Grantee Research Project Results
2013 Progress Report: Producing and Characterizing Bacillus Subtilis Biosurfactants with Potentially Lower Environmental Impact for Salt Water Applications
EPA Grant Number: R835181Title: Producing and Characterizing Bacillus Subtilis Biosurfactants with Potentially Lower Environmental Impact for Salt Water Applications
Investigators: Lamsal, Buddhi , Somasundaran, P. , Nyman, John A. , Green, Christopher C. , LeBlanc, Brian D.
Institution: Iowa State University , Columbia University in the City of New York , Louisiana State University - Baton Rouge
EPA Project Officer: Aja, Hayley
Project Period: April 1, 2012 through March 31, 2015 (Extended to March 31, 2016)
Project Period Covered by this Report: April 1, 2013 through March 31,2014
Project Amount: $500,000
RFA: Environmental Impact and Mitigation of Oil Spills (2011) RFA Text | Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Aquatic Ecosystems
Objective:
Specific Objective #1: Evaluate fermentation and purification strategies to produce effective and low-toxicity biosurfactants, their isoforms, and elucidate their structure: This objective entails optimizing fermentation conditions by utilizing different energy sources for Bacillus subtillis, such as fibrous feedstocks like switchgrass, bagasse, etc. We hypothesize that different fermentation strategies and operational parameters can affect isoforms of biosurfactant produced resulting in different surface properties.
Specific Objective #2: Evaluate dispersant activity of biosurfactants and their isoforms: This objective entails studying the structural aspects of biosurfactants and their isoforms produced by Bacillus subtillis (surfactin and fatty acyl glutamate (Fa-Glu)) to understand the structure-property relationships of various Fa-Glu isoforms and developing formulations that potentially could have applications in oil-spill cleanup.
Specific Objective #3: Evaluate environmental effects of biosurfactants and their isoforms: This objective involves analysis of toxicity of biosurfactants produced by Bacillus subtillis (Fa-Glu and surfactin) on small fish (Fundulus grandis) common to estuaries on the northern Gulf of Mexico.
Progress Summary:
Specific Objective #1:
Accomplished: Pretreatment conditions were optimized for six fibrous feedstocks and bacterial growth was analyzed for media containing hyrdolysates from fibrous biomasses compared to glucose based media. Three 15-L surfactin fermentations were conducted on glucose-based S-7 media for isoform fractionation experiments.
1.1. Optimization of pretreatment
- For this objective, a pretreatment study on six different fibrous biomasses for utilization of cellulosic and lignocellulosic carbohydrates, as carbon source for the growth of biosurfactants, produced by Bacillus subtillis was conducted.
- Six fibrous feedstocks including, (a) switchgrass, (b) alfalfa, (c) soy hulls, (d) soy fiber, (e) distillers dry grain solubles (DDGS), and (f) bagasse were pretreated with two different pretreatment techniques: (i) liquid ammonia pretreatment (ii) ultrasonication at varying combinations of concentration, time, temperature and amplitude.
- Post- pretreatment enzymatic hydrolysis for all pretreatment combinations biomass samples was conducted to determine the optimum pretreatment conditions, which were found to be 60 C, 12 hr, for switchgrass, alfalfa, soy hulls and bagasse and 3 min, 100% amplitude ultrasonication for soy fiber and DDGS.
1.2. Bacterial growth on fibrous biomass hydrolysates
- After determination of best pretreatment conditions, pretreatments were done on 30g (dry weight) of all six fibrous biomasses to produce sufficient carbohydrate from each feedstock to substitute glucose as a carbon source at 2% concentration in 50 mL S-7 fermentation media for production of FA-Glu and surfactin and fermentation growth as a function of absorbance at 650 nm was analyzed for all feedstocks. (Fig. 1a, 1b) This study on pretreatment and utilization of carbohydrates from fibrous biomass has shown that biosurfactant producing bacterial strain can grow better or equally well compared to glucose based media which provided a new direction towards a potentially cheaper and environmentally sustainable energy source for biosurfactant production as utilization of fibrous biomass adds value to the sustainable bio-refinery models.
1.3. Large-scale surfactin fermentation for isoform fractionation
- We conducted three, 15-L, fermentations for Bacillus subtillis on glucose based media to produce surfactin in a larger quantity for isoform separation and structure function characterization for toxicity profile and dispersion activity. We successfully separated surfactin isoforms on the analytical HPLC scale and have been working on large scale preparatory HPLC to fractionate pure isoforms for comparative studies of composite and individual isoform properties. Completion of this part of the project will provide us strong data to analyze the structure and property of individual molecules (isoforms) and will provide a clear picture for dosage, activity and function of FA-Glu and surfactin.
Specific Objective #2:
Accomplished: Critical micelle concentration of FA-Glu was determined.
Critical micelle concentration (CMC) of Fa-Glu was determined using Wilhelmy vertical plate technique with a sandblasted platinum plate as the sensor at 25±1°C. The surface tension was measured at using the Cahn microbalance kept in a draft-free enclosure. The CMC was obtained from the intersection of the sloped and plateau portions of the surface tension vs. concentration curve. The slope of the curve was used to calculate the adsorption density at the interface with the Gibbs equation. The CMC of FA-Glu in double de-ionized distilled water (DDDW) was ~0.35 g/L which is higher than one of the earlier batches received (0.1g/L) (Fig. 2).
