Open Access Original Research Article

Production of Fungal Laccase Using Orange Peelings as Substrate by Submerged Static Fermentation

Francis Sopuruchukwu Ire, Eze Frank Ahuekwe

Microbiology Research Journal International, Page 1-19
DOI: 10.9734/BMRJ/2016/27257

Aims: Laccases are diphenol oxidases that have numerous applications in biotechnological processes. In this work, the production of fungal laccase using organic and inorganic feed substrates in submerged static fermentation was investigated.

Study Design: One-factor-at-a-time strategy was adopted to optimize the cultural parameters for enhanced laccase production.

Place and Duration of Study: Department of Microbiology, Faculty of Science, University of Port Harcourt, Nigeria, between October 2014 and November 2015.

Methodology: A total of nine fungal isolates were obtained from wood decaying sites of University of Port Harcourt forest areas and subjected to laccase screening with 2,2-Azinobis-3-ethyl(benzthiazoline-6-sulphonate) (ABTS). The influence of medium components using the basal medium at pH 5.0 as base was evaluated and these cultural parameters include carbon sources (glycerol, rice bran, glucose and ground orange peelings), nitrogen sources (yeast extract, potassium nitrate, peptone and ammonium chloride), metal ions (copper sulfate and manganese sulfate) and inducer compounds (ABTS, Tween 80 and soya oil). Time course study was conducted with the unoptimized and optimized cultural medium.

Results: Out of nine cultures tested, seven were found to be laccase-positive with isolates CF-1 and CF-2 being the best potential cultures. Isolate CF-1 which had the highest laccase activity was identified as Pleurotus ostreatus and was chosen for further studies. Ground orange peelings (0.1% w/v) and NH4Cl (0.1% w/v) were the most suitable carbon and nitrogen source for laccase production by the fungus. Maximum laccase production was obtained with Cu2+ at a concentration of 0.05%w/v among other metal ions. Soya oil at concentration of 0.05% (v/v) was the best inducer of the enzyme. The highest laccase production was achieved at an Initial pH 4.5. Under optimal culture medium, the maximum laccase activity was determined to be 7.21 U ml-1 on the 7th day of cultivation; which was approximately three times higher than that in basal medium (2.5 U ml-1). The results obtained indicate that the extracellular laccase production is dependent on various cultural parameters.

Conclusion: One-factor-at-a-time strategy adopted in this study proved that the optimum conditions enhanced laccase production by three folds using orange peelings. The results obtained are very interesting since orange peelings are common agricultural wastes in several countries and imply that their re-utilization in the production of enzymes would help solve pollution problems caused by their improper disposal. In addition, Pleurotus ostreatus having shown promise for laccase production using low-cost lignocellulosic substrates could be suggested as a prospective candidate for higher laccase production for several biotechnological applications.

Open Access Original Research Article

Evaluating Efficacy of Biosurfactants from Bacterial Isolates in Conferring Protection against Rhizoctonia and Sclerotium Infection in Wheat and Peanut Plants

Janki Fulwala, Shraddha Prabhu

Microbiology Research Journal International, Page 1-13
DOI: 10.9734/BMRJ/2016/27097

Aims: To isolate biosurfactant producers from natural habitat and to test the antimicrobial activity of the extracted biosurfactant against fungal plant pathogens.

Study Design: The present study was designed to achieve the following objectives:

1. Isolation, screening and identification of potent biosurfactant producers.
2. Production, Extraction and Characterization of biosurfactant.
3. Testing the antimicrobial activity of the biosurfactant in vitro.
4. In vivo evaluation of the ability of biosurfactant to protect plants from fungal diseases.

Place and Duration of Study: Department of Microbiology, Sophia College, University of Mumbai, Maharashtra, India, between June 2014 and May 2015. 

Methodology: The bacterial isolates obtained from natural habitat were screened for biosurfactant producers using hemolytic plate assay, drop collapse test, penetration assay and emulsification activity. Potent biosurfactant producing isolates were biochemically characterized and identified up to genus level using Bergey’s manual. Biosurfactant was extracted by chloroform: Methanol method. Characterization of extracted biosurfactant was done using blue agar plate and orcinol assay. Agar well diffusion method was used to test antimicrobial activity of biosurfactants. Ability of the biosurfactants to provide protection against fungal plant pathogens was demonstrated in vivo using wheat and peanut plant seedlings.

