MC Chem Wiki - User contributions [en]

Chemical, Enzymatic, and Electrochemical Oxidation of Biophenols

2022-05-14T01:46:29Z

Ssimonson: /* Materials and Methods */

==Abstract==

The redox properties of phenols can be studied using chemical, enzymatic, and electrochemical techniques. Whereas chemical and enzymatic methods have fixed oxidation potentials, electrochemical methods allow for control of the oxidation potential. Here, the oxidation product outcomes from KMnO4 oxidation, peroxidase/HRP oxidation, and bulk electrolysis of 4-hydroxyphenylacetic acid (HPA) are presented and compared with HPLC. ESR data is also shown. Similar data is presented for other biologically relevant phenols.

==Introduction==
HPA is a monocarboxilic acid and a member of the phenol family. In industry, HPA serves as a common precursor to the synthesis of drugs and pesticides due to its simplicity in structure (figure 1) (1,2). HPA is an important metabolite for anaerobic and aerobic bacteria alike....(3,4)
As a phenol, HPA is able to undergo a one electron oxidation with an appropriate oxidizing agent to generate the radical shown in figure 2. This oxidative radical will couple to other molecules to form dimers, trimmers, and heavier polymers. HPA will readily undergo radical polymerization when met with the appropriate conditions. Common oxidation techniques employed to generate the radical include doing so chemically, electrochemically, and enzymatically.

Electrochemical, enzymatic, and chemical oxidation data is shown for HPA in this paper and the oxidation product outcomes are compared. Initial data for the more complex tyrosine and N-acetyl tyrosine is also presented.

==Materials and Methods==

===Electrochemical Oxidation===

===Chemical Oxidation===

Chemical Oxidation of 2.00 mM HPA (pH 5) in H2O was carried out with KMnO4 at various concentrations ranging from 0.33 mM to 4.00 mM. Reactions were carried out in 5 mL reaction vials.

===Beaker Enzymatic Oxidation===

Enzymatic Oxidation of 2.00 mM HPA (pH 5) in H2O was carried out with horseradish peroxidase (HRP) and various concentrations of H2O2 ranging from 0.25 mM to 2.00 mM. HRP concentration was kept constant at 500 nM for each reaction. Reactions were carried out in 5 mL reaction vials. Enzymatic oxidation of N-acetyl tyrosine was carried out under the same conditions described for HPA. Enzymatic oxidation of tyrosine was completed with the same experimental parameters as decribed for HPA and NAY; however, tyrosine was studied at 6.00 mM and solubilized with 1 mL 0.1 M NaOH prior to addition to pH 7.4 H20 solution due to solubility issues.

===Immobilized Enzymatic Oxidation===

===High Performance Liquid Chromatography ( HPLC) Methodology===

===Electron Spin Resonance===

==Results==

==Discussion==

One of the main takeaways from the oxidation data of HPA is the oxidation product outcomes are not only dependent on generation of the radical; this is also dependent on the oxidation technique employed. If product outcome was only depent on generation of the radical, then the oxidation product distribution would look the same gor each method, but this is not the case.

==References==

==Page History==

This page was created by Sara L. Simonson in the fall of 2021

Chemical, Enzymatic, and Electrochemical Oxidation of Biophenols

2022-05-13T23:47:40Z

Ssimonson: /* Discussion */

==Abstract==

The redox properties of phenols can be studied using chemical, enzymatic, and electrochemical techniques. Whereas chemical and enzymatic methods have fixed oxidation potentials, electrochemical methods allow for control of the oxidation potential. Here, the oxidation product outcomes from KMnO4 oxidation, peroxidase/HRP oxidation, and bulk electrolysis of 4-hydroxyphenylacetic acid (HPA) are presented and compared with HPLC. ESR data is also shown. Similar data is presented for other biologically relevant phenols.

==Introduction==
HPA is a monocarboxilic acid and a member of the phenol family. In industry, HPA serves as a common precursor to the synthesis of drugs and pesticides due to its simplicity in structure (figure 1) (1,2). HPA is an important metabolite for anaerobic and aerobic bacteria alike....(3,4)
As a phenol, HPA is able to undergo a one electron oxidation with an appropriate oxidizing agent to generate the radical shown in figure 2. This oxidative radical will couple to other molecules to form dimers, trimmers, and heavier polymers. HPA will readily undergo radical polymerization when met with the appropriate conditions. Common oxidation techniques employed to generate the radical include doing so chemically, electrochemically, and enzymatically.

Electrochemical, enzymatic, and chemical oxidation data is shown for HPA in this paper and the oxidation product outcomes are compared. Initial data for the more complex tyrosine and N-acetyl tyrosine is also presented.

==Materials and Methods==

===Electrochemical Oxidation===

===Chemical Oxidation===

===Beaker Enzymatic Oxidation===

===Immobilized Enzymatic Oxidation===

===High Performance Liquid Chromatography ( HPLC) Methodology===

===Electron Spin Resonance===

==Results==

==Discussion==

One of the main takeaways from the oxidation data of HPA is the oxidation product outcomes are not only dependent on generation of the radical; this is also dependent on the oxidation technique employed. If product outcome was only depent on generation of the radical, then the oxidation product distribution would look the same gor each method, but this is not the case.

==References==

==Page History==

This page was created by Sara L. Simonson in the fall of 2021

Chemical, Enzymatic, and Electrochemical Oxidation of Biophenols

2022-05-13T23:42:20Z

Ssimonson: /* Materials and Methods */

2021-10-10T15:16:19Z

Ssimonson:

'''Electrochemical and enzymatic evaluation of biophenols.'''
:''Sara Simmonson and Bradley E. Sturgeon''

The redox properties of phenols can be studied using chemical, enzymatic, and electrochemical methods. Whereas chemical and enzymatic methods have a fixed oxidation potential, electrochemical methods allow for control of the oxidation potential. The redox properties of 4-hydroxyphenylacetic acid will be evaluated using cyclic voltammetry. In addition, oxidation product outcomes (HPLC) from KMnO4 oxidation, peroxidase/HRP oxidation, and bulk electrolysis will be presented. Similar data will be presented for tyrosine, N-acetyltyrosine, and acetaminophen.

''submitted to 2022 ACS National Meeting, San Diego CA, March 20-24''

File:AEM.00179-18.pdf

2021-09-21T22:09:01Z

Ssimonson: Ssimonson uploaded a new version of File:AEM.00179-18.pdf

File:AEM.00179-18.pdf

2021-09-21T22:06:32Z

Ssimonson:

Freshwater Aquarium Microbiome

2021-09-21T22:04:59Z

Ssimonson: /* References */

==Introduction==
Microbiomes, the communities of microorganisms living together in particular habitats, are vital for maintaining ecological balance. The habitat hosting the microbial communities can be anything from the gastrointestinal tract of a cow to the soils of the land (1). Of recent particular interest are those of aquatic ecosystems, which contain diverse arrays of bacterial communities. The fish themselves oftentimes possess more microbial cells than fish cells in their bodies (2). Of increasing use in identifying the microbes present in these microbiomes is 16S amplicon sequencing. The 16S rRNA gene encodes for 16S rRNA, which is an important constituent of the prokaryotic ribosome 30S subunit. It is noteworthy to point out that this gene is only found in bacteria and archaea (3). The 16S gene has a number of conserved regions, which enables the 16S gene to be recognized across many different microbes through only a few sets of primers catered to this conserved region. The variable regions in the gene are indicative to which specific microbes are products of the amplification of the microbiomes. Up until 2012, there have not been very many studies pertaining to the characterization of the microbiome pertaining to the water associated with freshwater ornamental fishes (4)...This experiment aims to obtain a report on the bacterial community composition of the freshwater fish tank housing four generations of convict cichlids and on the saltwater fish tank, located on the 3rd floor of CSB. To accomplish this, a protocol modeled after Smith et al. and Patin et al. will be established (4,5). 600 mL of aquarium water will be filtered through a 0.22 um filter unit. The concentrated microbial biomass trapped on the filter will be treated with a Puregene Qiagen kit with some modifications from the protocol. Afterwards amplification of the bacterial 16S rRNA gene will be carried out and sequencing at the University of Illinois will ensue.