Specific Objective #3:
Accomplished: Toxicity tests for unfractionated FA-Glu was conducted on Gulf killfish.
Unfractionated samples of FA-Glu were utilized to artificially spawn sexually mature Gulf killifish (Fundulus grandis) within October and November in order to provide 8-day old larvae for dose-response acute bioassays at salinities between 10 and 13 g/L. Three 96-hr acute toxicity bioassays have been performed on FA-Glu to date. The results of these assays indicate a 48-hr median lethal concentration of 114.5 mg/L for FA-Glu with upper and lower 95% confidence intervals of 87.03 mg/L and 156.72 mg/L, respectively.
Fig 1a) Growth of Surfactin producing Bacillus subtillis on glucose and pretreated hydrolysates of
switchgrass (SW), alfalfa (AA), Soy hulls (SH), BG (Bagasse).
Fig 1b) Growth of FA-GLu producing Bacillus subtillis on glucose and pretreated hydrolysates of switchgrass (SW), alfalfa (AA),
Soy hulls (SH), BG (Bagasse)
Fig 2). Critcal micelle concentration (CMC) of FA-GLu in double distilled deionized
water. The solution was maintained at around pH 7.
Outreach Activity:
Accomplished: Social media pages have been set up to collect feedback from general public regarding developing safer or more eco-friendly alternatives to oil spill cleanup and control
Evaluation of peer institution programs related to extension education and outreach on principles of toxicology are being reviewed by graduate students to determine successful programs and curricula elements that will effectively demonstrate the results of this research and broader concepts of environmental toxicology.
The outreach component to date has involved the following actions.
- A summary of the intended research and outreach project has been written and posted on the Louisiana Sea Grant website under the link Oil Spill Dispersant Research. (http://www.laseagrant.org/)
- Work performed in Dr. Greens laboratory at the LSU Agricultural Centers Aquaculture Research Station was highlighted in a press release and local NPR story describing goals of biosurfactants.
- Seven Sea Grant Marine Extension agents in costal Louisiana periodically discuss and link the article(s) on their individual Marine Extension social media pages (Facebook and Twitter).
- A draft publication entitled Principles of Toxicology has been developed by graduate student of Dr. Chris Green.
Outcomes:
Comments on social media pages from citizens in coastal communities have been overwhelmingly positive about attempts to develop safer or more eco-friendly alternatives to oil spill cleanup and control. Although no spills are preferred, the public is concerned that many current oil-spill clean-up products are only slightly better than the spill effects alone. The also express concerns about the overall toxicity of the ecosystems they live and work in.
Future Activities:
Specific Objective # 1
- With the preparatory scale fractionation of surfactin and Fa-Glu isoforms, more comprehensive analysis of structure and function of surfactin and Fa-Glu would be conducted that will include, toxicity profiles and dispersion activity of composite and individual isoforms of surfactin and Fa-Glu.
- Media modification of for enhanced growth and higher yield of FA-Glu and surfactin producing strains will be conducted.
Specific Objective # 2
- Surface-active property, for example CMC, of FA-Glu will be evaluated under saline conditions, with conditions typically representing that of sea conditions. Similarly, CMC of surfactin will also be determined both in DDDW and under saline conditions. The Dispersant-to-Oil Ratio (DOR) of these surfactants will be determined with hexane as model oil. The CMC values for mixtures having both surfactin and Fa-Glu will also be studied. The emphasis of this study - mixed system will be to elucidated and synergistic interactions between the two surfactants. Properties related to performance will also include emulsion stability and packing of surfactants at the oil water interface. Packing of surfactants will be studied using Electro Spin resonance spectroscopy and fluorescence spectroscopic techniques.
Specific Objective # 3
- The future activities planned pertaining to this objective are to continue to generate 48 and 96-hr lethal concentration for 50% survival (LC50) values from acute toxicity bioassays from materials created at cooperative institutions. The principle organism of study remains the Gulf killifish (Fundulus grandis) and we will still be using 8-10 day old larvae. Specifically, assays will be conducted at numerous salinities as we believe that salinity plays a critical role in the toxicity of these biosurfactants due to their ability to alter ion regulation at the gill epithelium. To this end, we also plan to investigate the physiological role of these biosurfactants and reference surfactants to alter osmo and ion-regulation. Our ability to conduct these investigations are contingent upon receipt of fractionated samples from our cooperative institutions; therefore, delays in the production and fractionation of these biosurfactants will correspondingly delay our testing and the development of these dose-response curves. Bioassays using unfractionated surfactin with appropriately aged larvae will be conducted between December 22 and 30. To date, we have not had to alter our methods or research approach for this project.
Journal Articles:
No journal articles submitted with this report: View all 5 publications for this projectSupplemental Keywords:
Biosurfactants, surfactin, Bacillus subtillis, isoforms, oil spill, dispersant, critical micelle concentrationRelevant Websites:
Louisiana Sea Grant Exit
Facebook: Alan Matherne Exit
Facebook: Thomas Hymel Exit
Facebook: Thu Bui Exit
Facebook: Mark Shirley Exit
Facebook: Julie J Falgout Exit
Facebook: Kevin A. Savoie Exit
Facebook: Twyla Herrington Cheatwood Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.