Environmental Microbiology Laboratory Research Articles

In-vitro Antibacterial Potentials of Essential oil from Citrus limon against Selected Pathogenic Bacteria Isolated from Cultured Nile Tilapia (Oreochromis niloticus)

Essential oils (EOs) from medicinal plants like lemon (Citrus limon) are considered promising to replace synthetic antibiotics in aquaculture production due to their ability to improve fish health. The aim of the research was to investigate the in vitro antibacterial potentials of varying levels of lemon EO against Streptococcus agalactiae, Escherichia coli, Staphylococcus aureus and Aeromonas hydrophila isolated from Nile Tilapia. This research was conducted at the Microbiology unit., Kenyan Marine and Fisheries Research Institute, Mombasa, Kenya and Environmental Microbiology and Biotechnology Laboratory, Department of Microbiology, University of Ibadan, Ibadan, Nigeria in December 2021. Extraction of EO from lemon peel was performed using steam hydro distillation. The quantitative and qualitative profiling of extracted oil was performed using Gas Chromatography-Mass Spectrometry. The antibacterial potential of lemon EO was tested against some pathogens using the agar well diffusion method while the Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentration (MBC) were determined by the broth dilution method. The most abundant chemical was D-Limonene (37.56%), 3-Carene (15.88%) and Terpinen-4-ol (93.4%). The lemon EO at 100% and 50% concentration respectively showed the highest antibacterial activity against S. agalactiae (26.00mm and 28.33mm) and E. coli (33.00mm and 30.00 mm); while the least activity was observed against A. hydrophila (8.33mm and 6.67 mm) at P = .05. The MIC and MBC values revealed that 50% lemon EO inhibited bacterial growth for S. agalactiae whereas it was greater than 50% for S. aureus, E. coli and A. hydrophila. Lemon EO has potential antibacterial properties against tested pathogens that affect cultured fish and could be considered a good candidate for further research to develop new antibacterial drugs in aquaculture.

Utilization of metal-resistant bacteria in the detoxification of contaminated dumpsites

The potentials of four metal-resistant bacterial strains to reduce the concentration of selected metals in dumpsite leachate were studied. The Environmental Microbiology and Biotechnology laboratory's culture collection of bacteria was used to extract the bacteria, which were then exposed to progressively higher metal concentrations in a medium enriched with metals. After the bacterial cells had been separated from the leachate by centrifugation at 10000 rpm for 15 min, the bacteria were then added to a batch culture biosorption set-up containing the culture medium and tyndallized leachate. The residual metal concentration was then determined after a 14-day incubation period using the atomic absorption spectrophotometer (AAS). The bacterial strains demonstrated high resistance to the four selected heavy metals [lead (Pb), chromium (Cr), cadmium (Cd) and nickel (Ni)] and their combination. The Minimum Inhibitory Concentration (MIC) value for the strains on the metal-incorporated medium for all the selected metals ranged from 700-1500 µg/ml. The resistance to the metals was in the order: Pb ˃ Ni ˃ Cr ˃ Cd. Pseudomonas aeruginosa had the highest MIC to the metal combination (1300 µg/ml) while the lowest was Proteus mirabilis (800 µg/ml); Paenalcaligenes faecalis and Bordetella petrii had MIC values of 1000 µg/ml and 1200 µg/ml respectively. Bordetella petrii removed the highest concentration of Cd and Ni from the leachate, with values of 32.81 percent and 34.91 percent, respectively, according to the biosorption setup, while Paenalcaligenes hominis had a higher percentage reduction for Pb in the leachate with a reduction of 35.77 percent. However, when the leachate was treated with a combination of the four bacterial strains, the largest percentage decrease for Cr (32.54 percent) was seen. According to this research, these metal-resistant bacteria might be highly helpful in the biological treatment of wastewater that contains metals.

Photovoltaic concentrator potential and its inactivation rate of indicator microorganism in point of use water treatment system.

Objective:Unsafe drinking water has a significant health impact all over the world especially, in developing countries. Household water treatments become more affordable than conventional ones for many reasons. Photovoltaic concentrator is environmentally sound and effective inactivation method by converting light energy to electricity. This study aimed to assess the inactivation potential of photovoltaic concentrator on drinking water quality indicator microorganisms at different solar exposure times.Methods:A laboratory-based experimental study was conducted at Jimma University’s environmental microbiology laboratory to measure the disinfection potential of the photovoltaic concentrator for months in uneven weather conditions. A membrane filtration, a pour plate count method, and a calibrated clear sky calculator were used.Results:Among indicator microorganisms, Escherichia coli was completely inactivated (standard deviation = 11.3°C, R2 = 0.80) at 2 h of solar exposure whereas heterotrophic plate count was measured as (SD = 12.2°C, R2 = 0.82) at 35°C for 48 h in the sample that contains 2.81 NTU.Conclusion:Based on the findings, photovoltaic concentrator was one of the most effective inactivation technologies for E. coli and total coliform.