==Materials and Methods==
===Water Sampling===
Sterivex Filters
:1 L of water from the surface of the freshwater fish tank and saltwater fish tank were filtered through separate 0.22 um sterivex filter units via a peristaltic pump. Air was pushed through following filtration to initiate the removal of residual water contained in the filter units.

Nanopure Filtration
: Due to the low concentration of DNA extracted from filtering 1 L of water through the sterivex filter unit, 2 L of water from the freshwater and saltwater fish tanks were filtered through the free 0.22 um filter membranes via a reusable filter funnel and with the aid of a vacuum source.
[https://waterra.com/product/spectra-field-pro-peristaltic-pump/ Spectra Field-Pro Peristaltic Pump (~$2600)]
:- [https://youtu.be/XBaSQcmXc0s eDNA Sampler Usage]

===DNA Extraction===

DNA extraction from the 0.22 um sterivex filter unit followed the protocol outlined in the DNeasy Powerwater Sterivex Kit with no noteworthy deviations from this protocol (6). As expected, 100 ul of product was obtained. 2 ul were tested on the nanodrop to assess the quantity and quality of the DNA obtained. The DNA extracted from the freshwater sterivex filter unit absorbed at a concentration of 3.6 ng/ul at 260 nm, which was a lower concentration of DNA than expected. Its A260/A280 ratio was 1.80, which indicates that the protein and inhibitor removal steps were successful. The recorded A260/A230 ration was very low at 0.04, which may indicate the presence of excess salt left in solution, for most of the salts used in this DNA extraction protocol absorb at 230 nm. This low ratio suggests that improvements to the overall quantity of DNA obtained may be higher than 3.6 ng/ul through purification techniques aimed to get the excess salt out.

[[:File:PowerWater_Sterivex.pdf]]

===Primer Construction===

Forward primer design: 5'-AATGATACGGCGACCACCGAGATCTACAC-ATCGTACG-TATGGTAATT-GT-GTGCCAGCMGCCGCGGTAA-3' | 5'-adaptor-barcode-pad-linker-V4 region primer-3'

Reverse primer design: 5'-CAAGCAGGAAGACGGCATACGAGAT-AGTCAGTCAG-CC-GGACTACHVGGGTWTCTAAT-3' | 5'-adaptor-pad-linker-V4 region primer-3'

The pads serve the purpose of preventing hairpin formation and manipulating the melting point of the primers, for the adaptors need to anneal to the flow cell, a glass slide with lanes of two different types of oligo (complementary to the adaptors), at 65°C in order for sequencing to ensue (7). The linkers are noncomplementary to the 16S gene so that alignment for bioinformatics processing can be carried out efficiently (7). The adaptors, pads, and linkers are needed on both the forward and reverse primers because bridge amplification is utilized in Illumina sequencing platforms. During this process, one side of the adaptor sequences hybridizes to the flow cell, a complement of this original fragment is produced by a polymerase, the double strand of DNA is denatured and washed away, and then the single strand folds over and hybridizes to the second type of oligo on the flow cell (8). Polymerases generate the complementary strand, and this double stranded bridge is denatured, which results in two single stranded copies tethered to the flow cell (8). This process is repeated many times. After bridge amplification, the reverse strands are cleaved and cut off, which is why the barcode is only important for the forward primer.

===PCR Amplification===

===Sequencing===

===Bioinformatics===

==Results==

==Discussion==

==References==
1. Fierer, N. Embracing the unknown: disentangling the complexities of the soil microbiome. Nature Reviews Microbiology. 2017. 15. 579-590.

2. Savage, D. C. [[Media:32_300.pdf|Microbial ecology of the gastrointestinal tract.]] Ann Rev Microbiol. '''1977'''. ''31''. 107-133.

3.

4. Smith, K. F.; Schmidt, V.; Rosen, G. E.; Amaral-Zettler, L. [[Media:pone.0039971.pdf|Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water.]] ''Public Library of Science One.'' '''2012'''. ''7''.

5. Patin, N. V.; Pratte, Z. A.; Regensburger, M.; Hall, E.; Gilde, K.; Dove, A. D. M.; Stewart, F. J. [[Media:AEM.00179-18.pdf|Microbiome Dynamics in a Large Artificial Seawater Aquarium. Applied and Environmental Microbiology.]] 2018. 84 (10).

6. [[Media:HB-2266-002_HB_DNY_PowerWater_Sterivex_0519_WW%20(2).pdf|DNEASY PowerWater Sterivex Kit Handbook]]

7. Kozich, J. J.; Estcott, S. L.; Baxter, N. T.; Highlander, S. K.; Schloss, P. D. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Applied and Environmental Microbiology. 2013. 79 (17). 5112-5120.

8. [https://www.youtube.com/watch?v=womKfikWlxM Illumina Sequencing Technology - Illumina YouTube Video]

==Page History==
This page was created by Sara L Simonson in the spring of 2021

Nitrate in the Freshwater Fish tank

2021-09-21T04:54:37Z

Ssimonson: /* Materials and Methods */

==Introduction==

Nitrates in surface water primarily result from the decay of autotrophs and from chemoheterotrophic life, for nitrates are a byproduct of the waste produced from the consumption of organic molecules as an energy source. Fish are the primary chemoheterotrophs in aquatic ecosystems; therefore, they are accountable for the majority of nitrogen found in water. Ammonia is the main component of fish waste, which is a very toxic compound of nitrogen, but aerobic ammonia oxidizers convert ammonia to nitrite via the intermediate hydroxylamine, which requires aid from ammonia monooxygenase and hydroxylamine dehydrogenase (1). Nitrite is an even more toxic form of nitrogen, but this is further oxidized to nitrate via another type of nitrogen bacteria with the enzyme nitrite oxidoreductase(1). In nature, nitrates are regulated to a certain extent through aquatic plant life, for it is essential in the production of chlorophyll (2). In fish tanks, however, algae is the only natural nitrate regulator, but unlike more complex and nutrient dependent plants, algae is able to grow beyond the capacity of a given ecosystem with ease. Algae cannot convert nitrogen to a gas that can escape the system, so when it dies, nitrogen is released back into the system (2). In closed systems, like that of a fish tank, mechanical work such as changing out volumes of water, being mindful in not overfeeding the fish, and cleaning off the algae and filtration systems, is required in order to ensure nitrate levels are kept at a safe and constant concentration.

