11.soil solution changes affected by biosolids and aluminum
Studying the Effects of the Soil Supplement, Biochar,
1. 1
Cooley, Heather; Gleick, Peter; Wilkinson, Robert. (June 2014) Agricultural Water Conservation and Efficiency Potential in
California. National Resources Conservation Service, Accessed 5/1/2016. Web: https://www.nrdc.org/sites/default/files/ca-water-
supply -solutions- ag-efficiency-IB.pdf
Weidner, Simone; Koller, Robert; Latz, Ellen; Kowalchuk, George; Bonkowski, Michael; Scheu, Stefan. (October 2015) Alexandre
Jousset Bacterial diversity amplifies nutrient-based plant–soil feedbacks. Functional Ecology, 29, 10, 1341–1349,
Delzer, G.C.; McKenzie, S.W. (November 2003) Five-Day Biochemical Oxygen Demand. Texas Water Resources Institute’s Book, 9,
A7, 3 rd ed. 1-21.
X600 Image of Biochar [Online Image] Retrieved 5/2/2016 from http://www.charborn.com/
Cell Count Results
References
The occurrence of record drought conditions in Southern California has significantly impacted the local
environment, the agricultural industry, and the economy as a whole. The persistence of these conditions
necessitates the development of new methods devoted to the conservation of water. The state’s agricultural
activities, which account for forty percent of the state’s water usage, are an excellent target for water
conservation (Cooley et. al., 2014). One such method to be taken into consideration is the supplementation of
crop soil with biochar.
Biochar is charcoal that has been modified via pyrolysis, which is a treatment that decomposes substances
via extremely high temperatures in anoxic conditions. This process creates a porous matrix that, when mixed
with soil, allows for increased water retention capacity. In addition to improved water preservation, biochar
is thought to expand the soils ability to retain vital soil nutrients, as well as function has microenvironments
for the soil microbiota. It is proposed that these factors have the potential to improve microbial population
densities, diversity, and activity. These factors also have the potential to improve overall plant growth
through the symbiotic relationships between plants and soil microorganisms (Weidner et al. 2015). In order
to test the hypothesis that biochar will have positive effects on soil microbiota both quantitative and
qualitative measurement were taken. Our cohorts, in their poster titled The Effects of Biochar on Plant
Growth, monitored the direct effect of biochar on the growth of the plants, while this poster focuses on the
effect of the same amendments on the soil microbiota.
The effects on the microbial community were measured by simultaneously running two
distinct analytical processes. The first method included performing cell counts, which is a technique that
directly measures the microbial communities’ population. The second technique measured microbial activity
by quantifying the biological oxygen demand (BOD) of an enclosed environment containing a sample of the
soil. Microbes consume oxygen via metabolic processes, and as result changes in the dissolved oxygen
content of the water present in the system correlates to microbial activity (Delzer and McKenzie 2003).
Analysis of colony morphology differences was also performed in order to observe microbial diversity.
Soil Preparation
4 treatment groups of 4 kg of Miracle Gro potting mix were created: Autoclaved with biochar supplement (A+B+), autoclaved
without biochar supplement (A+B-), non-autoclaved with biochar supplement (A-B+) and non-autoclaved without biochar
supplement (A-B-). The autoclaved treatment groups were autoclaved for 30 minutes. Biochar treatment groups were supplemented
with CoolTerratm biochar in accordance to manufacturer guidelines: 1:4 parts biochar to soil.
Colony Counts
10g of soil (wet weight) was dispersed in 240ml of phosphate buffer in a 2L Erlenmeyer flask. Samples were shaken in a New
Bruinswick Scientific I 24 incubator Shaker for 1 hour. Serial dilutions of 10-4 to10-7 were prepared from the extraction mixture. 1ml
of diluted extraction mixture was placed in a petri dish. Molten (~40ºC) nutrient broth agar was poured over the diluted extraction
mixture. Plates were incubated at room temperature for 2 days before counting.
Phosphate buffer
Phosphate buffer was prepared with this formulation:1.18g Na2HPO4, 0.22g NaH2PO4, 8.5g NaCl, fill to 1L.
Morphological Differences
The cell extraction mixture from colony counts was spread plated over LB agar. Differences in colony morphology were assessed by
size, shape, spreading pattern, and color of colonies.
