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The Effects of Trehalose on the Bioluminescence and Pigmentation of the Phase I Variant of Photorhabdus luminescens

Journal of Life Sciences 2012年第2期6卷 119-129页

作者： Floyd L. Inman III leonard D. holmes Sartorius-Stedim Biotechnology Laboratory Biotechnology Research and Training Center University of North Carolina PembrokeNC 28372 USA

Photorhabdus luminescens is a Gram-negative, bioluminescent, pigment producing enteric bacterium, which is pathogenic to insects and has the capability to undergo phase variation. The phase I variant of P. luminescens exists as a mutualistic symbiont where it plays a critical role in the life-cycle of the soil-dwelling nematode, Heterorhabditis bacteriophora. Both the bacterium and the nematode receive their nutritional requirements from the bioconversion of the insect host which is rich in many macromolecules such as the disaccharide, trehalose. Trehalose is a non-reducing disaccharide of glucose that is formed by an a-1,1-glycosidic bond and is associated with the physiology of many bacteria, insects and nematodes. Trehalose has been shown to be the most abundant storage sugar found within insect hemolymph （1%-2%）. The physicochemical properties of trehalose allow this carbohydrate to act as a stress protectant where it has been implicated with thermal stress, dehydration, and osmotic protection of many microorganisms. Due to these properties, trehalose may allow culture stability of the phase I variant in vitro and in vivo. Traits of the phase I variant that were studied in this work include bioluminescence and the production of the red anthroquinone-derived pigment. The carbohydrates that were utilized in this study were glucose and trehalose; where shake flask cultures of the phase I variant were cultured at room temperature for up to six days in carbohydrate supplemented basal media with increasing carbohydrate concentrations of 0. 1%, 0.5% and 1.0% （v/v）. Relative luminosity, pigmentation and pH were graphed as a function of time, carbohydrate used, and carbohydrate concentration. Data obtained from this study suggests that the supplementation of 1.0% trehalose, when culturing the phase I variant ofP. luminescens, can maintain bioluminosity and pigmentation over extended periods of time （five days） as compared to basal media and basal media supplemented with

关键词： Trehalose Photorhabdus luminescens pigmentation bioluminescence Heterorhabditis bacteriophora.

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Remote Control of Fed-Batch Fermentation Systems

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Journal of Chemistry and Chemical Engineering 2011年第10期5卷 897-902页

作者： Eric Moreau Floyd Inman, III Sunita Singh Heather Walters leonard holmes Biotechnology Research and Training Center University of North Carolina at Pembroke Pembroke NC USA Central Institute of Agricultural Engineering Bhopal Madhya Pradesh India Universite de Picardie Jules Verne Amiens France

Bioreactor operation requires continuous monitoring of fermentation parameters and real-time control over bioreactor devices. Remote monitoring and control of the bioreactor＇s computer via the Internet avoids the necessity of personnel being continually onsite during operation. A two liter Sartorius-stedim Biostat~ A Plus fermentation system was networked and interfaced with the commercial software from GoToMyPC to allow remote control of the fermentation system utilizing the internet. The fermentation vessel was equipped with hardware calibrated for monitoring and controlling culture parameters during experimentations. The uniform resource locator controlled night-vision web camera allowed continuous monitoring of the glass fermentation vessel during the day and at night. The main window screen of the laboratory computer can be securely accessed from any portable device （i.e. laptop） capable of establishing an Internet connection and executing the commercial software from GoToMyPC. The secured internet protocol address, provided by GoToMyPC, assures that the system can only be controlled by authorized users who have been given access to the account. This interface permits the remote control of the Biostat A Plus fermentation system and possibly other automated or potentially automated culturing systems at the convenience of the user（s）.

关键词： Fermentation Fed-batch Remote-monitoring Remote-controlled fermentation systems

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Bioluminescence and Chitinase Production during Chitin Fermentation by Vibrio harveyi

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Journal of Life Sciences 2013年第5期7卷 491-494页

作者： Badireddy Madhusudana Rao Floyd L. Inman Ⅲ leonard D. holmes Sartorius Stedim Biotechnology Laboratory Biotechnology Research and Training Center University of North Carolina NorthCarolina 28472 USA Central Institute of Fisheries Technology Visakhapatnam Research Centre Andhra University Visakhapatnam 530003 India