This experiment aims to quantify the rise in daily nitrate levels from the few sources of nitrate in a closed off fish tank system by way of using the Thermo Fisher ScientificTM DionexTM AquionTM Ion Chromatography System with the built in Chromeleon 7 Chromatography Data System software. While there are no direct regulations published by the EPA for nitrate and nitrite levels in a fish tank, this experiment utilized the EPA's recommendations for surface water systems. The EPA has set different guidelines for surface water that is just a reservoir for aquatic life and water that is used for human consumption. The maximum contaminant level for nitrates and nitrites in drinking water in the U.S.A. are 10.0 ppm and 1.0 ppm respectively (3). For water systems not being used as drinking water or connected to public water systems, such as isolated ponds, the EPA recommends that nitrate levels do not exceed 60 ppm and that nitrite levels should be kept as close to 0.0 ppm as possible (3).

With a focus on nitrates and nitrites, chloride and sulfate concentrations were also investigated in this experiment. To serve as a comparative analysis, freshwater samples from a connected creek and pond system and ground water samples from a closed off underground spring were also run on the Ion Chromatographer. The EPA defined the maximum Contaminant level for chloride in drinking water as 250 ppm and interprets chlorides as chronically toxic to freshwater aquatic life at 230 ppm and acutely toxic at 860 ppm (4).

==Materials and Methods==

''Instrumentation''

Data was collected on the Thermo Fisher ScientificTM DionexTM AquionTM Ion Chromatography System with the built in Chromeleon 7 Chromatography Data System software. Analysis of the desired anions was carried out using the (name of column) column with a 4.5 mM sodium carbonate and 1.4 mM sodium bicarbonate mobile phase and the flow rate kept constant at 1.2 mL/min. Currently the software is programmed to detect eight common anions (chloride, fluoride, acetate, sulfate, nitrate, nitrite, phosphate, bromide), but for this study, standards were only made to analyze nitrate, nitrite, chloride, and sulfate.

''Calibration Standards''

The anion calibration standards were prepared by dissolving 0.06620 g KNO3, 0.06100 g Na2NO3, 0.05970 g Na2SO4, and 0.05970 g NaCl into four separate 100 mL volumetric flasks to yield 400 ppm stock solutions for all four anions. With appropriate dilutions, these stock solutions were combined to give rise to 10, 20, 40, 80, and 100 ppm stock solutions of calibration standards.

''Fish Tank Set-Up''

The closed system for which data was collected consisted of a 29 gallon freshwater fish tank with convict cichlids. An Aquatop Canister Filter was connected to the fish tank via a hose and cleaned before sample collection took place. A water top off system was put to use in which a float water level sensor was calibrated at the 29 gallon mark to ensure the water level remained constant in order to eliminate nitrate increments due to evaporation. A feeding mechanism with the Zacro Automated Fish Feeder was devised so that 1.0 gram of TetraCichlid Floating Cichlid Pellets would be delivered twice a day. This was accomplished by setting the automated fish feeder to release 1 meal portion at 8:30 and 16:30.

''Fish Tank Samples''

In order to see how mainly nitrate and nitrite levels change from day to day in a closed system, 11 water samples were collected throughout the span of 28 days at roughly the same time each day (17:00 - 30 minutes after the second feeding cycle) and stored at 4°C before analysis. One sample was collected 76 days after the first sample was collected in order to gauge whether nitrate levels continue to rise at the same rate or if they level off eventually. After filtering these samples with 0.45 um hydrophilic filter units and a one in five dilution, these samples were run on the Thermo Fisher ScientificTM DionexTM AquionTM Ion Chromatography System with the built in Chromeleon 7 Chromatography Data System software, with two sets of nitrate, nitrite, and chloride calibration standards of 10, 40, and 100 ppm.

''Fish Food Analysis''

In order to see how much nitrate entered the system directly from the food source being supplied to the tank, a portion size of 1 gram of the TetraCichlid Floating Cichlid Pellets was diluted in 1 L of milipore water, centrifuged for 5 minutes at 300 RPM and analyzed on the ion chromatographer with the same standards as the evaporative fish tank samples were.

''Pond and Creek Water Samples''

Freshwater samples from a creek and a pond were collected from the locations displayed in figure 1. After a one in five dilution, they were analyzed on the same Ion Chromatography System as the fish tank samples, with nitrate, nitrite, chloride, and sulfate standards of 10, 40, and 100 ppm.

[[File:watermap.png|300px|thumb|left|Figure 1. The red tag displays where the pond sample was collected and the gray tag shows where the creek sample was collected during late September]]

''Ground Water Samples''

A water sample from a private well that draws directly from a natural underground spring was collected along with a sample from the well source ran through a water softener. Ground water samples were not diluted prior to analysis.

==Results==

''Fish Tank System''

[[File:nitrateincreasefwft2.png|300px|thumb|Left|Figure 3: The data here relates the nitrate increments due to metabolic activity of the fish, algal modulation, and dissolved fish food pellets over a span of 72 days.]]

[[File:nitrateincreasefwft.png|300px|thumb|Left|Figure 4: The data here is the same presented in Figure 3 excluding the last data point collected on day 72.]]

The rate of daily nitrate concentration was determined to be 2.5912 ppm/day. This is due to metabolic activity by way of the fish, algae interference, and mainly evaporation (figure 3). The portion size of fish pellets dissolved in 1 Liter was determined to have a nitrate concentration of 2.8564 ppm. Considering that the fish were fed twice a day by this same amount and that the tank is filled to 29 gallons when in the system this becomes a nitrate concentration of 0.0520 ppm. It was determined that nitrate increments due to the direct food source could be excluded since the amount of nitrate in the food is negligible compared to other causes.

''Ground and Surface Water Analysis''

Table 1 displays the results of the varying anion concentrations in the creek, pond, and ground water samples.

{| class="wikitable"
|-
! Anion !! Creek Water Sample !! Pond Water Sample !! Well Water Sample !! Well Water Sample w/Softener
|-
| Chloride || 108.055 ppm || 33.8505 ppm || 18.467 ppm || Example
|-
| Nitrite || 0 ppm || 0 ppm || 0 ppm || Example
|-
| Nitrate || 13.079 ppm || 10.875 ppm || 11.9325 ppm || Example
|-
| Sulfate || 39.737 ppm || 16.8185 ppm || 13.1475 ppm || Example
|}

==Discussion==

Figure 3 demonstrates that nitrate concentrations do end up leveling off when they become high, for a linear relationship is only observed for days 1 - 28 and not for day 76. This may be due to increased algae growth due to nutrient abundance and hence increased algal nitrate uptake. Keeping this in mind, the formula can only serve a good model for when the daily rise in nitrate demonstrates a linear relationship (day 1 - 28); therefore, calculated water exchanges via the equation below should take place within this key period.

Even though the rise in daily nitrate concentrations was quantified over a 28 day span in this experiment, this rate does not serve as an absolute representation of daily nitrate increments for all year round. Sample collection took place in mid winter; therefore, during periods of increased metabolic activity, such as nesting and breeding seasons, the rate of daily rises in nitrate concentration is expected to be higher. Another factor to keep in mind is the population of the fish tank.