Biological Oxygen Demand of Soils
The soil samples were incubated in an air-tight container, a Ball mason jar, which was additionally sealed with para-film tape to
ensure an enclosed environment. A 25g soil sample (wet weight) was measured out in an oxygen permeable membrane (Signature
coffee filter) and placed aside. DI water was added to a separate container to be subsequently aliquoted. The dissolved oxygen
content of this water was then equilibrated to atmospheric oxygen levels through rigorous shaking. A graduated cylinder was used to
measure out 250ml of this water and added to a mason jar. A Hach DO probe was used to take the initial dissolved oxygen reading.
The coffee filter containing the soil sample was placed on top of a plastic column suspending it above the water (Figure 7). After
being left to incubate, out of direct sunlight, for 3 days the samples were recovered and measured for the final reading (Figure 8).
Dry Weight Analysis
12 g of each soil (wet weight) sample was measured on a weigh boat and then incubated for 3 days at 60 °C in a Boekel Incubator.
After the incubation period the soil samples were retrieved and weighed shortly after.
Soil Bacteria Densities
• There was exponential growth of bacterial populations over the course of the experiment.
• Sterilization of the soils by autoclaving decreased bacterial population densities by about 90%.
• Differences in bacterial densities between treatments disappeared by week 7.
Soil Bacteria Diversity
• Variations were observed in colony morphology between treatment groups.
• Non-autoclaved soil supplemented with biochar exhibited greatest diversity of colony
morphology.
• Non-autoclaved soil without biochar uniquely contained agar degraders.
• Both autoclaved soil treatment groups’ agar contained unidentified swarming bacteria.
Soil Bacteria Activity
• Biological oxygen demand was highly variable between treatments and time points.
• There was no correlation between BOD and bacterial density.
• Variations in BOD are suspect to be the result of differences in soil water content between
samples.
• There was an apparent positive correlation between water content and BOD.
Soil Bacteria and Plant Growth
• Plants grew better in the absence of biochar, regardless of soil sterilization.
• This effect was most likely not due to variations in bacterial soil densities.
We would like to thank Lissette Gutierrez, Cathy Hutchinson, and Mike Mahoney for their assistance as the biology
department technicians.
● The Miracle Gro potting soil included large fibrous wood particles and some recycled plastics thus
increasing variation between samples. In future experiments, a more homogenous soil will be used.
● The change in microbial populations quickly outpaced our dilution series. The prepared dilutions should
change (increasing the dilution factor) as the experiment continues. By week 7 a 10-7 dilution was
warranted and by week 9 a 10-8 dilution was necessary.
● T-RFLP analysis of the genetic differences of the populations in each treatment group would provide a
better quantitative assessment of diversity than colony morphology.
● A temperature controlled environment for the plants may prevent microbial die-off.
● A temperature controlled area for mason jars could also reduce variability.
● A set watering and sampling regimen that allows for soil samples with closer water content.
● Increase the period of incubation to 5 days or more for larger difference in BOD.
Studying the Effects of the Soil Supplement, Biochar,
on Microbial Communities
Brianna DeMirci, Chet Moneypenny, Scott Souza, Christian Usher, David Walsh • Dr. Erich Fleming
a. A-B+ c. A-B-b. A+B+ d. A+B-
Figure 3. Number of distinct colony morphologies in each treatment group
Figure 9. Scanning electron micrograph of biochar. 600x magnificationIntroduction
Discussion
Figure 2. Cell Counts of soil samples by treatment group
Figure 2. Colony diversity in a) non-autoclaved soil with biochar, b) autoclaved soil with biochar, c) non-autoclaved soil without biochar & d) autoclaved soil without biochar.
BOD Results
Figure 5. Water content of soil samples. Figure 6. Biological oxygen demand of soils by treatment group.
Figure 7. a) Individual components of the BOD measuring apparatus. b) The five experimental
set ups in their respective assembled apparatus. c) Assembled apparatus containing soil sample
Figure 8. Probe measuring the change in oxygen content in the water to determine the biological
oxygen demand of the sample
a
c
b
Future Directions
Acknowledgements
Figure 1. Cross-section of the root system of a cherry tomato plant.