Results: Three isolates BMW1, BMW2 and BPS1 showing good biosurfactant activity were selected for biosurfactant production. They belonged to genus Pseudomonas, Bacillus and Micrococcus. Extraction of culture supernatant gave white residue which was used in further studies as biosurfactant. The biosurfactant produced by isolates BMW1 and BPS1 was glycolipid anionic biosurfactant while CTAB medium indicated non-ionic nature of biosurfactant from BMW2. Biosurfactant extracted from all three isolates showed good antimicrobial activity. Biosurfactant produced by BMW1 and BPS1 most effectively protected peanut plantlets from Sclerotium rolfsii infection and wheat plantlets from Rhizoctonia solaniinfection respectively.

Conclusion: Our study suggested a strategy for eliminating plant pathogenic fungi by using microbial biosurfactants.

Open Access Original Research Article

Isolation and Characterization of Cellulase Producing Bacterial Strains from an Amazonian Geothermal Spring in Peru

Yolanda Cortez, Sujay Paul, Gretty K. Villena, Marcel Gutiérrez- Correa

Microbiology Research Journal International, Page 1-8
DOI: 10.9734/BMRJ/2016/27520

Aim: Aguas Calientes (AC) is an isolated geothermal spring located deep into the Peruvian Amazon rainforest. The principal aim of this study was to isolate and characterize thermophilic lignocellulolytic bacterial strains from AC.

Study Design: Bacterial strains were cultured in laboratory and screened for cellulase activity. Superior lignocellulolytic strains were characterized by 16S rRNA sequences.

Place and Duration of Study: Samples were collected from Aguas Calientes (7°21'12'' S, 75°00'54'' W). The duration of the study was 2013-2016.

Methodology: Primary isolation of lignocelluloytic bacterial strains was carried out by direct plating, filtration and water enrichment analysis techniques. Secondary screening was performed by semi quantitative plate clearing assay. In both primary and secondary screening assay the growth media contained carboxymethylcellulose (CMC).  CMC hydrolysis by bacterial colonies was evaluated by Congo red solution. Selected strains with higher cellulase activity were characterized by 16s rRNA gene sequencing. 

Results: A total of 22 best performing strains were finally selected from 110 cellulolytic bacterial strains isolated in this study. Thirteen strains were selected from water sample (CW) while 9 selected from soil sample (CS). Water isolate LMB-AC10 showed highest cellulase activity (9.5 U ml-1) at pH 7.4 among all selected strains while LMB-AC3 was found to be the most consistent one (activity was between 6.5 U ml-1 to 7.5 U ml-1) throughout different alkaline pH levels. In CS, the highest cellulase activity (5.2 U ml-1) was showed by strain LMB-SC4 at pH 7.4. Sequence homology analysis of selected bacterial 16S RNA revealed that most of our strains are similar with different reported strains of Geobacillus, more precisely with Geobacillus thermoleovorans and Geobacillus kaustophilus.

Conclusion: For the first time several thermophilic lignocellulolytic bacterial strains were isolated from a remote Amazonian geothermal spring and some of them were found very promising.

Open Access Original Research Article

Bacteriological and Pathological Studies of Mammary Glands Affections in Camels (Camelus dromedarius) at Tumbool Abattoir, Sudan

A. M. Abeer, A. M. Zakia, E. A. Muna, Y. A. Sabiel

Microbiology Research Journal International, Page 1-8
DOI: 10.9734/BMRJ/2016/25966

Aims of the Study: To isolate and identify the bacteria associated with mastitis and study the pathology of udder tissue.

Study Design: Out of 353 culled she-camels (from Tumbool abattoir), 105 (29, 7%) sections of udders (including 31 milk samples) showing mastitis lesions were collected. Isolation and characterization of bacteria and histopathological pictures were reported.

Place of Study: This study was undertaken in the Departments of Bacteriology and Pathology, Central Veterinary Research Laboratories, Ministry of Animal Resources and Fisheries, Khartoum during Jan-Dec/ 2009.

Methodology: The isolates were characterized using two techniques: Api kits and automated system Vitek 2 Compact and the histopathological method done according to standard method.