Though natural regulation proves to be useful in open surface water systems, there are a lot more variables in how nitrates and nitrites can enter the system, therefore, . Although mainly byproducts from aquatic life, nitrate and nitrite levels in surface and ground water can also result from agricultural runoff (fertilizers), or improper disposal of human and animal waste (). Considering the pond and creek are interconnected to each other and have equal exposure to potential agricultural runoff, it makes sense that these two systems have very similar nitrate concentrations as seen by table 1. While the creek and water samples collected came from a reservoir for aquatic life, it is likely the nitrate levels are also a result from the heavy agricultural activity surrounding the open system. Even so, the nitrate and nitrite levels are well below the EPA's guidelines for surface water not serving the purpose as drinking water.
Road-salt runoff, which primarily consists of NaCl, can pose a major threat to surface water quality and aquatic life. Even in warm weather months, when roads are not being salted, chloride levels from winter salt applications persisted in a study carried out in Milwaukee Massachusetts (5). Road salt is the primary reason why the creek water sample has a higher chloride concentration than the pond water sample (figure 1).

==References==

1. Harms, G.; Layton, A. C.; Dionsi, H. M.; Gregory, I. R.; Garrett, V. M.; Hawkins, S. A.; Robinson, K. G.; Slayer, G. S. Real-Time PCR Quantification of Nitrifying Bacteria in a Municipal Wastewater Treatment Plant. ''Am. Chem. Soc.'' '''2002''', ''37'', 343-351.

2. Ullrich, W. R.; Lazarova, J.; Ullrich, C. I.; Witt, F. G.; Aparcio, P. J. Nitrate Uptake and Extracellular Alkalinization by the Green Algae Hydrodictyon ''reticulatum'' in Blue and Red Light. ''Journal of Experimental Botany.'' '''1998''', ''49'', 1157-1162.

3. U.S. Environmental Protection Agency. https://www.epa.gov/

4. U.S. Environmental Protection Agency. Ambient Water Quality Criteria for Chloride. ''Office of Water Regulations and Standards Criteria and Standards Division''. '''1988'''.

5. Corsi, S. R.; Graczyk, D. J.; Geis, S. W.; Booth, N. L.; Richards, K. D. A Fresh Look at Road Salt: Aquatic Toxicity and Water-Quality Impacts on Local, Regional, and National Scales. ''Environmental Science & Technology''. '''2010''', ''44'', 7376-7382.

==Page History==

This page was created by Sara L Simonson in the fall of 2020

Nitrate in the Freshwater Fish tank

2021-09-21T04:51:56Z

Ssimonson: /* Materials and Methods */

==Introduction==

Nitrates in surface water primarily result from the decay of autotrophs and from chemoheterotrophic life, for nitrates are a byproduct of the waste produced from the consumption of organic molecules as an energy source. Fish are the primary chemoheterotrophs in aquatic ecosystems; therefore, they are accountable for the majority of nitrogen found in water. Ammonia is the main component of fish waste, which is a very toxic compound of nitrogen, but aerobic ammonia oxidizers convert ammonia to nitrite via the intermediate hydroxylamine, which requires aid from ammonia monooxygenase and hydroxylamine dehydrogenase (1). Nitrite is an even more toxic form of nitrogen, but this is further oxidized to nitrate via another type of nitrogen bacteria with the enzyme nitrite oxidoreductase(1). In nature, nitrates are regulated to a certain extent through aquatic plant life, for it is essential in the production of chlorophyll (2). In fish tanks, however, algae is the only natural nitrate regulator, but unlike more complex and nutrient dependent plants, algae is able to grow beyond the capacity of a given ecosystem with ease. Algae cannot convert nitrogen to a gas that can escape the system, so when it dies, nitrogen is released back into the system (2). In closed systems, like that of a fish tank, mechanical work such as changing out volumes of water, being mindful in not overfeeding the fish, and cleaning off the algae and filtration systems, is required in order to ensure nitrate levels are kept at a safe and constant concentration.

This experiment aims to quantify the rise in daily nitrate levels from the few sources of nitrate in a closed off fish tank system by way of using the Thermo Fisher ScientificTM DionexTM AquionTM Ion Chromatography System with the built in Chromeleon 7 Chromatography Data System software. While there are no direct regulations published by the EPA for nitrate and nitrite levels in a fish tank, this experiment utilized the EPA's recommendations for surface water systems. The EPA has set different guidelines for surface water that is just a reservoir for aquatic life and water that is used for human consumption. The maximum contaminant level for nitrates and nitrites in drinking water in the U.S.A. are 10.0 ppm and 1.0 ppm respectively (3). For water systems not being used as drinking water or connected to public water systems, such as isolated ponds, the EPA recommends that nitrate levels do not exceed 60 ppm and that nitrite levels should be kept as close to 0.0 ppm as possible (3).

With a focus on nitrates and nitrites, chloride and sulfate concentrations were also investigated in this experiment. To serve as a comparative analysis, freshwater samples from a connected creek and pond system and ground water samples from a closed off underground spring were also run on the Ion Chromatographer. The EPA defined the maximum Contaminant level for chloride in drinking water as 250 ppm and interprets chlorides as chronically toxic to freshwater aquatic life at 230 ppm and acutely toxic at 860 ppm (4).

==Materials and Methods==

''Instrumentation''

Data was collected on the Thermo Fisher ScientificTM DionexTM AquionTM Ion Chromatography System with the built in Chromeleon 7 Chromatography Data System software. Analysis of the desired anions was carried out using the (name of column) column with a 4.5 mM sodium carbonate and 1.4 mM sodium bicarbonate mobile phase and the flow rate kept constant at 1.2 mL/min. Currently the software is programmed to detect eight common anions (chloride, fluoride, acetate, sulfate, nitrate, nitrite, phosphate, bromide), but for this study, standards were only made to analyze nitrate, nitrite, chloride, and sulfate.

''Calibration Standards''

The anion calibration standards were prepared by dissolving 0.06620 g KNO3, 0.06100 g Na2NO3, 0.05970 g Na2SO4, and 0.05970 g NaCl into four separate 100 mL volumetric flasks to yield 400 ppm stock solutions for all four anions. With appropriate dilutions, these stock solutions were combined to give rise to 10, 20, 40, 80, and 100 ppm stock solutions of calibration standards.

''Fish Tank Set-Up''

The closed system for which data was collected consisted of a 29 gallon freshwater fish tank with convict cichlids. An Aquatop Canister Filter was connected to the fish tank via a hose and cleaned before sample collection took place. A water top off system was put to use in which a float water level sensor was calibrated at the 29 gallon mark to ensure the water level remained constant in order to eliminate nitrate increments due to evaporation. A feeding mechanism with the Zacro Automated Fish Feeder was devised so that 1.0 gram of TetraCichlid Floating Cichlid Pellets would be delivered twice a day. This was accomplished by setting the automated fish feeder to release 1 meal portion at 8:30 and 16:30.

