A STUDY TO EVALUATE THE BIO‐REMEDIATION EFFECTS OF THE AQUASPHEREPRO BY BIOVERSE, INC IN IRREGATION PONDS Dr. Conrad B. Schmidt, CTO, Bioverse, Inc., Pipestone, Minnesota. Bioverse, Inc., of Pipestone, Minnesota, commissioned a 12‐week bio‐remediation study to evaluate the beneficial effects of treating golf course irrigation ponds with the AquaSpherePRO from Bioverse, Inc. The study was conducted at the Elmwood Golf Course, Sioux Falls, SD. The 27‐ hole public golf course utilizes two irrigation ponds of equal capacity that irrigate separate sections of the course individually. Each pond holds roughly 1.5 million gallons of water with an average depth of six feet. Additionally, the two trial ponds contained a thick accumulated layer of sludge and high levels of both submersible and emerged vegetation. A longitudinal study was conducted for the evaluation of the all‐natural microbial formulations found in the 24/7/30 bio‐degradable AquaSpherePRO water remediation delivery system. The AquaSpherePRO has been used successfully to provide a safe, effective and all‐natural delivery system for improving pond water health through microbial bio‐remediation. The objectives of the study were to monitor water and sludge inoculation from the AquaSpherePRO over a 12‐week period. During this time, weekly samples of surface water, mid‐level water and sludge were collected for validation. The study established monitoring levels of the total number of aerobic bacteria on the pond water’s surface, the total number of anaerobic bacteria present in mid‐level water and the total number of anaerobic bacteria inoculated and contained in the sludge layers (CFU/ml). Testing methodology and sample testing was completed by Rural Technology, Inc. of Brookings, SD. A site assessment of the two ponds was also done to clinically evaluate the trial ponds as to overall water quality and esthetics during the 12‐week test. The trial was initiated on July 14th, 2009 after the two test ponds reached a mid‐day temperature of 20 degrees Celsius for one week. This temperature allowed both ponds to develop similar biodiversity patterns of microbial populations and water conditions. Each of the two test ponds served separate fairways and greens on the golf course. The treatments (control or treated) were selected randomly. The treatment (test) pond received a dosage that is recommended for an irrigation pond which is one 500,000 gallon, AquaSpherePRO / 250,000 gallons of irrigation capacity. All spheres were placed evenly throughout the sludge base of the test pond. The AquaSpherePRO spheres were replaced on August 12th, 2009 and September 9th, 2009. A total of six spheres were added to the test pond every 30 days. No spheres or other pond additives and/or products were added to the control pond during the study period. The trial concluded on September 30th, 2009. Trial Responses: pH: There was very little change in the pH levels during the study of the two ponds. The pH ranged between 7.5 and 8 for both ponds. Conclusion: Pond water pH levels between 7.5 and 8 are appropriate for Bioverse formulated water bio‐
remediation products. Surface Water Anaerobes: Control pond: For the control pond, there was an increase in the CFU/ml in Week 3 (9,000 CFU/ml). This declined the following week (Week 4). From Weeks 5 to 8, there was a gradual increase in the aerobic population which peaked in Week 8, followed by a sharp decline in Week 9, another increase in Week 10, and followed by another decline in Week 11. Treatment Pond: During the first four weeks, the treated pond maintained a constant level of surface water aerobic concentration. On Week 4, the pond received a second set of treatment spheres. The treated pond surface aerobes increased gradually and steadily, peaking at Week 7 at 11,000 CFU/ml. The third sphere treatment was provided on Week 8. During Week 9, the levels increased again and gradually fell through Week 12 as the water temperature continued to decline due to weather changes (late summer). Conclusion: During Weeks 1‐4, the surface pond aerobic levels were similar with the exception of a sharp increase during Week 3 in the control pond and then absent again in Week 4. As the pond water temperatures increased from Weeks 4 – 8, both ponds increased their surface levels of active aerobic isolates. The CFU/ml did not appear to be statistically different between the two ponds. There was also no statistical difference in the recorded levels between the control and treated ponds from Weeks 9‐12. Both ponds saw a similar reduction of surface aerobes as the water temperature began to decline. The study validated that surface water temperature has a primary action on the levels of surface water pond aerobes. The treatment pond surface aerobic levels increased within 7 days of inoculation and peaked during Week 3. Declining aerobic surface levels were seen starting 21 days after application of the spheres. This further validates the 24/7/30 day treatment schedule for the AquaSpherePRO. In this study, the control pond had increased spikes and faster declines of aerobic CFU/ml counts than the treated pond. The treated pond saw more consistent CFU/ml levels on the water surface. Mid – level Anaerobes: Control and Treatment Ponds: The mid‐level anaerobic CFU/ml for the control and the AquaSpherePRO treated ponds were similar. During the first four weeks, mid‐level anaerobes were detected. Further characterization of the anaerobic CFU/ml for the two ponds revealed that during Weeks 4‐8, the CFU/ml reached 3,500 CFU/ml levels, and during