Open Access Original Research Article

Proteolytic Activity of Bacillus amyloliquefaciens UEF01 Endophytic to Carnivorous Plant Utricularia exoleta R. Br.

Madhubanti Chaudhuri, Sayantani Biswas, Arundhati Pal, A. K. Paul

Biotechnology Journal International, Page 1-11
DOI: 10.9734/BJI/2017/30475

Aims: The endosphere of the carnivorous plant Utricularia exoleta R. Br. represents a unique niche for the study of microbial diversity. This study was aimed at to isolate and enumerate the protease producing potential of bacteria endophytic to U. exoleta R. Br.

Study Design: Extracellular proteolytic activity of the cell-free culture filtrate of the bacterial endophyte was determined following standard colorimetic assay using casein as the substrate.

Place and Duration of Study: The study was undertaken in the Microbiology Laboratory, Department of Botany, University of Calcutta during September 2015 and March 2016.

Methodology: A total of 36 phenotypically distinguishable bacteria endophytic to leaf, stem, bladder and fruit of U. exoleta were isolated and evaluated for protease production. The best protease producing isolate, UEF01 was selected, characterized and the conditions for protease activity were optimized.

Results: The selected isolate was characterized following morphological, physio-biochemical and 16S rRNA gene sequence analysis and identified as Bacillus amyloliquefaciens UEF01 (GenBank Accession No. KX816572). The crude protease of the cell-free culture filtrate of UEF01 was maximum at 35° C, pH 9 with 1.5% (w/v) casein. The enzyme appeared to be thermolabile with loss of >80% activity at 70°C (15 min incubation). Kinetic studies indicated the Km and Vmax values as 9.21 mg/ml and 71.43 U/mg of protein respectively. The enzyme was sensitive to Na and Mn ions as well as some selective protease inhibitors such as phenyl methyl sulfonyl fluoride, β-mercaptoethanol, and EDTA.

Conclusion: This proteolytic study will help in understanding the role of endophytes in digestion of prey within the bladders of the carnivorous plants.


Open Access Original Research Article

Co-production of Coprinus cinereus (Schaeff.) S. Gray. s. lato Mushrooms and Biogas from Palm Oil Wastes in Tanzania

Mariam Said Mmanywa, Anthony Manoni Mshandete

Biotechnology Journal International, Page 1-13
DOI: 10.9734/BJI/2017/30479

Aims: The aim of this study was to investigate on the effect of: (a) mixed fractions of palm oil wastes on Coprinus cinereus edible and medicinal mushroom yield using solid state fermentation plastic bioreactor and (b) of spent mushrooms substrate (SMS) on the extent of methane yield in batch anaerobic bioreactors using cow dung manure as an inoculum. Only one research reported on pre-treatment of palm oil wastes and biogas production in Tanzania.

Study Design: Palm oil wastes were obtained from Bagamoyo district, Pwani Region, in Tanzania. Coprinus cinereus mushroom production was performed in solid state fermentation plastic bioreactor while biogas production was carried out in batch anaerobic bioreactors.

Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Dar es Salaam, between September 2012 and July 2013.

Methodology: Completely randomized design was used for this study where by nine different fractions of blended palm oil wastes were prepared, Spawn rate (mushroom seed of Coprinus cinereus) employed was 7% based on wet weight of the substrate (about 35 g per 500 g moist weight substrate), then placed in solid state fermentation plastic bioreactor and incubated in mushroom house. Time was recorded in days for the completion of growth of mycelium on substrates (/spawn running/vegetative growth), appearance of pinheads (pinning) and fruiting bodies (fructification). The data for the yield number, fresh weight of fruiting bodies and biological efficiency worked out against the dry weight of each substrate were also recorded. The substrates for biogas production used were biological treated palm oil wastes i.e. the spent mushroom substrates obtained after harvesting mushrooms and cow dung manure was as an inoculum.  Anaerobic digestion of spent palm oil wastes mushroom substrates for biogas production was carried out in batch scale (500 ml E-Flask) under anaerobic condition at ambient temperature ranged from 28-30°C and at various mixing palm oil wastes composition fraction in triplicates. Determining both biogas volume and methane content monitored the performance of the anaerobic digestion process in the batch anaerobic bioreactors.