Indoor Air Quality of Selected Lecture Theatres in Faculty of Life Sciences, University of Benin

The presence of airborne microbes can be detrimental to mankind. Research on bacteriological pollution of indoor air in selected lecture theatres of Life Sciences Faculty, University of Benin, was carried out. Surveys were conducted in the period of July-September, 2019. Microbiology lecture Theatre, Environmental Management and Toxicology Lecture Theatre, Animal and Environmental Biology (200 Level) Laboratory, Microbiology (400 level) Laboratory, Plant Biology and Biotechnology (200 level) Laboratory and Microbiology Postgraduate Laboratory were the sampling areas. Air samples were obtained using passive sedimentation method (settle plate method). Visible colonies were expressed in colony forming units per meter cube (cfu/m3) and pure discrete colonies were enumerated and characterized. Bacteria counts ranged from 1.83 + 0.35 – 18.34 + 2.83 (x102 cfu/m3) in July 2019. In August 2019, Bacteria counts ranged from 3.14 + 0.71 –33.27 + 65 (x102 cfu/m3). In September 2019, values ranged from 3.67 + 0.71 – 29.60+ 6.01 (x102 cfu/m3). The evaluation of air quality in the designated sampling points based on the sanitary standards formulated by the European commission revealed that 8 of the sampling points showed high level of pollution (44.44%) and 4 sampling points had very high level of contamination (22.33%). Three bacterial isolates were identified as Staphylococcus epidermis, Staphylococcus aureus and Bacillus cereus and they are of public health importance. High influx of students, architectural design of rooms and systems of ventilation are factors that can influence the indoor airborne bacterial population.

Response surface methodology and optimization of the processes for bioethanol production from Calabash (Crescentia cujete) Using Cronobacter malonaticus

Aim: Response surface methodology (RSM) model was applied to optimize ethanol production from Calabash (Crescentia cujete) pulp juice using Cronobacter malonaticus. Study Design: The Calabash pulp was squeezed with muslin cloth, and vacuum filtered to clear solution before use. The clear juice was tested for reducing sugars using the Dinitrosalicylic acid (DNS) method. Twenty three (23) runs, including 3 controls, of the fermentation was conducted at varying temperatures, pH, and volumes of inoculum. The process parameters (input variables): volumes of inoculum, temperature, and pH were subjected to response surface model, using the Central Composite Design (CCD). Place and Duration of Study: This study was carried out in the Environmental Microbiology Laboratory, University of Port Harcourt for six months. Methodology: Fermentation was done in conical flasks covered with cotton wool and foil in a stationary incubator for four days (96 hours). Active stock culture of Cronobacter malonaticus was used, with inoculum developed using Marcfaland’s method. Samples were collected every 24 hours, centrifuged, filtered and analyzed for measurement of the output variables: Reducing sugar, cell density and ethanol concentration. Results: The concentration of reducing sugars from Calabash pulp was 3.2 mg/ml. Results obtained also revealed that the fermentation can take place on a wide range of temperature 28-32°C. The optimal pH range for performance of C.malonaticus for the fermentation process was pH 5.95-6.5. The optimum volume of inoculum was 10%v/v (i.e. 10 ml in 90 ml juice). The optimized process using the RSM model gave 5.08% v/v bioethanol, being the highest achieved at pH6.08 and 28oC . Conclusion: The bioethanol yield from Calabash substrate is reasonable considering that the bacterium used is not known for ethanol production. Also the concentration of reducing sugars in the substrate and the duration of fermentation could be responsible for the yield.

First Report of Powdery Mildew Caused by Erysiphe viciae-unijugae on Vicia sativa subsp. nigra in Korea.

