Medicine, University of Ibadan, Nigeria; 2 months.

Methodology: Soft drink extract (SDE) was prepared by dissolving ground plant materials (200g) in 1 L seven up (7 UP®) for 48 h, filtered, lyophilized and then used for the pharmacological investigations. Standard phytochemical methods were used to test for the presence of phytoactive compounds in the plant. Acute toxicity was carried out in mice to determine safe doses for use. The anti-inflammatory activities were conducted using carrageenan and histamine to induce oedema in rats while analgesic activities were embarked upon using acetic acid- induced writhing test and formalin-induced paw lick test.

Results: The extract in doses of 100 and 200 mg/kg at 3 hr showed 15.1 and 16.4% inhibition of histamine induced-paw oedema respectively while ibuprofen caused 9.6% inhibition at the same period. In the case of carrageenan induced paw oedema, the extract in doses of 100 and 200 mg/kg at 4 hr showed 10.5 and 12.0% inhibition respectively while ibuprofen only caused 3% inhibition. In the acetic acid- induced writhing test, the extract showed a good analgesic activity characterized by a significant reduction in the number of writhes with 100 and 200 mg/kg doses used when compared to the control group. The result was also similar to the formalin-induced paw lick test.

Conclusion: The soft drink leaf extract of Phyllanthus amarus has both analgesic and anti-inflammatory potential. The activities of this extract were comparable to that of ibuprofen, the reference drug used in this study.

Open Access Original Research Article

Effect of Argon Laser Curing on the Shear Bond Strength of Composite Resin Restorative Material

Ibrahim M. Hammouda, Mohammed M. Beyari

Biotechnology Journal International, Page 205-212
DOI: 10.9734/BBJ/2013/2889

Aim: The aim of this in vitro study was to analyze the shear bond strength of composite resin using two different curing light sources: halogen light (control group) for 20 or 40s, and Argon laser (test group) for 10 or 15s.

Study Design: This study was carried in the Department of Dental Biomaterials, Faculty of Dentistry and Ophalmolgy Hospital, Mansura University between January 2011 and August 2012.

Methodology: Sixty freshly extracted human molars were prepared to receive composite resin samples in four groups "n=15". The teeth were centrally horizontally mounted in plastic molds with cold cure acrylic resin. Flat occlusal surfaces were prepared and smoothed. One-step self-etching dental adhesive (Xeno®111) was applied to the dentine surface and cured. Composite resin (Spectrum universal composite) was inserted into standardized Perspex mold and polymerized with a halogen light and an argon laser curing units. Specimens were stored in deionized water at 37oC for 48 hours. The specimens were stressed under shear force at a crosshead speed of 0.5 mm/min using Lloyd testing machine. The shear bond strength was calculated to the four groups and statistical analysis performed (One-way ANOVA and LSD tests) at the level of significance at p<0.05.

Results: LSD tests indicated that there was significantly higher shear bond strength for the composite cured with argon laser compared with the halogen light curing method. There was no statistical significant difference between curing times of the same light source.

Conclusions: The argon laser is a promising source for optimal initiation of polymerization of composite resins. The use of argon laser has been suggested as a new alternative for polymerization of restorative materials. The shear bond strength of composite resin cured with argon laser was superior to that was cured with halogen light even with short curing times.

Open Access Original Research Article

Diversity of Bacterial Community in Fermentation of African Oil Bean Seeds (Pentaciethra macrophylla Benth) by comparison of 16S rRNA Gene Fragments

C. N. Opara, P. I. Okolie, S. V. A. Uzochukwu

Biotechnology Journal International, Page 213-220
DOI: 10.9734/BBJ/2013/2518

Aims: The microbial diversity, fermentation dynamic and the predominant micro-organisms involved in the fermentation of African oil bean (Pentaciethra macrophylla Benth) seeds to “Ugba” traditional African food in Eastern Nigeria were investigated by analyzing the microbial community DNA of the food using sequences of their 16S rRNA genes fragment analysis.

Study Design: Universal bacterial conserved 16S rRNA gene region was used to study bacterial dynamics as well as the diversity during fermentation stages. Predominant microorganisms were investigated with the view to establishing the best possible starter culture for the production of high flavoured “Ugba”.

Place and Duration of Study: Biotechnology Centre of Federal University of Agriculture, Abeokuta, Ogun State, Nigeria, between January 2007 and May 2009.

Methodology: Raw seeds were boiled for two hours for easy removal of the seed coats. Peeled seed cotyledons were sliced, cooked for 4hrs until softened. Sliced cotyledons were washed, wrapped in local leafs for fermentation for a period of 96hrs. Sampling for analysis was performed, at every 24 hours interval. Bacterial Community of freshly fermenting “Ugba” was obtained by washing seeds at room temperature in 0.40% NaCl salt solution for 15 minutes. The supernatant was used for streaking on both Nutrient agar and “Ugba” agar plates and for Community DNA extraction. DNA extraction was carried out from community DNA extracts and culture isolates grown in LB (Luria – Bertani) broth at 37°C for 24 hours using Promega DNA extraction kit. Partial 16S rRNA genes of isolates DNA and entire microbial community DNA were amplified using 16S rRNA primers. Amplified fragments were cloned using the PCR-TRAP. The transformed clones were sequenced and aligned with reference sequences in the NCBI data base for identification.

Results: This analysis indicated that from community DNA, seventeen clones were identified as Bacillus subtilis, Nine as Bacillus pumilus, four as Bacillus licheniformis, two as Bacillaceae bacterium, two as Bacillus sp Van 22, and two as Staphylococcus spp. Also, of the ten sequenced cloned isolates from the cultural technique, eight were identified as Bacillus subtilis, while two sequences were identified as Bacillus pumilus. The percentage abundance revealed that Bacillus subtilis had the highest abundance of 47.2% followed by Bacillus pumilus with 25%.

Conclusion: Bacillus subtilis is the predominant species in Ugba fermentation as it had high percentage abundance throughout the fermentation period. This study indicated that molecular analysis of community DNA provides a more accurate picture of diversity and dynamics of microbial communities.