0
EVALUATION OF ENHANCED BIOLOGICAL
WASTEWATER TREATMENT PRODUCT
FOR LIVESTOCK OPERATIONS Submitted to Environment Depot
ABSTRACT Evaluation of dairy manure over a four-month period to
determine changing nutrient levels, solids profile,
microbiology, and plant growth after the continuous
addition of a bioactive compound.
Carien Vandenberg M.Sc & Ruth Elvestad December 12, 2014
1
1.0 ACKNOWLEDGEMENTS ........................................................................................................................... 2
2.0 EXECUTIVE SUMMARY ............................................................................................................................ 2
3.0 PROJECT BACKGROUND .......................................................................................................................... 3
3.1 Objectives............................................................................................................................................ 3
4.0 MATERIALS AND METHODS .................................................................................................................... 3
4.1 Lagoon Study ....................................................................................................................................... 3
4.1.1 Baseline Testing ........................................................................................................................... 4
4.1.2 Acti-Zyme Addition ...................................................................................................................... 4
4.1.3 Testing Schedule .......................................................................................................................... 4
4.1.4 Parameters Measured ................................................................................................................. 6
4.2 Controlled Study ............................................................................................................................... 10
4.2 1 Trial Design ................................................................................................................................. 10
4.3 Greenhouse Study ............................................................................................................................. 12
5.0 RESULTS................................................................................................................................................. 13
5.1 Lagoon Study ..................................................................................................................................... 13
5.2 Controlled Study ............................................................................................................................... 16
5.3 Greenhouse Study ............................................................................................................................. 18
6.0 CONCLUSIONS ....................................................................................................................................... 22
7.0 RECOMMENDATIONS............................................................................................................................ 22
8.0 REFERENCES .......................................................................................................................................... 24
9.0 APPENDIX .............................................................................................................................................. 25
Appendix A – Extractable Nitrate, Phosphate and Potassium in Soil ..................................................... 26
Appendix B –Extractable Ammonium and Nitrate .................................................................................. 28
Appendix C - Biochemical Oxygen Demand ............................................................................................ 30
Appendix D – Total Dissolved Solids ....................................................................................................... 31
Appendix E – Total Suspended Solids ..................................................................................................... 33
Appendix F - Moisture ............................................................................................................................. 34
Appendix G – Exova Lagoon Study Data Results ..................................................................................... 35
Appendix H - Exova Controlled Study Data Results ................................................................................ 48
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1.0 ACKNOWLEDGEMENTS
The authors would like to thank the work of summer research assistants Sydel Falkenburger, Cesar
Reyes-Bernabe, and Chen-Hsun (Tom) Yang for their work shipping samples, managing data files, and
organizing data for the report. Our gratitude goes out to Sietze Sietzema and his farm team for their
support in this research by allowing access to his lagoon and incorporating the product in his dairy
manure system.
2.0 EXECUTIVE SUMMARY
The project was designed to evaluate the changes in manure nutrient profile, physical parameters
Including various water quality tests, and plant growth trials when Acti-Zyme was added to the dairy
manure system.
The lagoon study investigated the use of Acti-Zyme in an applied agricultural setting. Although used
extensively in many lagoon systems across Canada, this was the first objective look at manure nutrient
and biological activity over a four month period. As in all applied research and in situ applied research,
various factors have an effect on outcome; rainfall events, fluctuations in temperature and feeding
regime, additives, and wash water just to name a few. Overall the farmer noted that the lagoon
pumped out well at the conclusion of the study and did not negatively affect manure agitation.
Additionally, a bench-scale controlled study comparing manure samples with the Acti-Zyme to a control
(no Acti-Zyme added) was also completed which eliminated various influencing factors that the lagoon
trial could not. The study showed trends of reducing Biochemical Oxygen Demand (BOD) and shifts in
dissolved solids to suspended solids over time, however trends were noted in both the treated and
untreated manure samples.
Finally, the growth trials in the greenhouse provided a preliminary look at the impact of manure on plant
germination and growth. In some cases, germination was delayed which resulted in smaller plants at
day 15 post planting. The general observation is that rate of application does affect growth perhaps due
to moisture content and the coverage the manure had on seeds germinating which may have impeded
emergence. Overall, initial trial results show that manure, regardless of treatment or rate, was equal to
or better than the control suggesting manures did not negatively impact germination and growth.
Considerations for future research include investigating dose response and impact of various factors in
an applied open dairy lagoon setting such as wash water, rain events, agitation and stratification and
oxygen levels at varying depths of manure. This requires multiple experimental units to ensure
replication of various treatments and compare results.
Further greenhouse and field studies using the manure treated with Acti-Zyme and studying the
resulting germination, plant growth, and soil microbial levels, can confirm the relationship between the
bioactive compound and agricultural applications.
