Exploring the Effects ofExploring the Effects ofAntimicrobial Material on the Antimicrobial Material on the

Process of Biofouling in an Process of Biofouling in an Aquatic EnvironmentAquatic Environment

Ms. Kathleen TunneyBurleigh Manor Middle School, Howard County, MDUMCES~Sea Grant Summer Research Fellow 2002

Mr. J. Adam FrederickMD Sea Grant Extension/ COMBBaltimore, MD

I worked with J. Adam Frederick of the MD Sea Grant Extension Program and Frank Vavra of Maxxam Industries, Baltimore, MD,

as part of the Summer Research Fellowship at the Center of Marine Biotechnology (COMB). The research project concerned the effectiveness of an industrial anti-microbial material on the

prevention of biofouling.

Research Project

Classroom ProjectOne of my tasks during the summer research fellowship was to

develop an application for the classroom based upon my research project. The activity I developed is entitled,

“Culturing Biofilms and Exploring Biodiversity in Classroom Aquaria”.

Research ProjectResearch ProjectMr. J. Adam FrederickMr. J. Adam FrederickMs. Kathleen TunneyMs. Kathleen Tunney

““Exploring the Effects ofExploring the Effects ofAntimicrobial Material on the Antimicrobial Material on the

Process of Biofouling in an Process of Biofouling in an Aquatic EnvironmentAquatic Environment””

Adapted from an educational outreach project by, J. Adam Frederick, D. Adapted from an educational outreach project by, J. Adam Frederick, D. Jacobs, and W.R. Jones, Journal of Industrial Microbiology and Biotechnology Jacobs, and W.R. Jones, Journal of Industrial Microbiology and Biotechnology (2000) 24, 334-338. http://www.nature.com/jim(2000) 24, 334-338. http://www.nature.com/jim

I worked with J. Adam Frederick at the Center of Marine Biotechnology as part of the Summer Research Fellowship in 2002. Mr. Frederick’s work in the Inner Harbor in Baltimore, MD, concerns the development of interactive educational tools on the web related to biofouling and biodiversity.

COMB is situated on Pier 6 of the Baltimore Inner Harbor and provides easy access to the water for the study of biofilms and the process of biofouling.

A biofilm is a “coating or covering on the surface of a living or nonliving substrate. It is composed of organisms like bacteria, protozoa, algae, and invertebrate animals.” (Maryland Sea Grant, Biofilms and Biodiversity lesson, http://www.mdsg.umd.edu/Education/biofilm/index.htm).Biodiversity is the number of different species of organisms in a particular environment and can change over time as conditions change.

The research project during the summer of 2002 involved the study of biofilms occurring on a variety of materials submerged in Baltimore’s Inner Harbor. Biofilm development and biodiversity on these materials were studied for two consecutive 21-day periods: June 27 through July 17 and July 18 through August 7, 2002. Water quality tests and weather conditions were monitored throughout each period.

Experimental Set-up

Test materials were in the form of 10cm diameter discs. The materials were provided by Maxxam Industries in Baltimore, MD, and were manufactured with a patented EPA approved anti-microbial material. The material was either coated on the 10 cm discs or embedded in the discs.

Test materials included control material (no anti-microbial compound), 2% and 4% concentrations of anti-microbial material applied as coatings on discs, and 2% and 4% embedded in the discs. Six discs of identical test material were stacked on a PVC post or rack including controls. Four PVC racks of various test material were hung vertically from a PVC pipe. Four of these racks were suspended from Pier 6 in the brackish water of Baltimore’s Inner Harbor.

Control and Experimental Samples

Collection of Sample Test Material

Racks were removed from the water every Monday and Wednesday over a 21-day period. Two discs of each type (control and experimental) were removed from the racks during each sampling day and taken to the COMB lab for analysis. Collected discs were viewed in the lab for biofilm development and biodiversity with stereomicroscopes and compound microscopes.Racks were returned to the water after discs were removed for sampling. Water quality and weather conditions were recorded each day.