Methodology
Figure 3. Cell counts of soil samples per gram of soil (dry weight) Figure 4. Number of distinct colony morphologies of soil samples present on LB agar
Week 0 Week 4 Week 7 Week 9
Week 0 Week 4 Week 7 Week 9
![1
Cooley, Heather; Gleick, Peter; Wilkinson, Robert. (June 2014) Agricultural Water Conservation and Efficiency Potential in
California. National Resources Conservation Service, Accessed 5/1/2016. Web: https://www.nrdc.org/sites/default/files/ca-water-
supply -solutions- ag-efficiency-IB.pdf
Weidner, Simone; Koller, Robert; Latz, Ellen; Kowalchuk, George; Bonkowski, Michael; Scheu, Stefan. (October 2015) Alexandre
Jousset Bacterial diversity amplifies nutrient-based plant–soil feedbacks. Functional Ecology, 29, 10, 1341–1349,
Delzer, G.C.; McKenzie, S.W. (November 2003) Five-Day Biochemical Oxygen Demand. Texas Water Resources Institute’s Book, 9,
A7, 3 rd ed. 1-21.
X600 Image of Biochar [Online Image] Retrieved 5/2/2016 from http://www.charborn.com/
Cell Count Results
References
The occurrence of record drought conditions in Southern California has significantly impacted the local
environment, the agricultural industry, and the economy as a whole. The persistence of these conditions
necessitates the development of new methods devoted to the conservation of water. The state’s agricultural
activities, which account for forty percent of the state’s water usage, are an excellent target for water
conservation (Cooley et. al., 2014). One such method to be taken into consideration is the supplementation of
crop soil with biochar.
Biochar is charcoal that has been modified via pyrolysis, which is a treatment that decomposes substances
via extremely high temperatures in anoxic conditions. This process creates a porous matrix that, when mixed
with soil, allows for increased water retention capacity. In addition to improved water preservation, biochar
is thought to expand the soils ability to retain vital soil nutrients, as well as function has microenvironments
for the soil microbiota. It is proposed that these factors have the potential to improve microbial population
densities, diversity, and activity. These factors also have the potential to improve overall plant growth
through the symbiotic relationships between plants and soil microorganisms (Weidner et al. 2015). In order
to test the hypothesis that biochar will have positive effects on soil microbiota both quantitative and
qualitative measurement were taken. Our cohorts, in their poster titled The Effects of Biochar on Plant
Growth, monitored the direct effect of biochar on the growth of the plants, while this poster focuses on the
effect of the same amendments on the soil microbiota.
The effects on the microbial community were measured by simultaneously running two
distinct analytical processes. The first method included performing cell counts, which is a technique that
directly measures the microbial communities’ population. The second technique measured microbial activity
by quantifying the biological oxygen demand (BOD) of an enclosed environment containing a sample of the
soil. Microbes consume oxygen via metabolic processes, and as result changes in the dissolved oxygen
content of the water present in the system correlates to microbial activity (Delzer and McKenzie 2003).
Analysis of colony morphology differences was also performed in order to observe microbial diversity.
Soil Preparation
4 treatment groups of 4 kg of Miracle Gro potting mix were created: Autoclaved with biochar supplement (A+B+), autoclaved
without biochar supplement (A+B-), non-autoclaved with biochar supplement (A-B+) and non-autoclaved without biochar
supplement (A-B-). The autoclaved treatment groups were autoclaved for 30 minutes. Biochar treatment groups were supplemented
with CoolTerratm biochar in accordance to manufacturer guidelines: 1:4 parts biochar to soil.
Colony Counts
10g of soil (wet weight) was dispersed in 240ml of phosphate buffer in a 2L Erlenmeyer flask. Samples were shaken in a New
Bruinswick Scientific I 24 incubator Shaker for 1 hour. Serial dilutions of 10-4 to10-7 were prepared from the extraction mixture. 1ml
of diluted extraction mixture was placed in a petri dish. Molten (~40ºC) nutrient broth agar was poured over the diluted extraction
mixture. Plates were incubated at room temperature for 2 days before counting.
Phosphate buffer
Phosphate buffer was prepared with this formulation:1.18g Na2HPO4, 0.22g NaH2PO4, 8.5g NaCl, fill to 1L.
Morphological Differences
The cell extraction mixture from colony counts was spread plated over LB agar. Differences in colony morphology were assessed by
size, shape, spreading pattern, and color of colonies.