Vicia sativa subsp. nigra, also known as narrow-leaved vetch, belongs to the family Fabaceae and is distributed in different countries.Vetch is one of the most important annual forage legumes in the world; it is used as hay, grain, silage, green manure, and livestock fodder. In Korea, this plant is often used as cattle feed. In early May of 2019 and 2020, a powdery mildew was observed on leaves of V. sativa subsp. nigra plants in a garden of Chonnam National University, Gwangju, Korea. The first symptom appeared on the upper surface of the leaves as a small white spot. Within two weeks, the white spots of powdery mildew spread and covered the entire leaf. Voucher specimens were deposited at the Environmental Microbiology Laboratory Herbarium, Chonnam National University, Gwangju, Korea, under the accession numbers CNUFC PWV1 and CNUFC PWV2. The hyphal appressoria of the fungus were nipple-shaped, moderately lobed or multilobed, single or in opposite pairs. Measurements were made of 35 conidiophores and 55 conidia. Conidiophores arising from the upper part of mother cells, were cylindrical, 60.5 to 117.5 × 7.5 to 9.5 μm. The foot cells of the conidiophores were cylindrical, straight to slightly curved at the base, 23.5 to 38.7 × 7.3 to 9.2 μm, followed by 2-3 shorter cells. Conidia were formed singly, subcylindrical to oval, 32.5 to 48.7 × 13.5 to 19.7 μm. Conidia produced short to long germ tubes, terminating in lobed or unlobed appressoria. No chasmothecia were observed. The morphological characteristics of conidiophores and conidia were consistent with the description of Erysiphe viciae-unijugae in Shin (2000). However, it was not possible to identify the fungus only based on the asexual morph. In addition, the conidiophore and conidium shapes of E. baeumleri on the known host V. sativa were similar to those of E. viciae-unijugae (Braun and Cook 2012). Therefore, genomic DNA was extracted directly from the mycelia and conidia using a DNA prep kit (Solgent, Daejeon, Korea). The internal transcribed spacer (ITS) regions ofribosomal DNA (rDNA) and 28S rDNA from the two specimens were amplified using the primers ITS5 and PM6 (Takamatsu and Kano 2001), and LROR and LR3 (Vilgalys and Hester 1990), respectively. A BLASTn search with the ITS sequences indicated that CNUFC PWV1 (GenBank accession no. MT509395) and CNUFC PWV2 (MT509396) were 100% (503/503 bp) and 99.8% (502/503 bp) identical to those of E. viciae-unijugae (LC010086 and LC009962), respectively. The 28S sequences of CNUFC PWV1 (MT705629) and CNUFC PWV2 (MT705630) showed 99.8% (507/508 bp) and 99.4% (505/508 bp) identity values with E. viciae-unijugae (LC009976 and LC010029), respectively. Phylogenetic analyses based on the combined ITS and 28S rDNA sequences indicated that the two specimens, CNUFC PWV1 and CNUFC PWV2, formed a monophyletic clade together with sequences retrieved from E. viciae-unijugae. Therefore, the causal pathogen was identified as E. viciae-unijugae based on the results of morphological and molecular analyses. Pathogenicity tests were performed by gently pressing the infected leaves onto 10 young leaves of three healthy plants. A non-inoculated plant was used as a control. The plants were maintained in a greenhouse at 22 to 25°C, with relative humidity around 75%, and natural light. Powdery mildew symptoms were observed on the inoculated leaves after 10-14 days, whereas no symptoms were observed on the leaves of the control. The fungi on the inoculated leaves were morphologically identical to the fungi on the original infected leaves. Erysiphe viciae-unijugae has been reported to occur on Lathyrus spp. and Vicia spp. (Farr and Rossman 2020). To our knowledge, this is the first report of powdery mildew caused by E. viciae-unijugae on V. sativa subsp. nigra in Korea. The weather conditions, especially during May, are favorable for disease outbreaks. This disease may be a potential threat to feed production in Korea.

Benefit of Dietary Supplementation with Bacillus subtilis BYS2 on Growth Performance, Immune Response, and Disease Resistance of Broilers.

A strain of Bacillus subtilis (B. subtilis) BYS2 was previously isolated from Mount Tai, which is located in Tai'an City in the Shandong Province of China. The strain was then stored in the Environmental Microbiology Laboratory at Shandong Agricultural University. To evaluate the effect of the bacterium preparation in broiler production, we fed the bacterium (106CFU/g) to 1-day-old broilers and continued this feeding for 6weeks to analyze its effect on growth and immune performance. We found that the average weight of the bacterium-fed group increased by 17.19% at weeks 5 compared to the control group (P < 0.05). The height of the villi in the duodenum and jejunum and the ratio of villi to crypt were significantly increased in the bacterium-fed group at weeks 5 (P < 0.05). Also, the IgG in the serum of broilers in the experimental group increased by 31.60% (P < 0.05) and IgM 30.52% (P < 0.05) compared with those in the control group. The expressions of the major pattern recognition receptors (PRRs), antiviral proteins, pro-inflammatory cytokines, and β-defensins were significantly higher than those in the control group (P < 0.05). Meanwhile, the bursa immune organ indices of broilers in the experimental group were significantly higher than those in the control group (P < 0.05). Also, after 5weeks of continuous feeding, when infected with Escherichia coli (E. coli) O1K1 and Newcastle disease virus (NDV) F48E8, the content of bacteria and virus in tissues and organs of the experimental group decreased significantly, and the survival rate of infected chickens increased by 31.1% and 17.7%, respectively (P < 0.05). These results show that the anti-infective B. subtilis BYS2 could, to some extent, replace antibiotics to promote growth, improve innate immunity, and enhance disease resistance in broilers.

Formulation and Viability of Consortia LIBeM Using Protective Agent Skim Milk Subjected to Freeze Drying Method for Degradation of Oil Sludge Contaminated Soil

The formulation and viability of locally-isolated beneficial microorganisms (LIBeM) consortia was conducted by using protective agent skim milk at 30% (w/v). A 10% (v/v) of inoculum consists of mixed cultures C. tropicalis-RETL-Cr1+ C. violaceum-MAB-Cr1 + P. aeruginosa-BAS-Cr1 (RETL-Cr1+ MAB-Cr1+BAS-Cr1) were obtained from the Environmental Microbiology Laboratory, Universiti Malaysia Sabah and tested for biodegradation of oil sludge using the live cell powdered form. LIBeM formulation was carried out by using lyophilization under vacuum conditions for 24 hours. The viability of the initial cell loads on the final recovery after freeze drying process was measured weekly by heterotrophic plate counts (HPC). Surface morphological of LIBeM powder (LiBeM-POW) was observed under scanning electron microscopy (SEM) to show any changes on the surface cell structures. The results showed that the use of 10% (v/v) of inoculum with 108 CFU/ mL as initial cell concentrations and 30% (w/v) skim milk as protective agent yielded 31.9% maximum viability. The findings also indicate that the survival rate of LIBeM cells have potential to survive and functionality even after 13 weeks of the storage with 106 CFU/ mL recorded. The effectiveness of microbial formulation of LiBeM-POW was done by carrying out the biodegradation experiment in ASP-bioreactor system for 56 days. Two sets of ASP- bioreactor system containing 2 kg of soil mixed with 20% (v/v) of oil sludge were prepared as T1: treatment with consortia LIBeM-POW and T2: natural attenuation as a control plot. The results indicated that LIBeM-POW has potential to be applied in bioremediation of oil sludge contaminated environments with 92% degradation of TPH as compared to natural attenuation 26% degradation. This finding suggests the possibility of producing freeze dried powder of LIBeM consortia with high viability for prior used in bioremediation study. The comparison of other protective agent used by previous studied was also discussed in this paper.