''Fish Tank Samples''

In order to see how mainly nitrate and nitrite levels change from day to day in a closed system, 11 water samples were collected throughout the span of 28 days at roughly the same time each day (17:00 - 30 minutes after the second feeding cycle) and stored at 4°C before analysis. One sample was collected 76 days after the first sample was collected in order to gauge whether nitrate levels continue to rise at the same rate or if they level off eventually. After filtering these samples with 0.45 um hydrophilic filter units and a one in five dilution, these samples were run on the Thermo Fisher ScientificTM DionexTM AquionTM Ion Chromatography System with the built in Chromeleon 7 Chromatography Data System software, with two sets of nitrate, nitrite, and chloride calibration standards of 10, 40, and 100 ppm.

''Fish Food Analysis''

In order to see how much nitrate entered the system directly from the food source being supplied to the tank, a portion size of 1 gram of the TetraCichlid Floating Cichlid Pellets was diluted in 1 L of milipore water, centrifuged for 5 minutes at 300 RPM and analyzed on the ion chromatographer with the same standards as the evaporative fish tank samples were.

''Pond and Creek Water Samples''

Freshwater samples from a creek and a pond were collected from the locations displayed in figure 1. After a one in five dilution, they were analyzed on the same Ion Chromatography System as the fish tank samples, with nitrate, nitrite, chloride, and sulfate standards of 10, 40, and 100 ppm.

[[File:watermap.png|300px|thumb|left|Figure 1. The red tag displays where the pond sample was collected and the gray tag shows where the creek sample was collected during late September]]

''Ground Water Samples''

A water sample from a private well that draws directly from a natural underground spring was collected along with a sample from the well source ran through a water softener.

==Results==

''Fish Tank System''

[[File:nitrateincreasefwft2.png|300px|thumb|Left|Figure 3: The data here relates the nitrate increments due to metabolic activity of the fish, algal modulation, and dissolved fish food pellets over a span of 72 days.]]

[[File:nitrateincreasefwft.png|300px|thumb|Left|Figure 4: The data here is the same presented in Figure 3 excluding the last data point collected on day 72.]]

The rate of daily nitrate concentration was determined to be 2.5912 ppm/day. This is due to metabolic activity by way of the fish, algae interference, and mainly evaporation (figure 3). The portion size of fish pellets dissolved in 1 Liter was determined to have a nitrate concentration of 2.8564 ppm. Considering that the fish were fed twice a day by this same amount and that the tank is filled to 29 gallons when in the system this becomes a nitrate concentration of 0.0520 ppm. It was determined that nitrate increments due to the direct food source could be excluded since the amount of nitrate in the food is negligible compared to other causes.

''Ground and Surface Water Analysis''

Table 1 displays the results of the varying anion concentrations in the creek, pond, and ground water samples.

{| class="wikitable"
|-
! Anion !! Creek Water Sample !! Pond Water Sample !! Well Water Sample !! Well Water Sample w/Softener
|-
| Chloride || 108.055 ppm || 33.8505 ppm || 18.467 ppm || Example
|-
| Nitrite || 0 ppm || 0 ppm || 0 ppm || Example
|-
| Nitrate || 13.079 ppm || 10.875 ppm || 11.9325 ppm || Example
|-
| Sulfate || 39.737 ppm || 16.8185 ppm || 13.1475 ppm || Example
|}

==Discussion==

Figure 3 demonstrates that nitrate concentrations do end up leveling off when they become high, for a linear relationship is only observed for days 1 - 28 and not for day 76. This may be due to increased algae growth due to nutrient abundance and hence increased algal nitrate uptake. Keeping this in mind, the formula can only serve a good model for when the daily rise in nitrate demonstrates a linear relationship (day 1 - 28); therefore, calculated water exchanges via the equation below should take place within this key period.

Even though the rise in daily nitrate concentrations was quantified over a 28 day span in this experiment, this rate does not serve as an absolute representation of daily nitrate increments for all year round. Sample collection took place in mid winter; therefore, during periods of increased metabolic activity, such as nesting and breeding seasons, the rate of daily rises in nitrate concentration is expected to be higher. Another factor to keep in mind is the population of the fish tank.

Though natural regulation proves to be useful in open surface water systems, there are a lot more variables in how nitrates and nitrites can enter the system, therefore, . Although mainly byproducts from aquatic life, nitrate and nitrite levels in surface and ground water can also result from agricultural runoff (fertilizers), or improper disposal of human and animal waste (). Considering the pond and creek are interconnected to each other and have equal exposure to potential agricultural runoff, it makes sense that these two systems have very similar nitrate concentrations as seen by table 1. While the creek and water samples collected came from a reservoir for aquatic life, it is likely the nitrate levels are also a result from the heavy agricultural activity surrounding the open system. Even so, the nitrate and nitrite levels are well below the EPA's guidelines for surface water not serving the purpose as drinking water.
Road-salt runoff, which primarily consists of NaCl, can pose a major threat to surface water quality and aquatic life. Even in warm weather months, when roads are not being salted, chloride levels from winter salt applications persisted in a study carried out in Milwaukee Massachusetts (5). Road salt is the primary reason why the creek water sample has a higher chloride concentration than the pond water sample (figure 1).

==References==

1. Harms, G.; Layton, A. C.; Dionsi, H. M.; Gregory, I. R.; Garrett, V. M.; Hawkins, S. A.; Robinson, K. G.; Slayer, G. S. Real-Time PCR Quantification of Nitrifying Bacteria in a Municipal Wastewater Treatment Plant. ''Am. Chem. Soc.'' '''2002''', ''37'', 343-351.

2. Ullrich, W. R.; Lazarova, J.; Ullrich, C. I.; Witt, F. G.; Aparcio, P. J. Nitrate Uptake and Extracellular Alkalinization by the Green Algae Hydrodictyon ''reticulatum'' in Blue and Red Light. ''Journal of Experimental Botany.'' '''1998''', ''49'', 1157-1162.

3. U.S. Environmental Protection Agency. https://www.epa.gov/

4. U.S. Environmental Protection Agency. Ambient Water Quality Criteria for Chloride. ''Office of Water Regulations and Standards Criteria and Standards Division''. '''1988'''.

5. Corsi, S. R.; Graczyk, D. J.; Geis, S. W.; Booth, N. L.; Richards, K. D. A Fresh Look at Road Salt: Aquatic Toxicity and Water-Quality Impacts on Local, Regional, and National Scales. ''Environmental Science & Technology''. '''2010''', ''44'', 7376-7382.

