There is little literature available on subsoil microbial communities because these areas were believed to be mainly devoid of life. Despite this dearth of information, it has been reported that over 30% of soil microbes exist in the subsurface (below 25 cm), where they play an important role in soil formation, ecosystem biogeochemistry, contaminant degradation and maintaining ground water quality (Fierer et al. 2003, Kieft 1999). One aspect not studied is their role in regolith (or saprolite) formation. Saprolite is the layer of loose rock which is resting on the intact bedrock. The heterotrophic aerobic bacteria (HAB) in the soil play an important role in biodegradation (Droycon Bioconcepts 2004), and therefore the enumeration of these organisms provides a good indicator of soil quality (BBC Laboratories 1999). The HAB are typically found in large numbers in microbial communities, and so detecting and quantifying their presence at the bedrock interface zone, where the intact rock is broken down into saprolite, is an important first step in understanding what role microbes play in the weathering of rock. The Rio Icacos watershed in the Luquillo Experimental Forest (LEF) of Puerto Rico exhibits one of the world’s fastest rates of saprolite formation (Buss et al submitted to Geobiology), and so is an ideal model to study this process. INTRODUCTION CONCLUSIONS LITERATURE MICROBIAL ANALYSIS OF HETEROTROPHIC AEROBIC BACTERIAL COMMUNITIES IN SOIL FROM THE LUQUILLO MOUNTAINS, PUERTO RICO by Ashley Fink Mentor: Dr. Mathur ACKNOWLEDGMENTS RESULTS OBJECTIVES RESULTS The primary goal of this study is to determine the role the bacterial community of the LEF plays in granitoid rock degradation. To employ two different methods for the study of HAB in subsoil communities. BBC Laboratories, Inc Functional Groups: A summary guide for the microbiological analysis of soil and compost. Available from: Accessed 2005 May 25. Buss, H.L., Schultz, M.J., Bruns, M.A., Moore, J., Mathur, C.F. and Brantley, S.L Microbial ecology and biogeochemical iron cycling in deep regolith, Luquillo Mountains, Puerto Rico. Submitted to Geobiology. Droycon Bioconcepts, Inc (DBI) Available from: Accessed 2004 June 28. Fierer, N., Schimel, J.P. and Holden, P.A Variations in microbial community composition through two soil depth profiles. Soil Biology and Biochemistry 35: Kieft, T.L Microbial Ecology of the Vadose Zone. Subsurface Microbial Ecology in Bell, C.R., Brylinsky, M. and Johnson-Green, P. (eds.). Microbial Biosystems: New Frontiers: Proceedings of the 8th International Symposium on Microbial Ecology, Halifax, Canada. Department of Geosciences, Penn State University There was a large amount of variation in cell density and morphology of the cells with depth. Cell counts ranged from 4.02x10 2 cfu g -1 to 2.01x10 10 cfu g -1. All HAB-BART™ vials bleached clear within 24 hours indicating that the HAB populations are larger than the test is able to detect. An increase in cell density was seen at the bedrock-saprolite interface. HAB-BART™ METHOD Bacterial Suspension 0.5g soil in 15mL PBS buffer, vortex HAB-BART™ Tester Inoculation Pour suspension into smaller vial containing HAB-selective media and invert for 60 sec to distribute methylene blue dye throughout media Incubation Store at room temperature, out of direct sunlight Observations and Measurements Made daily for four days (Experiment extended to look at biofilms, Bioflim Heights measured in cm) Comparison Time for color change (Time Lag) compared to known values to determine the approximate concentration in sample STANDARD PLATE COUNT METHOD Soil Sample Collection Puerto Rico, Summer 2004 Bacterial Extraction 0.5g soil, PBS buffer, glass beads vortex SPREAD PLATEPOUR PLATE 10 -3,10 -5, 10 -7,10 -8 (upper 3m) 10 -1,10 -2 (below 3m) 10 1 (upper 3m) Observations Observe colonies/growth for 3 weeks, using cell counts to calculate cfu g -1 Plating Plate dilutions of samples onto R2A Agar The increase in overall cell density and iron oxidizing bacteria at the bedrock weathering interface seems to be a bacterial response to the increase in bioavailable iron at this level. The patchy distribution of microbial communities within the soil is most likely due to variations of nutrients, water, pore space, etc. within the soil profile. The correlation between biofilm height and cell density below 2 m suggest that it is more advantageous for microbial communities to form biofilms at this depth. Fig 1. Microbial concentrations determined by Standard Plate Count and HAB-BART™ testers. Fig 2. Photograph of BART™ vials taken on Day 1 of the control, 0.5ft (0.15m), and 8.0ft (2.44m) vials, showing a comparison of the depths. Fig 3. Microbial concentration compared to highest measured biofilm growth. Fig 4. Photograph of BART™ vials taken on Day 7 of 1ft (0.30m), 3.5ft (1.07m) and 11.0ft (3.35m) vials, showing a comparison of the biofilm growths.