Biological Oxygen Demand of Soils
The soil samples were incubated in an air-tight container, a Ball mason jar, which was additionally sealed with para-film tape to
ensure an enclosed environment. A 25g soil sample (wet weight) was measured out in an oxygen permeable membrane (Signature
coffee filter) and placed aside. DI water was added to a separate container to be subsequently aliquoted. The dissolved oxygen
content of this water was then equilibrated to atmospheric oxygen levels through rigorous shaking. A graduated cylinder was used to
measure out 250ml of this water and added to a mason jar. A Hach DO probe was used to take the initial dissolved oxygen reading.
The coffee filter containing the soil sample was placed on top of a plastic column suspending it above the water (Figure 7). After
being left to incubate, out of direct sunlight, for 3 days the samples were recovered and measured for the final reading (Figure 8).
Dry Weight Analysis
12 g of each soil (wet weight) sample was measured on a weigh boat and then incubated for 3 days at 60 °C in a Boekel Incubator.
After the incubation period the soil samples were retrieved and weighed shortly after.
Soil Bacteria Densities
• There was exponential growth of bacterial populations over the course of the experiment.
• Sterilization of the soils by autoclaving decreased bacterial population densities by about 90%.
• Differences in bacterial densities between treatments disappeared by week 7.
Soil Bacteria Diversity
• Variations were observed in colony morphology between treatment groups.
• Non-autoclaved soil supplemented with biochar exhibited greatest diversity of colony
morphology.
• Non-autoclaved soil without biochar uniquely contained agar degraders.
• Both autoclaved soil treatment groups’ agar contained unidentified swarming bacteria.
Soil Bacteria Activity
• Biological oxygen demand was highly variable between treatments and time points.
• There was no correlation between BOD and bacterial density.
• Variations in BOD are suspect to be the result of differences in soil water content between
samples.
• There was an apparent positive correlation between water content and BOD.
Soil Bacteria and Plant Growth
• Plants grew better in the absence of biochar, regardless of soil sterilization.
• This effect was most likely not due to variations in bacterial soil densities.
We would like to thank Lissette Gutierrez, Cathy Hutchinson, and Mike Mahoney for their assistance as the biology
department technicians.
● The Miracle Gro potting soil included large fibrous wood particles and some recycled plastics thus
increasing variation between samples. In future experiments, a more homogenous soil will be used.
● The change in microbial populations quickly outpaced our dilution series. The prepared dilutions should
change (increasing the dilution factor) as the experiment continues. By week 7 a 10-7 dilution was
warranted and by week 9 a 10-8 dilution was necessary.
● T-RFLP analysis of the genetic differences of the populations in each treatment group would provide a
better quantitative assessment of diversity than colony morphology.
● A temperature controlled environment for the plants may prevent microbial die-off.
● A temperature controlled area for mason jars could also reduce variability.
● A set watering and sampling regimen that allows for soil samples with closer water content.
● Increase the period of incubation to 5 days or more for larger difference in BOD.
Studying the Effects of the Soil Supplement, Biochar,
on Microbial Communities
Brianna DeMirci, Chet Moneypenny, Scott Souza, Christian Usher, David Walsh • Dr. Erich Fleming
a. A-B+ c. A-B-b. A+B+ d. A+B-
Figure 3. Number of distinct colony morphologies in each treatment group
Figure 9. Scanning electron micrograph of biochar. 600x magnificationIntroduction
Discussion
Figure 2. Cell Counts of soil samples by treatment group
Figure 2. Colony diversity in a) non-autoclaved soil with biochar, b) autoclaved soil with biochar, c) non-autoclaved soil without biochar & d) autoclaved soil without biochar.
BOD Results
Figure 5. Water content of soil samples. Figure 6. Biological oxygen demand of soils by treatment group.
Figure 7. a) Individual components of the BOD measuring apparatus. b) The five experimental
set ups in their respective assembled apparatus. c) Assembled apparatus containing soil sample
Figure 8. Probe measuring the change in oxygen content in the water to determine the biological
oxygen demand of the sample
a
c
b
Future Directions
Acknowledgements
Figure 1. Cross-section of the root system of a cherry tomato plant.
Methodology
Figure 3. Cell counts of soil samples per gram of soil (dry weight) Figure 4. Number of distinct colony morphologies of soil samples present on LB agar
Week 0 Week 4 Week 7 Week 9
Week 0 Week 4 Week 7 Week 9](https://image.slidesharecdn.com/7e0497d1-59a7-473e-8e6c-93dcffc5ac55-161014173436/85/studying-the-effects-of-the-soil-supplement-biochar-1-320.jpg)