Results: In the result herein reported the bacteria isolated from mastitic she-camels udders (105) included (31) milk samples and they were: Staphylococcus spp 40 (38 %) Streptococcus spp 29 (27.6%) and Micrococcus spp 14 (10.5%) as major pathogens followed by Corynbacteirum spp 5 (4.8%), Enterococcusspp 5 (4.8%), E. coli 3 (2.9%), and a low percent of Aerococcus spp 2 (1.9%), Proteus 2 (1.9%), Mannhemia haemolytica 1 (0.95%), Klebsiellasp 1 (0.95%) Acintobacter sp 1 (0.95%), Bacillus cereus 1 (0.95%).

The chronic mastitis was characterized by accumulation of inflammatory cellular exudates that in certain areas replacing the normal lobular structures. In addition, desquamation of most of atopic secretary glandular tissue, necrosis, and disintegration of alveolar tissue and extensive proliferation of fibrous tissue forming areas of scarring intermingled with aggregates of granulomatous cells, macrophage, plasma and lymphoid cells. And acute characterized by odema and profuse infiltration of cellular exudates, composed of mononuclear cells intermixed with neutrophils, Micro-abscesses and lymphoid follicle like structure.

Conclusions: Mastitis cases reveal different type of pathogenic bacteria concerning public health.

Chronic mastitis represented the most commonly available type.

Open Access Original Research Article

Role of Human Papillomavirus Infection in the Development of Prostate Carcinoma and Benign Prostatic Hyperplasia

Khaled M. Hassanein, Hani Al- Shobaili, Marwa Salah Mostafa, Abdelaziz Al- Saloom, Taher Obaid Alshammari

Microbiology Research Journal International, Page 1-6
DOI: 10.9734/BMRJ/2016/27283

Aims: Human papillomavirus (HPV) infections are associated with benign and malignant lesions of the female and male anogenital tract. Currently, the possible role of HPV infections in prostate carcinogenesis is a subject of great controversy. The aim of this study is to investigate the role of HPV infections as a risk factor in the development of prostatic carcinoma.

Study Design: Case-control study.

Place and Duration of Study: Department of Medical Microbiology and Immunology and Department of Dermatology, College of Medicine, Qassim University, between January 2013 and December 2015.

Methodology: Qualitative detection of the HPV DNA was performed in 85 Saudi prostatic carcinoma (PC) male patients. They were compared to 55 patients with benign prostatic hyperplasia (BPH) and 15 normal controls.

Results: The positivity of HPV DNA was significantly higher in patients with PC (25.9%) compared to the control group (0%) (P = .026). The HPV DNA positivity was higher in patients with PC than in BPH patients but with non-significant difference.

Conclusion: The present study reveals that the presence of HPV DNA in the prostatic tissue was associated with a higher incidence of PC. HPV DNA was also associated with a relatively high risk of BPH. Further studies are recommended to disclose the association between HPV infection and BPH.

Open Access Original Research Article

Serological Characterization and Antimicrobial Sensitivity Profile of Haemophilus influenzae Serotypes Isolated from Aminu Kano Teaching Hospital, Kano, Nigeria

M. Umar, A. H. Arzai, G. Yusuf, J. O. Oko, D. Y. Jobbi

Microbiology Research Journal International, Page 1-10
DOI: 10.9734/BMRJ/2016/26779

Aim: The aim of this research study was to isolate and serologically characterize Haemophilus influenzaeserotypes with view to determine antibiotic susceptibility profile of the isolated bacterium and compare the effectiveness of the antibiotics used on the capsulated and unencapsulated serotypes.

Methodology: In this study, 200 samples from different sites (respiratory tract, blood and cerebrospinal fluid) were collected from patients 1—70 years old and were cultured on chocolate agar. The presence of encapsulated bacteria was identified by Fildes medium, Congo red staining and Slide Agglutination test. Serotyping was performed by Slide Agglutination test.

Results: The results showed that 14 (7.0%) cases were positive for Haemophilus influenzae of which 78.5% were encapsulated. A statistical relationship was found between unencapsulated H. influenzae and the age of the subjects. No significant differences were found between the incidence of the infections caused by the bacterium and the gender. Isolates were sensitive to Levofloxacin (85.7%), Azithromycin and Clarithromycin both by (71.4%). While, moderate sensitivity was found in Amoxicillin (28.6) and Co-trimoxazole (28.6) with least sensitivity rates in Cloxacillin (7.1) and Tetracycline (0). Unencapsulated strains were found to be more sensitive to the tested antimicrobials than their encapsulated counterparts.

Conclusion: Our research showed that age is among the risk factors for H. influenzae infections and unencapsulated strains were found to be more prevalent in the diagnosed sites.