Aerated Static Pile (ASP) Enhanced Bioaugmentation of Oil Sludge Contaminated Soil Using LIBEM Consortium

This paper focused on efficiency of selective locally isolated beneficial microorganisms (LIBeM) consortia in bioaugmentation of oil sludge contaminated soil on a laboratory scale. Five different selective microbial consortia LIBeM were obtained from Environmental Microbiology Laboratory, Universiti Malaysia Sabah. The arrangement of aerated static pile (ASP)-bioreactor made up of acrylic materials was developed act as continuous aeration to the soil. Six different sets of treatment containing 10 kg of soil mixed with 20% (v/v) oil sludge with five bacterial strains and natural attenuation consisted of indigenous microbial in the soil as a control plot was utilized. The rates of biodegradation of oil sludge were studied for a period of three months and periodic sampling of soil from each bioreactor was carried out at 7 days of interval. Analysis of Total Petroleum Hydrocarbon (TPH) was done along with physiochemical parameters such as pH, moisture content and microbial population (CFUs) in the soil. The results obtained revealed biodegradation of oil sludge contaminated soil in all set treatments were much higher than Natural attenuation by 12.1 fold. The bioreactor augmented with Consortia 4 proved to be a better performance with 94% of TPH degradation followed by Consortia 2 (91.6%), Consortia 5 (90.1%), Consortia 1 (90%), Consortia 3 (87.5%) and Natural attenuation (27.4%) respectively. The variations of physiochemical parameters that influence the reduction of oil sludge in the soil were also discussed in this paper. High degradation of long chain alkanes was observed between consortia treatment as compared to natural attenuation plots suggested that bioaugmentation using consortia LIBeM decrease the level of oil sludge in contaminated soil. It was noted that, the continuous supplied of oxygen from aerated static pile (ASP)-system into the soil showed a good prospect in bioremediation of oil sludge.

First Report of Powdery Mildew Caused by Erysiphe michikoae on Celtis sinensis in Korea

Celtis sinensis Pers., commonly known as the Chinese hackberry, belongs to the family Cannabaceae. In Korea, the leaves and bark of this tree are used for the treatment of gastrointestinal disorders and lung abscess. In October 2015 to 2018, symptoms of powdery mildew were observed on the leaves of C. sinensis in a garden at Chonnam National University, Gwangju (35°10′0″N, 126°55′0″E), Korea. The symptoms included white powdery colonies on the surfaces of young leaves and brown spots on older leaves. The three specimens were deposited in the Environmental Microbiology Laboratory Herbarium, Chonnam National University, under accession numbers CNUFC PWS1, CNUFC PWS2, and CNUFC PWS3, respectively. The chasmothecia were most abundant on the lower surfaces of the leaves. The hyphal appressoria were lobed, single, or opposite in pairs. The conidiophores were cylindrical, 65 to 87 × 9 to 12 μm, followed by two or three straight cells. The conidia were ellipsoidal or cylindrical, 11.5 to 14.5 × 29 to 33.5 μm. The chasmothecia were 64 to 94.5 × 76.5 to 98.5 μm, globose, and black to dark brown. Each chasmothecium had eight to 16 appendages and was 98.5 to 167.5 μm long and 5.5 to 7.5 μm wide, containing four asci; each ascus contained four to five ascospores. The ascospores were ellipsoidal, 10.5 to 14 × 15.5 to 24.5 μm. On the basis of these characteristics, the fungus is morphologically similar to Erysiphe michikoae, as described by Meeboon et al. (2013). To confirm the morphological identification, genomic DNA was extracted directly from the mycelia and chasmothecia, using a DNA prep kit (Solgent, Daejeon, Korea). The internal transcribed spacer (ITS) regions of the rDNA of three specimens were amplified using the primers ITS5 and PM6, as described by Lee and Nguyen (2016). The ITS sequences from CNUFC PWS1, CNUFC PWS2, and CNUFC PWS3 were deposited at GenBank under accession numbers MK757876, MK757877, and MK757878, respectively. BLASTn with the ITS sequences showed 99% similarity with the sequences of E. michikoae (AB693963) and E. kusanoi (AB475120). Phylogenetic analyses based on the ITS rDNA indicated that the three specimens were identical to E. michikoae. Therefore, the causal pathogen was identified as E. michikoae based on the morphological and molecular analyses. Pathogenicity tests were performed by gently pressing the infected leaves onto 10 healthy leaves of C. sinensis. Powdery mildew symptoms were observed on the inoculated leaves after 10 to 15 days, whereas no symptoms were found on the control. The fungus on the inoculated leaves was morphologically identical to that on the naturally infected plants. Powdery mildew on C. sinensis has been previously reported as E. kusanoi (synonyms: E. kusanoi var. kusanoi, Uncinula kusanoi, and U. kusanoi var. kusanoi) and Pleochaeta shiraiana (synonyms: Uncinula shiraiana and Uncinulopsis shiraiana) in Japan, China, Taiwan, Korea, and South Africa (Farr and Rossman 2019). E. michikoae has been found in only C. jessoensis from Japan (Meeboon et al. 2013). To our knowledge, this is the first report of powdery mildew caused by E. michikoae on C. sinensis in Korea as well as worldwide. This information indicates that E. michikoae is able to infect other Celtis species and may pose as a serious threat to the health of C. sinensis in Korea. Further studies on the host range of E. michikoae are required.