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3.0 PROJECT BACKGROUND
The management of manure is one of the most difficult, expensive and potentially limiting problems
facing the agricultural industry today. This problem is particularly prevalent among that portion of the
agricultural industry that pertains to confined animal feeding operations (CAFOs), such as those used for
dairy and swine. Typically the manure is stored in earthen ponds or lagoons and then used for land
fertilization (land spreading). Enclosed storage and treatment facilities for odor control, such as those
used at municipal treatment plants, are too expensive to be practical for an agricultural operation.
Environment Depot for Waste Water to Energy Ltd (ED), identified a nation-wide need for an economical
and environmental process for handling waste from CAFO’s and responded with the development of a
bioactive compound called Acti-Zyme for the rapid breakdown of organic matter in manure entrained
waters. This waste treatment system eliminates the reliance on manure storage lagoons which in turn
reduces offensive odors, as well as the discharge of harmful contaminants, pathogens and greenhouse
gases. The Acti-Zyme treatment process produces cleaner water reducing the recycling time of waste
water to meet the water needs of the farm operation, in turn reducing the burden on local water
resources for freshwater.
3.1 Objectives
The project was designed to evaluate the changes in manure nutrient profile, physical parameters
Including various water quality tests, and plant growth trials when Acti-Zyme was added to the dairy
manure system. Additionally, a bench-scale controlled study comparing manure samples with the Acti-
Zyme to a control (no Acti-Zyme added) was also completed.
The following objectives were achieved:
(1) Determination of the concentration of nutrients in the manure entrained wastewater including:
nitrogen (ammonia, nitrite/nitrate, total nitrogen), available phosphorus (phosphate), total phosphorus,
(2) Standard water quality tests Including: Biochemical Oxygen Demand (BOD), pH, Total Dissolved
Solids (TDS), Total Suspended Solids (TSS), fecal coliforms, and
(3) Plant growth tests involving the germination and vigor of various seeds in two types of media to
assess phytotoxicity and nutrient value of manure.
4.0 MATERIALS AND METHODS
4.1 Lagoon Study
Environment Depot (ED) identified and engaged a local dairy farmer with a suitable lagoon to conduct
the trial. Olds College Centre for Innovation (OCCI) sampled manure from a lagoon setting containing
manure and waste water from a 130 head dairy herd operation 10 Km North West of Olds, Alberta,
Canada. Samples were taken bi-monthly from the lagoon for biochemical, nutrient, and microbiological
analyses. The system was monitored for four months from May through to November, 2014.
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4.1.1 Baseline Testing
Baseline lagoon sampling was completed with three samples
collected over a 2 week period. Samples collected were a
homogenous sample of manure collected and stored over
winter in an outdoor, uncovered lagoon (see Figure 1).
Samples were collected on May 15 from the outdoor lagoon
after a period of agitation. Additional material was collected
from the lagoon at this time which provided baseline data
and manure to conduct the small-scale controlled study.
Additional baseline samples of manure were collected on
May 27 and June 2 in the lagoon near the outlet pipe. This
represented fresh manure that was flowing from the barn into
the lagoon. This was labelled as the North sampling site.
Subsequent samples collected in the lagoon at the dairy barn outlet pipe were also labelled as North Site
samples. A secondary sample was collected mid-way during the trial from the opposite end of the
lagoon which was labelled South Site sample and represented manure that had moved across the
lagoon and was hypothesised to have more activity time with the bioactive compound.
4.1.2 Acti-Zyme Addition
Following baseline testing, Acti-Zyme was added in 2 lb bag Increments (prepackaged bags were used
which were enveloped in a dissolvable bag) by tossing a bag into the scraper system inside the dairy
barn. The first bag was added on June 2, 2014 following baseline sampling. At this time, three
additional bags were added to the outdoor lagoon to each of the far corners of the lagoon – furthest
from the outlet pipe to kick-start activity in the outdoor lagoon. One bag was added to the scraper
system every other day thereafter which mixed with the manure collected in the barn scraper prior to
flowing into the outdoor lagoon.
A second large dose of the product was made Aug 19 (Day 78) when the client added an additional 5
bags (10 lbs) to the outdoor lagoon. The farm team continued to add product every other day in the
barn until the conclusion of the trial.
4.1.3 Testing Schedule
Sampling was scheduled to be performed bi-monthly
at the outlined interval times shown in Table 1. Initial
sampling occurred at the North Lagoon Site which
was located by the outlet pipe near the barn. As
sampling from this site continually collected manure
that was just recently introduced to the Acti-Zyme
Figure 1. Agitation of stored, aged dairy
manure on May 15, 2014.
Figure 2. Sampling of manure at the
lagoon’s north site on July 22 (Day 50).
5
product, additional samples were taken on the south side of the lagoon – opposite from the outlet pipe.