==Page History==

This page was created by Sara L Simonson in the fall of 2020

Freshwater Aquarium Microbiome

2021-09-21T04:13:56Z

Ssimonson: /* References */

==Introduction==
Microbiomes, the communities of microorganisms living together in particular habitats, are vital for maintaining ecological balance. The habitat hosting the microbial communities can be anything from the gastrointestinal tract of a cow to the soils of the land (1). Of recent particular interest are those of aquatic ecosystems, which contain diverse arrays of bacterial communities. The fish themselves oftentimes possess more microbial cells than fish cells in their bodies (2). Of increasing use in identifying the microbes present in these microbiomes is 16S amplicon sequencing. The 16S rRNA gene encodes for 16S rRNA, which is an important constituent of the prokaryotic ribosome 30S subunit. It is noteworthy to point out that this gene is only found in bacteria and archaea (3). The 16S gene has a number of conserved regions, which enables the 16S gene to be recognized across many different microbes through only a few sets of primers catered to this conserved region. The variable regions in the gene are indicative to which specific microbes are products of the amplification of the microbiomes. Up until 2012, there have not been very many studies pertaining to the characterization of the microbiome pertaining to the water associated with freshwater ornamental fishes (4)...This experiment aims to obtain a report on the bacterial community composition of the freshwater fish tank housing four generations of convict cichlids and on the saltwater fish tank, located on the 3rd floor of CSB. To accomplish this, a protocol modeled after Smith et al. and Patin et al. will be established (4,5). 600 mL of aquarium water will be filtered through a 0.22 um filter unit. The concentrated microbial biomass trapped on the filter will be treated with a Puregene Qiagen kit with some modifications from the protocol. Afterwards amplification of the bacterial 16S rRNA gene will be carried out and sequencing at the University of Illinois will ensue.

==Materials and Methods==
===Water Sampling===
Sterivex Filters
:1 L of water from the surface of the freshwater fish tank and saltwater fish tank were filtered through separate 0.22 um sterivex filter units via a peristaltic pump. Air was pushed through following filtration to initiate the removal of residual water contained in the filter units.

Nanopure Filtration
: Due to the low concentration of DNA extracted from filtering 1 L of water through the sterivex filter unit, 2 L of water from the freshwater and saltwater fish tanks were filtered through the free 0.22 um filter membranes via a reusable filter funnel and with the aid of a vacuum source.
[https://waterra.com/product/spectra-field-pro-peristaltic-pump/ Spectra Field-Pro Peristaltic Pump (~$2600)]
:- [https://youtu.be/XBaSQcmXc0s eDNA Sampler Usage]

===DNA Extraction===

DNA extraction from the 0.22 um sterivex filter unit followed the protocol outlined in the DNeasy Powerwater Sterivex Kit with no noteworthy deviations from this protocol (6). As expected, 100 ul of product was obtained. 2 ul were tested on the nanodrop to assess the quantity and quality of the DNA obtained. The DNA extracted from the freshwater sterivex filter unit absorbed at a concentration of 3.6 ng/ul at 260 nm, which was a lower concentration of DNA than expected. Its A260/A280 ratio was 1.80, which indicates that the protein and inhibitor removal steps were successful. The recorded A260/A230 ration was very low at 0.04, which may indicate the presence of excess salt left in solution, for most of the salts used in this DNA extraction protocol absorb at 230 nm. This low ratio suggests that improvements to the overall quantity of DNA obtained may be higher than 3.6 ng/ul through purification techniques aimed to get the excess salt out.

[[:File:PowerWater_Sterivex.pdf]]

===Primer Construction===

Forward primer design: 5'-AATGATACGGCGACCACCGAGATCTACAC-ATCGTACG-TATGGTAATT-GT-GTGCCAGCMGCCGCGGTAA-3' | 5'-adaptor-barcode-pad-linker-V4 region primer-3'

Reverse primer design: 5'-CAAGCAGGAAGACGGCATACGAGAT-AGTCAGTCAG-CC-GGACTACHVGGGTWTCTAAT-3' | 5'-adaptor-pad-linker-V4 region primer-3'

The pads serve the purpose of preventing hairpin formation and manipulating the melting point of the primers, for the adaptors need to anneal to the flow cell, a glass slide with lanes of two different types of oligo (complementary to the adaptors), at 65°C in order for sequencing to ensue (7). The linkers are noncomplementary to the 16S gene so that alignment for bioinformatics processing can be carried out efficiently (7). The adaptors, pads, and linkers are needed on both the forward and reverse primers because bridge amplification is utilized in Illumina sequencing platforms. During this process, one side of the adaptor sequences hybridizes to the flow cell, a complement of this original fragment is produced by a polymerase, the double strand of DNA is denatured and washed away, and then the single strand folds over and hybridizes to the second type of oligo on the flow cell (8). Polymerases generate the complementary strand, and this double stranded bridge is denatured, which results in two single stranded copies tethered to the flow cell (8). This process is repeated many times. After bridge amplification, the reverse strands are cleaved and cut off, which is why the barcode is only important for the forward primer.

===PCR Amplification===

===Sequencing===

===Bioinformatics===

==Results==

==Discussion==

==References==
1. Fierer, N. Embracing the unknown: disentangling the complexities of the soil microbiome. Nature Reviews Microbiology. 2017. 15. 579-590.

2. Savage, D. C. [[Media:32_300.pdf|Microbial ecology of the gastrointestinal tract.]] Ann Rev Microbiol. '''1977'''. ''31''. 107-133.

3. Smith, K. F.; Schmidt, V.; Rosen, G. E.; Amaral-Zettler, L. [[Media:pone.0039971.pdf|Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water.]] ''Public Library of Science One.'' '''2012'''. ''7''.

4.

5.

6. [[Media:HB-2266-002_HB_DNY_PowerWater_Sterivex_0519_WW%20(2).pdf|DNEASY PowerWater Sterivex Kit Handbook]]

7. Kozich, J. J.; Estcott, S. L.; Baxter, N. T.; Highlander, S. K.; Schloss, P. D. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Applied and Environmental Microbiology. 2013. 79 (17). 5112-5120.

8. [https://www.youtube.com/watch?v=womKfikWlxM Illumina Sequencing Technology - Illumina YouTube Video]

==Page History==
This page was created by Sara L Simonson in the spring of 2021

File:HB-2266-002 HB DNY PowerWater Sterivex 0519 WW (2).pdf

2021-09-21T04:05:20Z

Ssimonson: Ssimonson uploaded a new version of File:HB-2266-002 HB DNY PowerWater Sterivex 0519 WW (2).pdf

2021-09-20T02:36:43Z

Ssimonson: /* Primer Construction */

==Introduction==
Microbiomes, the communities of microorganisms living together in particular habitats, are vital for maintaining ecological balance. The habitat hosting the microbial communities can be anything from the gastrointestinal tract of a cow to the soils of the land (1). Of recent particular interest are those of aquatic ecosystems, which contain diverse arrays of bacterial communities. The fish themselves oftentimes possess more microbial cells than fish cells in their bodies (2). Of increasing use in identifying the microbes present in these microbiomes is 16S amplicon sequencing. The 16S rRNA gene encodes for 16S rRNA, which is an important constituent of the prokaryotic ribosome 30S subunit. It is noteworthy to point out that this gene is only found in bacteria and archaea (3). The 16S gene has a number of conserved regions, which enables the 16S gene to be recognized across many different microbes through only a few sets of primers catered to this conserved region. The variable regions in the gene are indicative to which specific microbes are products of the amplification of the microbiomes. Up until 2012, there have not been very many studies pertaining to the characterization of the microbiome pertaining to the water associated with freshwater ornamental fishes (4)...This experiment aims to obtain a report on the bacterial community composition of the freshwater fish tank housing four generations of convict cichlids and on the saltwater fish tank, located on the 3rd floor of CSB. To accomplish this, a protocol modeled after Smith et al. and Patin et al. will be established (4,5). 600 mL of aquarium water will be filtered through a 0.22 um filter unit. The concentrated microbial biomass trapped on the filter will be treated with a Puregene Qiagen kit with some modifications from the protocol. Afterwards amplification of the bacterial 16S rRNA gene will be carried out and sequencing at the University of Illinois will ensue.