2465. Healthcare-Associated Pediatric Cutaneous Mucormycosis at Texas Children’s Hospital, 2012–2019

BackgroundCutaneous mucormycosis in children is a rare fungal infection which primarily occurs in patients with underlying medical conditions and is associated with significant morbidity. We describe characteristics of pediatric patients with healthcare-associated (HCA) cutaneous mucormycosis at Texas Children’s Hospital (TCH) and results of an outbreak investigation.MethodsPatients at TCH were identified retrospectively through review of the TCH Microbiology Laboratory mycology culture reports from 1/1/2012–2/18/2019. Pediatric patients <21 years of age with cutaneous mucormycosis that developed during a hospitalization or was associated with a medical device were included. Demographic information was collected through review of the electronic medical record. Randomly amplified polymorphic DNA (RAPD) analysis were performed by the Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio. This study was approved through the Baylor College of Medicine Institutional Review Board.ResultsWe identified 12 patients with HCA cutaneous mucormycosis. The characteristics of these patients are detailed in the table. Six cases of Rhizopus infection occurred over a narrow 7 month period from March through September of 2017 prompting an outbreak investigation. Genotyping results revealed 4 molecular types from 5 available isolates suggesting that the isolates were not clonally related. Adhesive products were suspected as a potential source. Infection Control activities included site visits to the hospital supply warehouse and linen facility. Tape practices throughout the hospital were assessed through a nursing survey. Samples of adhesive products were cultured at an environmental microbiology laboratory and no Rhizopus spp. were isolated. A source was not identified.ConclusionMucormycosis is a life-threatening infection in children. Providers should have a low threshold of suspicion for cutaneous mucormycosis in patients with underlying medical conditions (malignancies and extreme prematurity) that develop skin lesions near medical device dressings or securement sites. Disclosures All authors: No reported disclosures.

Application of Response Surface Methodology for Optimizing the Production of Bioethanol from Calabash (Crescentia cujete) Substrate Using Saccharomyces cerevisiae

Aim: The study employed the Response surface methodology (RSM) model to optimize ethanol production from Calabash (Crescentia cujete) pulp juice using Saccharomyces cerevisiae.&#x0D; Study Design: The Calabash pulp was squeezed with muslin cloth, and vacuum filtered to clear solution before use. The clear juice was tested for reducing sugars using the Dinitrosalicylic acid (DNS) method. Twenty three (23) runs, including 3 controls, of the fermentation was conducted at varying temperatures, pH, and volumes of inoculum.The process parameters (input variables): volumes of inoculum, temperature,and pH were subjected to response surface model, using the Central Composite Design (CCD).&#x0D; Place and Duration of Study: This study was carried out in the Environmental Microbiology Laboratory, University of Port Harcourt for six months.&#x0D; Methodology: Fermentation was done in conical flasks covered with cotton wool and foil in a stationary incubator for four days (96 hours). Active stock culture of Saccharomyces cerevisiae was used, with inoculum developed using Marcfaland’s method. Samples were collected every 24 hours, centrifuged, filtered and analyzed for measurement of the output variables: Reducing sugar, cell density and ethanol concentration.&#x0D; Results: The concentration of reducing sugars from Calabash pulp was 3.2 mg/ml. Results obtained also revealed that the fermentation can take place on a wide range of temperature 25-40°C. The optimal pH range for performance of S. cerevisiae for the fermentation process was pH 5.0-6.5. The optimum volume of inoculum was 5.5%v/v (ie 5.5 ml in 94.5ml juice). The optimized process using the RSM model gave 6.19% v/v bioethanol.&#x0D; Control: The bioethanol yield from Calabash substrate is reasonable considering the concentration of reducing sugars obtained from the juice and the duration of the fermentation.