Results: Mushrooms grew well in different composition of palm oil wastes and days for completion of spawn running to fruiting body formation ranged from 13 to 23 days; the best substrate formulation was 98% EFB (Empty Fruit Bunches) +1% S (Sediment)+1% P (Pome) which had highest mushroom yield (189 g fresh mushrooms/kg moist substrate) and maximum biological efficiency (35%). When the Spent Mushroom Substrates (SMS) were employed in batch anaerobic bioreactors for biogas production. The best results 0.43 and 0.49 CH4 m3/kg Volatile Solids (VS) added was obtained from pretreated (SMS) 98% EFB+1% S+1% and 39% MF (Mesocarp Fibers)+39% EFB+ (20% PK (Palm Kernel )+ 1% P+1% S palm wastes substrates formulation, respectively. The highest methane yield were 1.3-1.4 fold higher compared to methane yields 0.33-0.34 CH4 m3/kg VS registered from corresponding non-pre-treated palm oil wastes substrates formulations, mean methane content of the biogas obtained from treated SMS was 82%, which was slightly higher than 79% recorded from untreated palm oil wastes substrates formulations.

Conclusion: In conclusion it is technically feasible to co-produce both food in the form of Coprinus cinereus edible and medicinal mushrooms and bio-energy in the form biogas rich methane from palm oil wastes while at the same time reducing environmental pollution.

Open Access Original Research Article

Molecular Detection, Biological Characterization and Evaluation of Protective Potentiality of a Velogenic Strain of Newcastle Disease Virus Isolate of Bangladesh

Md. Golzar Hossain, Sukumar Saha, Sharmin Akter, Md. Alimul Islam, Md. Mansurul Amin

Biotechnology Journal International, Page 1-9
DOI: 10.9734/BJI/2017/31123

Aims: The aim of this study was to isolate and characterize Newcastle disease virus (NDV) from recent outbreaks in Bangladesh and protective potentiality evaluation of a velogenic NDV strain.

Methodology: A total of 19 lung tissue samples were collected from dead layer chickens of clinically suspected Newcastle disease (ND) cases. Ten days old embryonated chicken eggs, day-old chick and six-week-old sero-negative chickens were used for the isolation and pathotype determination of the virus. Hemagglutination (HA), hemagglutination inhibition (HI) tests using anti-APMV-1 polyclonal serum were used for primary identification and reverse transcription polymerase chain reaction (RT-PCR) was carried out for the confirmation of the isolated viruses by amplification of F gene of NDV using gene specific primers. Three, out of eleven isolates of NDV were subjected to pathotype determination by mean death time (MDT), intracerebral pathogenecity index (ICPI) and intravenous pathogenecity index (IVPI). One of the isolates of NDV of the year 2012 was selected as vaccine candidate to determine its immunogenicity. ND vaccine was prepared by inactivating virulent Newcastle disease virus (vNDV) with 0.1% formaldehyde and adjuvanted with 40% aluminium hydroxide gel.

Results: Of the 19 samples total eleven isolates were initially identified as NDV by HA and HI tests and finally confirmed by RT-PCR. Results of MDT, ICPI and IVPI indices indicated that all the isolates of NDV of 2011 and 2012 were velogenic in nature. The inactivated vaccine produced satisfactory level of antibody titre at 72 days of post vaccination and revealed 100% protection during challenge experiment with 2 egg lethal dosage, ELD50/bird against both the velogenic strains of NDV.

Conclusion: The frequent outbreaks of ND caused by vNDV in Bangladesh could easily be controlled by using inactivated vaccine prepared with velogenic strain of NDV.