The samples from the South side of the lagoon (South Site) were helpful in quantifying how manure was
developing on the far side of the lagoon which had more time to engage with the Acti-Zyme product.
Samples were collected using a long handled scoop from the bank of the lagoon (see Figure 2) at an
average depth of 10cm below the manure surface.
Table 1: Lagoon Sampling Dates, Day of Trial, and Testing Type
Date Day Testing Type
May 15 Baseline Baseline-Full
May 26 Baseline Baseline-Full
June 2 Baseline Baseline-Full
PRODUCT ADDED- June 2
June 10 7 Sampling – Basic
June 24 22 Sampling - Full
July 8 36 Sampling – Basic
July 22 50 Sampling - Full
August 5 64 Sampling - Basic
August 19 78 Sampling - Full
September 2 92 Sampling – Basic
September 16 106 Sampling - Full
October 3 123 Sampling - Full
October 6 126 Sampling - Full
November 4 155 Sampling - Full
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Samples were sent for either full testing or basic testing at Exova Laboratories (Calgary, AB) which
included the parameters outlined in Table 2.
Table 2. Summary of Parameters in Full and Basic Testing
Test Parameter Full Testing Basic Testing
Biochemical Oxygen Demand (BOD) X
Total Dissolved Solids (TDS) X
Total Suspended Solids (TSS) X
Nitrate X X
Nitrite X X
Ammonia X X
Total Nitrogen X X
Total Phosphorus X X
Available Phosphorus X X
pH X
Fecal Coliforms X
4.1.4 Parameters Measured
A brief summary of the testing parameters and their activity and influence, particularly in water
environments, is outlined below.
Nitrogen
Nitrogen is important to all life. Nitrogen in the atmosphere or in the soil can go through many complex
chemical and biological changes. It can be combined into living and non-living material and return back
to the soil or air in a continuing cycle called the nitrogen cycle.
Nitrogen occurs in natural waters in various forms, Including nitrate, or NO3, nitrite, or NO2, and
ammonia, or NH3. Nitrate is the most common form tested. Test results are usually expressed as nitrate-
nitrogen, or NO3-N, which simply means nitrogen in the form of nitrate. Ammonia is the least stable
form of nitrogen and thus difficult to measure accurately. Nitrite is less stable and usually present in
much lower amounts than nitrate. These three compounds are interrelated through the process of
nitrification, the biological oxidation of ammonia to nitrate. In this process nitrite is produced as an
intermediate product.
Order of decreasing oxidation state:
Nitrate → Nitrite → Ammonia → Organic Nitrogen
(Stable) → → →→ → →→ → →(Unstable)
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In relatively stable, oxygenated natural water systems the oxidation of nitrite to nitrate is rapid, but the
conversion of NH3 to NO2- is the rate limiting step in the total process. (1991, Streamkeeper's Field
Guide: Watershed Inventory and Stream Monitoring Methods).
Nitrate
Nitrate, or NO3 generally occurs in trace quantities in surface water. It is the essential nutrient for many
photosynthetic autotrophs and has been identified as the growth limit nutrient. It is only found in small
amounts in fresh domestic wastewater. Nitrate is a less serious environmental problem, it can be found
in relatively high concentrations where it is relatively nontoxic to aquatic organisms. When nitrate
concentrations become excessive, however, and other essential nutrient factors are present,
eutrophication and associated algal blooms can be become a problem. See Appendix A and B for testing
methods.
Organic Nitrogen
Organic Nitrogen is the by-product of living organisms. It Includes such natural materials as proteins and
peptides, nucleic acids and urea, and numerous synthetic organic materials. Organic Nitrogen is
determined by taking Total Nitrogen and subtracting ammonia, nitrate and nitrite.
Ammonia, or NH3
It is one of the most important pollutants in the aquatic environment because of its relatively highly
toxic nature and its ubiquity in surface water systems. It is discharged in large quantities in industrial,
municipal and agricultural waste waters. In aqueous solutions, ammonia assumes two chemical forms:
NH4+ - ionized (less/nontoxic) and NH3 - unionized (toxic). See Appendix B for testing Methods.
Total NH3: Total ammonia is the sum of the NH3 and NH4+.
Nitrite
Nitrite, or NO2 is extremely toxic to aquatic life, however, is usually present only in trace amounts in
most natural freshwater systems because it is rapidly oxidized to nitrate. In sewage treatment plants
using nitrification process to convert ammonia to nitrate, the process may be impeded, causing
discharge of nitrite at elevated concentrations into receiving waters.