==Materials and Methods==
===Water Sampling===
Sterivex Filters
:1 L of water from the surface of the freshwater fish tank and saltwater fish tank were filtered through separate 0.22 um sterivex filter units via a peristaltic pump. Air was pushed through following filtration to initiate the removal of residual water contained in the filter units.

Nanopure Filtration
: Due to the low concentration of DNA extracted from filtering 1 L of water through the sterivex filter unit, 2 L of water from the freshwater and saltwater fish tanks were filtered through the free 0.22 um filter membranes via a reusable filter funnel and with the aid of a vacuum source.
[https://waterra.com/product/spectra-field-pro-peristaltic-pump/ Spectra Field-Pro Peristaltic Pump (~$2600)]
:- [https://youtu.be/XBaSQcmXc0s eDNA Sampler Usage]

===DNA Extraction===

DNA extraction from the 0.22 um sterivex filter unit followed the protocol outlined in the DNeasy Powerwater Sterivex Kit with no noteworthy deviations from this protocol (). As expected, 100 ul of product was obtained. 2 ul were tested on the nanodrop to assess the quantity and quality of the DNA obtained. The DNA extracted from the freshwater sterivex filter unit absorbed at a concentration of 3.6 ng/ul at 260 nm, which was a lower concentration of DNA than expected. Its A260/A280 ratio was 1.80, which indicates that the protein and inhibitor removal steps were successful. The recorded A260/A230 ration was very low at 0.04, which may indicate the presence of excess salt left in solution, for most of the salts used in this DNA extraction protocol absorb at 230 nm. This low ratio suggests that improvements to the overall quantity of DNA obtained may be higher than 3.6 ng/ul through purification techniques aimed to get the excess salt out.

[[:File:PowerWater_Sterivex.pdf]]

===Primer Construction===

Forward primer design: 5'-AATGATACGGCGACCACCGAGATCTACAC-ATCGTACG-TATGGTAATT-GT-GTGCCAGCMGCCGCGGTAA-3' | 5'-adaptor-barcode-pad-linker-V4 region primer-3'

Reverse primer design: 5'-CAAGCAGGAAGACGGCATACGAGAT-AGTCAGTCAG-CC-GGACTACHVGGGTWTCTAAT-3' | 5'-adaptor-pad-linker-V4 region primer-3'

The pads serve the purpose of preventing hairpin formation and manipulating the melting point of the primers, for the adaptors need to anneal to the flow cell, a glass slide with lanes of two different types of oligo (complementary to the adaptors), at 65°C in order for sequencing to ensue (). The linkers are noncomplementary to the 16S gene so that alignment for bioinformatics processing can be carried out efficiently (). The adaptors, pads, and linkers are needed on both the forward and reverse primers because bridge amplification is utilized in Illumina sequencing platforms. During this process, one side of the adaptor sequences hybridizes to the flow cell, a complement of this original fragment is produced by a polymerase, the double strand of DNA is denatured and washed away, and then the single strand folds over and hybridizes to the second type of oligo on the flow cell (). Polymerases generate the complementary strand, and this double stranded bridge is denatured, which results in two single stranded copies tethered to the flow cell (). This process is repeated many times. After bridge amplification, the reverse strands are cleaved and cut off, which is why the barcode is only important for the forward primer.

===PCR Amplification===

===Sequencing===

===Bioinformatics===

==Results==

==Discussion==

==References==

2. Savage, D. C. [[Media:32_300.pdf|Microbial ecology of the gastrointestinal tract.]] Ann Rev Microbiol. '''1977'''. ''31''. 107-133.

3. Smith, K. F.; Schmidt, V.; Rosen, G. E.; Amaral-Zettler, L. [[Media:pone.0039971.pdf|Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water.]] ''Public Library of Science One.'' '''2012'''. ''7''.

==Page History==
This page was created by Sara L Simonson in the spring of 2021

Freshwater Aquarium Microbiome

2021-09-20T01:38:04Z

Ssimonson: /* Primer Construction */

==Introduction==
Microbiomes, the communities of microorganisms living together in particular habitats, are vital for maintaining ecological balance. The habitat hosting the microbial communities can be anything from the gastrointestinal tract of a cow to the soils of the land (1). Of recent particular interest are those of aquatic ecosystems, which contain diverse arrays of bacterial communities. The fish themselves oftentimes possess more microbial cells than fish cells in their bodies (2). Of increasing use in identifying the microbes present in these microbiomes is 16S amplicon sequencing. The 16S rRNA gene encodes for 16S rRNA, which is an important constituent of the prokaryotic ribosome 30S subunit. It is noteworthy to point out that this gene is only found in bacteria and archaea (3). The 16S gene has a number of conserved regions, which enables the 16S gene to be recognized across many different microbes through only a few sets of primers catered to this conserved region. The variable regions in the gene are indicative to which specific microbes are products of the amplification of the microbiomes. Up until 2012, there have not been very many studies pertaining to the characterization of the microbiome pertaining to the water associated with freshwater ornamental fishes (4)...This experiment aims to obtain a report on the bacterial community composition of the freshwater fish tank housing four generations of convict cichlids and on the saltwater fish tank, located on the 3rd floor of CSB. To accomplish this, a protocol modeled after Smith et al. and Patin et al. will be established (4,5). 600 mL of aquarium water will be filtered through a 0.22 um filter unit. The concentrated microbial biomass trapped on the filter will be treated with a Puregene Qiagen kit with some modifications from the protocol. Afterwards amplification of the bacterial 16S rRNA gene will be carried out and sequencing at the University of Illinois will ensue.

==Materials and Methods==
===Water Sampling===
Sterivex Filters
:1 L of water from the surface of the freshwater fish tank and saltwater fish tank were filtered through separate 0.22 um sterivex filter units via a peristaltic pump. Air was pushed through following filtration to initiate the removal of residual water contained in the filter units.

Nanopure Filtration
: Due to the low concentration of DNA extracted from filtering 1 L of water through the sterivex filter unit, 2 L of water from the freshwater and saltwater fish tanks were filtered through the free 0.22 um filter membranes via a reusable filter funnel and with the aid of a vacuum source.
[https://waterra.com/product/spectra-field-pro-peristaltic-pump/ Spectra Field-Pro Peristaltic Pump (~$2600)]
:- [https://youtu.be/XBaSQcmXc0s eDNA Sampler Usage]

===DNA Extraction===

DNA extraction from the 0.22 um sterivex filter unit followed the protocol outlined in the DNeasy Powerwater Sterivex Kit with no noteworthy deviations from this protocol (). As expected, 100 ul of product was obtained. 2 ul were tested on the nanodrop to assess the quantity and quality of the DNA obtained. The DNA extracted from the freshwater sterivex filter unit absorbed at a concentration of 3.6 ng/ul at 260 nm, which was a lower concentration of DNA than expected. Its A260/A280 ratio was 1.80, which indicates that the protein and inhibitor removal steps were successful. The recorded A260/A230 ration was very low at 0.04, which may indicate the presence of excess salt left in solution, for most of the salts used in this DNA extraction protocol absorb at 230 nm. This low ratio suggests that improvements to the overall quantity of DNA obtained may be higher than 3.6 ng/ul through purification techniques aimed to get the excess salt out.