Feasibilitiy Study of Bioethanol Production: A Case of Spent Mushroom and Waste Paper as Potential Substrate

Aims: The aim of the present study is to produce ethanol from waste paper and spent mushroom using Saccharomyces cerevisiae as enzymes for the fermentation process.&#x0D; Study Design: Waste paper and spent mushroom samples were subjected to fermentation and hydrolysis by Aspergilus niger and Saccharomyces cerevisiae to produce bioethanol.&#x0D; Place and Duration of Study: This study was carried out in the Environmental Microbiology Laboratory, University of Port Harcourt, Nigeria, between May and October 2017.&#x0D; Methodology: Waste paper and spent mushrooms samples were hydrolyzed by Aspergillus niger, and the hydrolysate from each set up subjected to fermentation by Saccharomyces cerevisiae. Ethanol was extracted by fractional distillation, and qualitatively determined by Gas Chromatography with Mass Spectrometry.&#x0D; Results: After 8 days of fermentation, there was decrease in glucose content in waste paper hydrolysate ranging from (0.51-0.1 mg/l), and spent mushroom substrate (0.3-0.07 mg/l). Upon extraction of the bioethanol, the highest yield was recorded for waste paper hydrolysate which after characterization with GC-MS ethanol concentration was 28.01 mg/l, followed by spent mushroom hydrolysate 26.8 mg/l.&#x0D; Conclusion: This study revealed that bioethanol can be obtained from fermentation of waste paper using Saccharomyces cerevisiae and ethanol can be obtained after the paper has been used in growing edible mushroom; if adopted, this could be a way to achieving environmental sustainability.

Effectiveness of Single and Microbial Consortium of Locally Isolated Beneficial Microorganisms (LIBeM) in Bioaugmentation of Oil Sludge Contaminated Soil at Different Concentration Levels: A Laboratory Scale

An aerated static pile ASP-bioreactor system made up of acrylic material dimension (60 cm × 40 cm × 20 cm) was developed to examine the potential of single and microbial consortium of LIBeM to remediate oil sludge contaminated soil at different concentration levels. Three different strains of LIBeM namely P. aeruginosa-BAS-Cr1, S. paucimobilis-ReTOS-Cr1 and S. maltophilia-RAS-Cr1 were used in this study was obtained from Environmental Microbiology Laboratory, Universiti Malaysia Sabah. Five sets of experiment filled with 10 kg of soils contaminated with 5% and 10% of oil sludge were carried out as Treatment 1 (contaminated soil+P. aeruginosa-BAS-Cr1), Treatment 2 (contaminated soil+S. paucimobilis-ReTOS-Cr1), Treatment 3 (contaminated soil+S. maltophilia-RASCr1), Treatment 4 (contaminated soil+Microbial Consortium; P. aeruginosa-BAS-Cr1+S. paucimobilis-ReTOSCr1+ S. maltophilia-RAS-Cr1) and Treatment 5 (contaminated soil+indigenous bacteria in soil; NA). Their ability to degrade hydrocarbon in the soil was investigated during 60 days incubation periods. Physical and chemical analyses were carried out from each of the treatment and control plot on a weekly basis to check for pH, moisture content, temperatures and Total Petroleum Hydrocarbon (TPH). The results showed that single strain P. aeruginosa- BAS-Cr1 has the highest oil degrading capacity compared to microbial consortium with 80% and 85.2% at both concentration studied. The percentage of TPH removal by P. aeruginosa-BAS-Cr1 is 3-fold higher than NA, thus confirmed that the addition of oil selective degrading bacteria was much better than the control plot. High degradation of long chain alkanes were observed between the control and treatment plot suggested that bioaugmentation using single and microbial consortium had decrease the level of oil sludge in contaminated soil.

First Report of Powdery Mildew Caused by Erysiphe quercicola on Quercus mongolica in Korea

Mongolian oak (Quercus mongolica Fisch. Ex Ledeb), belonging to family Fagaceae, is mainly distributed in Asian countries including China, Japan, Korea, and Mongolia. From August to November 2016, a powdery mildew was observed on Q. mongolica on the Chilgap mountain located in Chungcheong nam-do and in a garden of the Chonnam National University, Gwangju, Korea. Diseased leaves showed irregular necrotic lesions on both sides. Two voucher specimens were deposited at the Environmental Microbiology Laboratory Fungarium, Chonnam National University, Gwangju, Korea, (EML-QMPW1, EML-QMPW2). Conidiophores were erect, 55.5–108.5 μm long and composed of 1 to 2 cells. Foot-cells of conidiophores were cylindrical. Conidia were formed singly, obovoid to ellipsoid, ends subtruncate, and measured 28.0–38.5 μm × 15.5–23.0 μm with a length/width ratio of 1.5 to 2.2. The morphological characteristics were consistent with the description of Erysiphe quercicola (Takamatsu et al. 2007; Lee et al. 2011). To confirm the morpho...

Environmental Soil Microbiology: A Novel Research-Oriented Laboratory Course for Undergraduate Students

Laboratory courses have a central and distinctive role in sci- ence education. The need of improving laboratory curricular materials in environmental soil microbiology education is a must more than ever as due to the followings: (i) emerg- ing of new types of soil biological problems associated with new manipulated environments, (ii) the intimacy of human, plants, and animals to soil microbial activities, and (iii) en- vironmental soil-connected issues is the area of current re- search interests. We present a novel research-oriented labo- ratory course for undergraduates in soil science, according to the need of reforming the laboratory curriculum as called by the National Science Education Standards to advocate in- quiry and cognitivity in teaching and learning. Our inquiry- based environmental soil microbiology laboratory course provides a good pedagogical opportunity in promoting criti- cal thinking, making predictions, proposing causative factors, and presenting consistent arguments to support a position through effective scientific writing. The novelty of our labora- tory curriculum relies on the integration of three main com- ponents namely the “skill learning”, “research experience”, and “effective writing skills” which ultimately may help in optimizing students thinking performance towards research oriented mindset. The course is divided into two modules (I&#x0D; &amp; II), where in module I students are introduced to the basic principles and techniques in soil microbiology. As for module II, students are assigned a research project to enhance their critical thinking and develop their conceptual skills in design- ing an experiment, problem solving, gathering and analy- sis of data, and scientific writing. The unit content can be modified to suit other specific laboratory curriculums in other branches of natural science without loss of students learning efficiency or impact.