Open Access Original Research Article

Antimicrobial Resistance and Detection of Biofilm in Staphylococcus aureus Isolates from Casablanca

Mohamed Achmit, Mohammed Rehhali, Taha Chouati, Khalid Katfy, Fouad Mellouki, Khalid Zerouali, Rajaa Ait Mhand, Naima Rhallabi

Biotechnology Journal International, Page 1-9
DOI: 10.9734/BJI/2017/31232

Aims: The ability of biofilm formation seems to play an essential role in the virulence of Staphylococcus aureus. The aims of the present study were to test the sensitivity of the clinical isolates of Staphylococcus aureus to antibiotics, detect the ability of these strains to form biofilm and evaluate the correlation between biofilm formation by clinical isolates and the resistance to antibiotics.

Place and Duration of Study: Laboratory of Virology, Microbiology and Quality/ Eco-toxicology and Biodiversity, Faculty of Sciences and Techniques Mohammedia, University Hassan II Casablanca and laboratory of Bacteriology, Virology and Hygiene, Ibn Rochd University Hospital, Casablanca during October 2015 and January 2016.

Methodology: A total of 117 clinical isolates of staphylococci were collected at the University Hospital Ibn Rochd of Casablanca, Morocco and examined for antimicrobial susceptibility, presence of mecA gene and biofilm formation. Staphylococci species identification and antibiogram were performed by standard procedures using disk diffusion method. The methicillin resistance was confirmed by PCR using mecA specific primers. The biofilm formation assay was realised by the tissue culture plate method (TCP).

Results: Among all strains collected, 74 were identified as Staphylococcus aureus. Out of 74 Staphylococcus aureus, 22 strains (29.7 %) were found methicillin resistant when tested with cefoxitin disc diffusion method. 20.3 %, 18.9 % and 13.5 % were classified as resistant to kanamicin, ciprofloxacin and erythromycin respectively. All strains were found resistant to penicillin G and sensitive to teicoplanin. All isolates resistant to methicillin by cefoxitin disc diffusion method were confirmed by presence the mecA gene by PCR. Of the 74 isolates 16 (21.6 %) were non adherent, 40 (54 %) weakly adherent, 12 (16.2 %) moderately adherent and 6 (8.1 %) strongly adherent.

Conclusion: The results of this study showed that there is a correlation between biofilm formation and resistance to all the antibiotics tested, except to teicoplanin, which was active against the all strains.

Open Access Review Article

Insights on Pharmacological Properties of Combretum leprosum Mart.

José Carlos da Silveira Pereira, Carlos Campos Câmara, Luana Cassandra Breitenbach Barroso Coelho, Michele Dalvina Correia da Silva

Biotechnology Journal International, Page 1-13
DOI: 10.9734/BJI/2017/31251

Combretum leprosum Mart. is a plant widely used in folk medicine; several studies revealed anti-inflammatory, antinociceptive, toxic, antiproliferative, antibacterial, antiparasitic, neuroprotective and gastroprotective effects, among other properties. However, there are no reports on evaluation of preparations obtained from this species’ tissues, exploring biological responses of macromolecular components, such as proteins, common active agents. Proteins, such as lectins, promote important pharmacological results due to specific interaction with carbohydrates or glycoconjugates. In this work, a protein fraction obtained from a saline extract of C. leprosum leaves was evaluated for anti-inflammatory potential in a paw edema assay induced by carrageenan in Wistar rats. A significant anti-edematous effect was observed after 3 h (100 mg protein/kg) and 6 h (30 mg/kg and 100 mg/kg). The saline extract and protein fractions showed lectin activity, inhibited by D-(+)-mannose, D-(+)-trehalose dihydrate, D-(+)-galactose and D-fructose. Conclusively, leaves of C. leprosum contained proteins and lectin(s), promising anti-inflammatory agents. A vision of C. leprosum pharmacological properties is also approached.