[[:File:PowerWater_Sterivex.pdf]]

===Primer Construction===

Forward primer design: 5'-AATGATACGGCGACCACCGAGATCTACAC-ATCGTACG-TATGGTAATT-GT-GTGCCAGCMGCCGCGGTAA-3' | 5'-adaptor-barcode-pad-linker-V4 region primer-3'

Reverse primer design: 5'-CAAGCAGGAAGACGGCATACGAGAT-AGTCAGTCAG-CC-GGACTACHVGGGTWTCTAAT-3' | 5'-adaptor-pad-linker-V4 region primer-3'

The pads serve the purpose of preventing hairpin formation and manipulating the melting point of the primers, for the adaptors need to anneal to the flow cell, a glass slide with lanes of two different types of oligo (complementary to the adaptors), at 65°C in order for sequencing to ensue (). The linkers are noncomplementary to the 16S gene so that alignment for bioinformatics processing can be carried out efficiently (). The adaptors, pads, and linkers are needed on both the forward and reverse primers because bridge amplification is utilized in Illumina sequencing platforms. During this process, one side of the adaptor sequences hybridizes to the flow cell, a complement of this original fragment is produced by a polymerase, the double strand of DNA is denatured and washed away, and then the single strand folds over and hybridizes to the second type of oligo on the flow cell (). Polymerases generate the complementary strand, and this double stranded bridge is denatured, which results in two single stranded copies tethered to the flow cell (). This process is repeated many times. After bridge amplification, the reverse strands are cleaved and cut off, which is why the barcode is only important for the forward primer.

===PCR Amplification===

===Sequencing===

===Bioinformatics===

==Results==

==Discussion==

==References==

2. Savage, D. C. [[Media:32_300.pdf|Microbial ecology of the gastrointestinal tract.]] Ann Rev Microbiol. '''1977'''. ''31''. 107-133.

3. Smith, K. F.; Schmidt, V.; Rosen, G. E.; Amaral-Zettler, L. [[Media:pone.0039971.pdf|Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water.]] ''Public Library of Science One.'' '''2012'''. ''7''.

==Page History==
This page was created by Sara L Simonson in the spring of 2021

Freshwater Aquarium Microbiome

2021-09-20T01:13:20Z

Ssimonson: /* Primer Construction */

==Introduction==
Microbiomes, the communities of microorganisms living together in particular habitats, are vital for maintaining ecological balance. The habitat hosting the microbial communities can be anything from the gastrointestinal tract of a cow to the soils of the land (1). Of recent particular interest are those of aquatic ecosystems, which contain diverse arrays of bacterial communities. The fish themselves oftentimes possess more microbial cells than fish cells in their bodies (2). Of increasing use in identifying the microbes present in these microbiomes is 16S amplicon sequencing. The 16S rRNA gene encodes for 16S rRNA, which is an important constituent of the prokaryotic ribosome 30S subunit. It is noteworthy to point out that this gene is only found in bacteria and archaea (3). The 16S gene has a number of conserved regions, which enables the 16S gene to be recognized across many different microbes through only a few sets of primers catered to this conserved region. The variable regions in the gene are indicative to which specific microbes are products of the amplification of the microbiomes. Up until 2012, there have not been very many studies pertaining to the characterization of the microbiome pertaining to the water associated with freshwater ornamental fishes (4)...This experiment aims to obtain a report on the bacterial community composition of the freshwater fish tank housing four generations of convict cichlids and on the saltwater fish tank, located on the 3rd floor of CSB. To accomplish this, a protocol modeled after Smith et al. and Patin et al. will be established (4,5). 600 mL of aquarium water will be filtered through a 0.22 um filter unit. The concentrated microbial biomass trapped on the filter will be treated with a Puregene Qiagen kit with some modifications from the protocol. Afterwards amplification of the bacterial 16S rRNA gene will be carried out and sequencing at the University of Illinois will ensue.

==Materials and Methods==
===Water Sampling===
Sterivex Filters
:1 L of water from the surface of the freshwater fish tank and saltwater fish tank were filtered through separate 0.22 um sterivex filter units via a peristaltic pump. Air was pushed through following filtration to initiate the removal of residual water contained in the filter units.

Nanopure Filtration
: Due to the low concentration of DNA extracted from filtering 1 L of water through the sterivex filter unit, 2 L of water from the freshwater and saltwater fish tanks were filtered through the free 0.22 um filter membranes via a reusable filter funnel and with the aid of a vacuum source.
[https://waterra.com/product/spectra-field-pro-peristaltic-pump/ Spectra Field-Pro Peristaltic Pump (~$2600)]
:- [https://youtu.be/XBaSQcmXc0s eDNA Sampler Usage]

===DNA Extraction===

DNA extraction from the 0.22 um sterivex filter unit followed the protocol outlined in the DNeasy Powerwater Sterivex Kit with no noteworthy deviations from this protocol (). As expected, 100 ul of product was obtained. 2 ul were tested on the nanodrop to assess the quantity and quality of the DNA obtained. The DNA extracted from the freshwater sterivex filter unit absorbed at a concentration of 3.6 ng/ul at 260 nm, which was a lower concentration of DNA than expected. Its A260/A280 ratio was 1.80, which indicates that the protein and inhibitor removal steps were successful. The recorded A260/A230 ration was very low at 0.04, which may indicate the presence of excess salt left in solution, for most of the salts used in this DNA extraction protocol absorb at 230 nm. This low ratio suggests that improvements to the overall quantity of DNA obtained may be higher than 3.6 ng/ul through purification techniques aimed to get the excess salt out.

[[:File:PowerWater_Sterivex.pdf]]

===Primer Construction===

Forward primer design: 5'-AATGATACGGCGACCACCGAGATCTACAC-ATCGTACG-TATGGTAATT-GT-GTGCCAGCMGCCGCGGTAA-3' | 5'-adaptor-barcode-pad-linker-V4 region primer-3'

Reverse primer design: 5'-CAAGCAGGAAGACGGCATACGAGAT-AGTCAGTCAG-CC-GGACTACHVGGGTWTCTAAT-3' | 5'-adaptor-pad-linker-V4 region primer-3'

The pads serve the purpose of preventing hairpin formation and manipulating the melting point of the primers, for the adaptors need to anneal to the flow cell at 65°C in order for sequencing to ensue. The linkers are noncomplementary to the 16S gene so that alignment for bioinformatics processing can be carried out efficiently.

===PCR Amplification===

===Sequencing===

===Bioinformatics===

==Results==

==Discussion==

==References==

2. Savage, D. C. [[Media:32_300.pdf|Microbial ecology of the gastrointestinal tract.]] Ann Rev Microbiol. '''1977'''. ''31''. 107-133.

3. Smith, K. F.; Schmidt, V.; Rosen, G. E.; Amaral-Zettler, L. [[Media:pone.0039971.pdf|Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water.]] ''Public Library of Science One.'' '''2012'''. ''7''.

==Page History==
This page was created by Sara L Simonson in the spring of 2021