First Report of Powdery Mildew Caused by Erysiphe alphitoides on Euonymus japonica in Korea

HomePlant DiseaseVol. 101, No. 2First Report of Powdery Mildew Caused by Erysiphe alphitoides on Euonymus japonica in Korea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Powdery Mildew Caused by Erysiphe alphitoides on Euonymus japonica in KoreaH. B. Lee and T. T. T. NguyenH. B. LeeSearch for more papers by this author and T. T. T. NguyenSearch for more papers by this authorAffiliationsAuthors and Affiliations H. B. Lee T. T. T. Nguyen , Division of Food Technology, Biotechnology & Agrochemistry, College of Agriculture & Life Sciences, Chonnam National University, Gwangju 61186, Korea. Published Online:16 Nov 2016https://doi.org/10.1094/PDIS-08-16-1128-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Euonymus japonica, family Celastraceae, is an evergreen shrub native to Korea, China, and Japan that is often used as an ornamental hedge plant. In October 2014, powdery mildew symptoms and signs were observed amphigenously on the leaves of E. japonica in a park in Seoul, Korea. Also, in June 2016, the same symptoms were found on E. japonica plants in a garden in Gwangju (35°10′N, 126°55′E), Korea, showing higher incidence and severity. Diseased leaves showed white colonies, associated with brown spots on the surface due to the presence of a hyperparasite, Ampelomyces quisqualis, and heavily infected young leaves. Voucher specimens were deposited at Environmental Microbiology Laboratory Fungarium, Chonnam National University, Gwangju, Korea, as EML-EJP1, EML-EJP2, and EML-EJP3. Appressoria of the fungus were lobed, and solitary or in opposite pairs. Measurements were made of 20 conidiophores and 50 conidia. Conidiophores were cylindrical, 45.0 to 77.0 × 6.5 to 10.5 μm. Foot cells of the conidiophores were straight to substraight to cylindrical, and followed by 1 to 3 shorter cells. Conidia were formed singly, cylindrical or ellipsoid-cylindrical, and measured 26.5 (–36.5) to 45 × 12 to 18.5 μm with a L/W ratio of 1.8 to 3.3. Short to moderately long germ tubes were produced terminating in lobed appressoria. The teleomorph was not found. Morphological characters were consistent with anamorphic Erysiphe species. To identify the fungus at the species level, mycelia and conidia of four samples from Euonymus japonica were collected with a sterile needle under a stereomicroscope. rDNA was extracted from mycelia and conidia using the Higene Genomic DNA Prep Kit for fungi (Biopact Corp., Korea). The internal transcribed spacers (ITS1 and ITS2) and 5.8S gene was amplified using primers ITS1F (5′-CTTGGTCATTTAGAGGAAGT-3′) and LR5F (5′-GCTATCCTGAGGGAAAC-3′) as by Lee (2013). A BLASTn search of the rDNA ITS sequences in GenBank indicated that EML-EJP1 (KP325487), EJP2 (KX495611), and EJP3 (KX495610) matched 100% (608/608 bp), 99.6% (766/769 bp), and 99.8% (497/498 bp) with Erysiphe alphitoides (Griffon & Maubl.) Braun & Takam (EF672350, AB257430, and AB292700, respectively). Phylogenetic analysis showed that our strains, EML-EJP1, EJP2, and EJP3, were placed within Alphitoides clade comprising E. alphitoides, E. akebiae, E. menispermi var. dahurica, E. wallrothii, E. sinomenii, and E. euonymicola. Interestingly, when the ITS sequences of our strain and some E. alphitoides were compared with E. euonymicola as a subclade within the Alphitoides clade, there was one base difference between them. The morphological characteristics of our strain completely matched E. alphitoides described by Takamatsu et al. (2007), except for a slight difference in width. The pathogenicity was confirmed by gently pressing the infected leaves onto five leaves of 5-year-old E. japonica plants. Five leaves were inoculated on each plant. The inoculated and noninoculated plants were maintained at 23 ± 2°C with a 12-h photoperiod and 80 to 85% RH. Ten days after inoculation, the symptoms were similar to those observed in the original diseased plants and the fungus on inoculated leaves was morphologically identical to that detected on their leaves, fulfilling Koch’s postulates. No disease developed on the controls. So far, E. euonymicola (formerly Oidium euonymi-japonici) has been found in only Euonymus spp. (Farr and Rossman 2016). In addition, Quercus spp. has been reported as the host of E. alphitoides. Recently, Sorbaria sorbifolia, Wisteria brachybotrys, and W. frutescens have been determined as additional hosts for E. alphitoides (Denton et al. 2016). This information indicates that E. alphitoides is able to infect a wide range of hosts and may pose a threat to multiple woody ornamental plant species. Further studies on the host and geographical ranges of E. alphitoides species are needed. To our knowledge, this is the first report of powdery mildew caused by E. alphitoides on E. japonica in Korea and in the world.