Weeks 9‐12, CFU/ml levels continued to increase slightly. Conclusion: Due to the shallow depths of the two ponds, mid‐level anaerobic concentrations were minimal in both. Further research is needed to continue to validate the expected increase in mid‐level anaerobes produced from the 24/7/30 day AquaSpherePRO application. Sludge Anaerobes: Control Pond: The control pond’s sludge anaerobic CFU/ml counts remained constant throughout the 12‐week study. Treatment Pond: The treatment pond’s anaerobic sludge levels remained similar to the control pond’s through Week 4. However, beginning at Week 5, the anaerobic population in the treated pond began to increase. There were increases in the anaerobic population of the treated pond during Weeks 6, 9, and 12. Also, beginning in Week 5, the CFU/ml level of inoculated sludge was substantially higher (14X) than the control sampling average through Weeks 5 – 12. Conclusions: The 12‐week study validated the benefits of sludge inoculation with the Bioverse AquaSpherePRO. The patented 7/24/30 delivery system was lodged in the sludge and allowed for the colonization of sludge‐reducing microbial components. Sludge was activated within 4‐5 weeks of sphere application and continued throughout the study. The treated pond had an average anaerobic sludge (CFU/ml) concentration that was three times greater than the control pond. Clinical Evaluations: A set of objective and subjective evaluation criteria was established by the Bioverse, Inc. research team. Key factors were developed based on the expectations of a golf course superintendent and golf course members for irrigation and golf course ponds. A subjective grading criteria was established to allow a weekly summary of the pond variations. During a 12 week irrigation pond study starting in July by Healthy Ponds by Bioverse at the Elmwood Golf Course in Sioux Falls, SD my staff and I noticed improvements in the treated pond within the first month of treatment. Both of our ponds are similar in size and filled from the same water source. Some of the improvements to water quality that were noticed in the treated pond were reduced algae, odor and greatly improved clarity. The water clarity and overall quality were the two things that we really noticed throughout this study. Norm Parsons Golf Course Superintendent Dakota Golf Management, Inc. Customer Comments: “ I looked at the two ponds today. One pond was crystal clear; I could see all the way to the bottom. The other pond just looked cloudy and unhealthy.” MF Subjective Evaluations: Water temperature: Direct effect on water health and conditions As water temperatures increased, both water health concerns and increased organic matter were seen. Dissolved Oxygen (DO): Indicator of healthy water for irrigation and the environment The treated pond had DO levels that were 65% higher than the control pond. Oxygen Saturation (OS): Used to verify oxygen stability and total amount of saturation The treated pond had OS levels that were 65% higher than the control pond. Water Clarity: A determinant of water health After four weeks, water clarity in the treated pond was greatly improved; it remained substantially improved during the last eight weeks of the trial. Water Aesthetics and Health: Sludge consistence in the treated pond was more condensed. Odor levels were similar. Floating organic matter was reduced in the treated pond. Water clarity in the treated pond remained clear. Trial Results and Conclusions: This 12‐week study validated the application of the AquaSpherePRO in stimulating and enhancing the quality and health of the treated pond. It also established a more favorable bio‐environment for healthy water. 1. The study demonstrated a substantial increase in overall water health and wellness. 2. The study validated a 300 % increase in the treated pond’s anaerobic sludge population (CFU/ml) compared to the control pond sludge. 3. The study revalidated that pond water treated with the AquaSpherePRO had dissolved oxygen concentrations that were over 65% greater in the treated pond versus the control pond. 4. Saturated oxygen levels remained higher in the treatment pond by 75%. 5. Water pH levels between 7.5 and 8 had no effect on the bio‐remediation effect of the AquaSpherePRO. 6. The highest pond water anaerobic CFU/ml counts of the AquaSpherePRO were noted from Weeks 4‐10 and gradually decreased as the water temperature declined in early fall. 7. The study also confirmed the preferred water treatment range of the AquaSpherePRO is from 55 degrees Fahrenheit or greater for optimal results. 8. The study demonstrated that the AquaSpherePRO has limited effects on surface water aerobes. 9. The improved water quality and clarity in the treatment ponds were enhanced by the AquaSpherePRO formulation, which is designed to remove excessive and unwanted nutrients and organic matter from retention and irrigated pond water basins. 10. Top dress applications of selected Bioverse, Inc. bio‐remediation bacterial supplements (VISTA, BLAST), clarifiers (RUSH) and Activators (AWAKE) can be a beneficial supplement to activate the natural bio‐remediation of unhealthy water. It is recommended that the study of natural microbial formulations for stagnant water retention systems should continue. The trial data for this study are on file at Bioverse, Inc., of Pipestone, Minnesota. Bioverse, Inc. extends our thanks to Dr. Elliot Stevens, Director of Rural Technology, Inc., of Brookings, South Dakota, and to Elmwood Golf Course in Sioux Falls, South Dakota, for their assistance with this trial.