Vibrio harveyi, like other luminescent bacteria, is capable of producing extracellular chitinases. Microbial chitinases are utilized to depolymerize chitin into chitooligosaccharides and N-acetylglucosamine for the acquisition of carbon and possibly nitrogen, needed for survival. For many luminous marine bacteria （Vibrio spp.）, quorum-sensing is highly speculated to be responsible for bioluminescence; however, in terrestrial species （Photorhabdus spp.） luminosity seems to be controlled through unknown mechanism of phase variation. In the present work, the correlation between bacterial luminosity and chitinase production of F. harveyi was studied. The utilization of bioluminescence could prove to be an easier and more convenient method to monitor chitin fermentations that employ luminous bacteria. Results from the fermentation study indicate that luminosity of F. harveyi inversely correlates with chitinase production. In other words, during chitin fermentation, chitinase production was seen to increase while luminosity decreased with respect to growth and growth conditions. Furthermore, the results also suggest that V. harveyi may utilize an alternate mechanism that can counter quorum-sensing mechanisms to ensure bacterial survival under deteriorating growth conditions. The inverse relationship observed in this study may lead to a basic understanding of monitoring and studying chitin fermentations and anti-quorum-sensing/phase variation mechanisms exhibited by luminous bacteria.

关键词： Vibrio harveyi bioluminescence chitinase chitin fermentation.

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A Novel Method for Determining Microbial Kinetics

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Journal of Life Sciences 2013年第8期7卷 787-790页

作者： Anne M. Talkington Floyd L. Inman III leonard D. holmes Sartorius Stedim-Biotechnology Laboratory Biotechnology Research and Training Center University of North Carolina atPembroke Pembroke North Carolina 28372 USA

Understanding microbial growth is essential to any research conducted in the fields of microbiology and biotechnology. Current methods of determining growth characteristics of microbes involve subjective graphical interpretations of linearized logarithmic data. Reducing error in logistical data decreases disparity between graphical and analytical predictions of microbial characteristics. In this study, a method has been developed to calculate the kinetics of microbial characteristics utilizing a modified Maclaurin series. Convergence of this series approaches the true kinetic value of microbial characteristics to include specific growth rates. In this research, a modified Maclaurin series is used to evaluate microbial kinetics in comparison to graphical determinations.

关键词： Microbial kinetics Maclaurin series microbial growth models series convergence.

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Selected Plant Extracts Show Antiviral Effects against Murine Norovirus Surrogate

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Advances in Microbiology 2019年第4期9卷 372-384页

作者： Uchenna Iloghalu Bryce holmes Janak Khatiwada leonard L. Williams North Carolina A&T State University Greensboro USA Center for Excellence in Post-Harvest Technologies Kannapolis USA

Noroviruses are the most common cause of acute gastroenteritis. Annually, 21 million Americans are infected with norovirus. Recent advances in molecular diagnostics have helped to establish norovirus as the most common cause of outbreaks of acute gastroenteritis across all ages. However, there is no effective or efficient treatment/control against norovirus infection. Conventional intervention techniques used to inactivate norovirus have shown lack of efficacy against human norovirus. Currently, effective treatment or control measures against human norovirus have not been identified. In this study, murine norovirus acts as a model to human norovirus to evaluate the inhibitory effects of crude extracts of Zanthoxylum armatum and Hibiscus sabdariffa. The study also separated, identified and quantified the selected compounds using the ultra-liquid chromatography (UPLC). To study the antiviral activities of crude extracts and its fractionated portions of Z. armatum and H. sabdariffa against norovirus surrogate, RAW 264.7 cells were infected with Murine norovirus surrogate virus of human norovirus and incubated at 37?C. Phytochemicals were extracted from the seeds and calyces of the plants using methanolic extraction. Fractionated portions of the crude extracts were subsequently used in both chromatographic and microbiological studies. Our data indicated that there was reduction of viruses, when treated with the 60% aqueous methanol extracts. Amongst the four selected phenolic compounds (myricetin, quercetin, kaempferol and luteolin), quercetin showed the most significant logarithmic viral reductions. These compounds were identified, purified and quantified using UPLC. Extracts of Zanthoxylum armatum and Hibiscus sabdariffa showed antiviral effects. Phenolic compounds are virucidal. Extracts of Hibiscus sabdariffa also exhibits anti-norovirus activities. The results are anticipated to control/prevent the human norovirus infections.

关键词： Plant Extracts Fractionation Noroviruses Plaque Assay

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