LeBIBIQBPP: a set of databases and a webtool for automatic phylogenetic analysis of prokaryotic sequences.

BackgroundEstimating the phylogenetic position of bacterial and archaeal organisms by genetic sequence comparisons is considered as the gold-standard in taxonomy. This is also a way to identify the species of origin of the sequence. The quality of the reference database used in such analyses is crucial: the database must reflect the up-to-date bacterial nomenclature and accurately indicate the species of origin of its sequences.DescriptionleBIBIQBPP is a web tool taking as input a series of nucleotide sequences belonging to one of a set of reference markers (e.g., SSU rRNA, rpoB, groEL2) and automatically retrieving closely related sequences, aligning them, and performing phylogenetic reconstruction using an approximate maximum likelihood approach. The system returns a set of quality parameters and, if possible, a suggested taxonomic assigment for the input sequences. The reference databases are extracted from GenBank and present four degrees of stringency, from the “superstringent” degree (one type strain per species) to the loosely parsed degree (“lax” database). A set of one hundred to more than a thousand sequences may be analyzed at a time. The speed of the process has been optimized through careful hardware selection and database design.ConclusionleBIBIQBPP is a powerful tool helping biologists to position bacterial or archaeal sequence commonly used markers in a phylogeny. It is a diagnostic tool for clinical, industrial and environmental microbiology laboratory, as well as an exploratory tool for more specialized laboratories. Its main advantages, relatively to comparable systems are: i) the use of a broad set of databases covering diverse markers with various degrees of stringency; ii) the use of an approximate Maximum Likelihood approach for phylogenetic reconstruction; iii) a speed compatible with on-line usage; and iv) providing fully documented results to help the user in decision making.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0692-z) contains supplementary material, which is available to authorized users.

Biodegradation of Total Petroleum Hydrocarbon by Aerobic Heterotrophic Bacteria Isolated from Crude Oil Contaminated Brackish Waters of Bodo Creek

Bodo creek is located in Gokana Local Government Area of Rivers state and is characterised by brackish water which is heavily contaminated with crude oil. Water samples collected from the creek were taken to Environmental Microbiology Laboratory of University of Portharcourt for isolation of aerobic heterotrophic bacteria using serial dilution spread plate technique. Isolation of morphologically distinct pure cultures was done and the isolates obtained were identified molecularly on the basis of 16S rRNA gene sequence analysis. 16S rRNA sequencing for identification of the isolates generated sequences ranging from 500 bp to 1700 bp and a 250 bp size PCR amplified fragment. The nucleotide sequence of the 16S rRNA gene was compared with published 16S rRNA sequences using BLAST search at the data base of National Centre for Biotechnology Information (NCBI). Biodegradation of petroleum hydrocarbon test on the isolates was conducted using Bony light crude oil obtained from Shell Petroleum Development Company and monitored using GC-FID (Agilent 6890 model) for 49 days. The initial quantity of TPH was 24091 and 6706 mg/l on day 0 for aerobic heterotrophic treatment and crude oil contaminated control and decreased progressively to 212.8 and 1174 mg/l respectively on day 49 indicating biodegradation in the treatment and control. Loss of TPH was statistically significant using one way analysis of variance (ANOVA) p<0.05 with time. Model for predicting TPH loss was developed and rate of biodegradation of the isolates determined using Trend line method of Microsoft Excel, 2010. The growth of bacteria cells increased progressively with decrease in TPH implying that the bacteria were responsible for biodegradation. Further application of bioremediation strategies in Bodo creek for biostimulation of the crude oil biodegrading bacteria for reclamation and restoration of an efficient ecosystem structure and function is suggested.

Molecular Characterization of Aerobic Heterohophic Bacteria Isolated from Petroleum Hydrocarbon Polluted Brackish Waters of Bodo Creeks, Rivers State Nigeria

Surface water sources in the oil producing Niger Delta region of Nigeria are highly susceptible to pollution by petroleum hydrocarbons and so it is important to understand the microbial diversity of such ecosystems. Water and sediment samples were collected between April-August, 2013 from Bodo creeks and taken to Environmental Microbiology laboratory of University of Portharcourt for analysis. A total of thirty aerobic heterotrophic bacterial strains isolated ranged from 3.0 - 7.0 × 104 cfu for surface water and 1.6 - 5.6 × 104 cfu for sediment samples of Bodo creek using serial dilution and spread plate technique. Pure cultures of bacteria were obtained on the basis of their morphological characteristics and subjected to biochemical tests and further classified on the basis of 16S rRNA gene sequence analysis. The DNA was isolated from size fractionated samples and the diversity of bacteria in each fraction was studied using PCR amplification of partial 16S rRNA. The sequences were submitted to NCBIGen bank for identification and assigning of accession numbers. The isolated aerobic heterotrophic bacteria belong to the families of Enterobacteriaceae, Bacilliceae, Alcaligenaceae, Pseudomonadaceae, Flavobactericeae and Planococcaceae.