Analysis of Sample Test Material

Discs were observed in the lab for types of species and numbers of individuals. Biofilms from each disc were scraped with a razor blade and weighed for wet biomass and then dried to measure dry biomass for comparison.Biodiversity was calculated using the Maryland Sea Grant’s online worksheet for calculating biodiversity after random sampling procedures were followed for enumerating species on each disc.Go to the following for more reading:http://www.mdsg.umd.edu/Education/biofilm/random.htmhttp://www.mdsg.umd.edu/Education/biofilm/studnt4.htm

Types of Organisms Identified•Whip mud worms•Flatworms•Roundworms•Tubeworms•Limpets•Barnacles•Crustaceans•Clutches of fish eggs•Bryozoans•Hydrozoans•Colonial ciliates•Stentor and other ciliate protists

bryozoans

barnacle

Photos by Kathleen Tunney with Olympus America 35 mm microscope system

In addition to various lab skills developed over the course of the seven-week program, the opportunity existed to photograph microscopic organisms while viewing them on the microscope. This version of 35mm photography was not as easy as it sounds and a greater appreciation for this area of photography occurred as a result. Only one out of ever fifty so photographs taken represented the organisms well. Digital photographs were taken in addition to 35mm pictures.

Microscopy & Photography

stentormussel

bryozoan

Representative Data Set5 7 12 14 19 21

Acrylic Control 0.13 0.03 0.66 0.79 1.59 1.97Blue Control 0.03 0.12 0.84 0.49 1.78 1.08Blue M1 0.08 0.09 0.79 0.7 2.7 1.94Blue M2 0.24 0.07 0.86 0.73 0.81 0.68Gray Control 0.12 0.08 0.86 0.7 1.28 1.14Gray M1 0.05 1.37 0.59 0.2 2.14 2.1Gray M2 0.07 0.09 0.74 0.34 2.31 1.51

Biomass Set 2

0

0.5

1

1.5

2

2.5

3

5 7 12 14 19 21

Time (Days)

Biomass (grams)

Acrylic ControlBlue ControlBlue M1Blue M2Gray ControlGray M1Gray M2

Dry Biomass of Biofouling Samples from Second Three Week Period.

Representative Data SetDry Biomass of Biofouling Samples from Second Three Week

Period

The representative data set on the previous slide illustrates the means of a large amount of raw data. Each data point on the graph for a particular sample is composed of a larger number of smaller samples which were analyzed and reduced to the mean value. The data illustrates that no particular sample had a greater amount of biofouling than another and that all samples followed a similar pattern of development related to total biomass. Additionally, the total amount of biomass was relatively low and resembled samples from the acrylic control discs.

Acrylic controls are known to be highly “attractive” to biofilm forming microorganisms and are used as a standard of comparison (http://www.mdsg.umd.edu/Education/biofilm/baier.htm#2).

Research Project Conclusions

Overall, much more work needs to be done in the field of biofouling prevention since there are few options other than toxic marine coatings available for the shipping industry, research vessels and recreational boating. However, proper licensing is required to use marine coatings.

The work we performed this summer also teaches that there are so many environmental variables that relate to biofouling that the process is very difficult to prevent without using toxics.

Credits

Ms. Kathleen Tunney, Burleigh Manor Middle SchoolDr. Dan Jacobs, Maryland Sea Grant

Mr. Frank Vavra, Maxxam Industries, Baltimore, MDMr. J. Adam Frederick, Maryland Sea Grant

ReferencesAmerican Society for MicrobiologyMicrobial Slime, http://www.microbe.org/experiment/biofilms.asp

Biofilms and Biodiversity web lesson, Maryland Sea Grant, http://www.mdsg.umd.edu/Education/biofilm/

Frederick, J. Adam, D. Jacobs, and W.R. Jones, Biofilms and Biodiversity: an interactive exploration of aquatic microbial biotechnology and ecology. Journal of Industrial Microbiology and Biotechnology (2000) 24, 334-338. http://www.nature.com/jim

Wood, Timothy S., Aquarium Culture of Freshwater Invertebrates, The American Biology Teacher, vol. 58, January 1996.