The conversion process is affected by several factors, Including pH, temperature and dissolved oxygen,
number of nitrifying bacteria and presence of inhibiting compounds. Total ammonia in wastewater
treatment systems consists of NH3- plus NH4+. If pH of the solution Increases either naturally or by
addition of a base, the concentration of unionized NH3 Increases. It impedes the conversion of nitrite to
nitrate, causing nitrite to accumulate. When the pH decreases, as NH4+ and NO2 are oxidized an Increase
in HNO2 concentration occurs. Nitrous acid inhibits both nitrobacteria and nitrosomonos bacteria – this
inhibition can result in an increase in nitrite.
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Phosphorus
Phosphorus is often the limiting nutrient for plant growth, meaning it is in short supply relative to
nitrogen. Phosphorus usually occurs in nature as phosphate, which is a phosphorous atom combined
with four oxygen atoms, or PO43-. Phosphate that is bound to plant or animal tissue is known as organic
phosphate. Phosphate that is not associated with organic material is known as inorganic phosphate.
Both forms are present in aquatic systems and may be either dissolved in water or suspended (attached
to particles in the water column). See Appendix A for testing methods.
Inorganic phosphate is often referred to as orthophosphate or reactive phosphorous. It is the form most
readily available to plants, and thus may be the most useful indicator of immediate potential problems
with excessive plant and algae growth. Testing for total phosphorous (both inorganic and organic
phosphate) provides you with a more complete measure of all the phosphorus that is actually in the
water (1991, Streamkeeper's Field Guide: Watershed Inventory and Stream Monitoring Methods).
Solids
Total Solids is a measure of the suspended and dissolved solids in a body of water. Thus, it is related to
both conductivity and turbidity. To measure total suspended and dissolved solids, a sample of water is
placed in a drying oven to evaporate the water, leaving the solids. To measure dissolved solids, the
sample is filtered before it is dried and weighed. To calculate the suspended solids, the weight of the
dissolved solids is subtracted from the total solids (1991, Streamkeeper's Field Guide: Watershed
Inventory and Stream Monitoring Methods). See Appendix D and E for testing methods.
Microbiology
Human and animal wastes carried to stream systems are sources of pathogenic or disease-causing,
bacteria and viruses. The disease causing organisms are accompanied by other common types of
nonpathogenic bacteria found in animal intestines, such as coliform bacteria which Include enterococci
bacteria and E. coli.
Fecal coliform bacteria are not usually disease-causing agents themselves. However, high concentrations
suggest the presence of disease-causing organisms. Fecal coliforms are used as indicator organisms;
they are indicative of the probability of finding pathogenic organisms in a stream.
To measure indicator bacteria, water samples must be collected in sterilized containers. The samples are
forced through a filter and Incubated at a specific temperature for a certain amount of time. The
resulting colonies that form during Incubation are counted and recorded as the number of colony
producing units per 100 mL of water (1991, Streamkeeper's Field Guide: Watershed Inventory and
Stream Monitoring Methods).
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pH
pH is an important limiting chemical factor for aquatic life. If the water in a stream is too acidic or basic,
the H+ or OH- ion activity may disrupt aquatic organisms’ biochemical reactions by either harming or
killing the stream organisms.
pH is expressed in a scale with ranges from 1 to 14. A solution with a pH less than 7 has more H+ activity
than OH-, and is considered acidic. A solution with a pH value greater than 7 has more OH- activity than
H+, and is considered basic. The pH scale is logarithmic, meaning that as you go up and down the scale,
the values change in factors of ten. A one-point pH change indicates the strength of the acid or base has
Increased or decreased tenfold.
Streams generally have pH values ranging between 6 and 9, depending upon the presence of dissolved
substances that come from bedrock, soils and other materials in the watershed.
Biochemical Oxygen Demand
The Biochemical or Biological Oxygen Demand (BOD) is the amount of oxygen consumed by bacteria in
the decomposition of organic material. Whereas Chemical Oxygen Demand (COD) is the oxygen required
for the oxidation of various chemicals in the water, such as sulfides, ferrous iron and ammonia. While a
dissolved oxygen test tells you how much oxygen is available, a BOD test tells you how much oxygen is
being consumed by organisms.
BOD is determined by measuring the dissolved oxygen level in a freshly collected sample and comparing
it to the dissolved oxygen level in a sample that was collected at the same time but Incubated under
specific conditions for a certain number of days. The difference in the oxygen readings between the two
samples in the BOD is recorded in units of mg/L.
Unpolluted, natural waters should have a BOD of 5 mg/L or less. Raw sewage may have BOD levels
ranging from 150 – 300 mg/L (1991, Streamkeeper's Field Guide: Watershed Inventory and Stream
Monitoring Methods). See Appendix C for testing methods.
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4.2 Controlled Study
A controlled study was performed because the open pit lagoon is both the sample site and experimental
site for this project. Because of this, there are various potential factors that can influence the data
generated during the study. These factors include weather, temperature, water additions, chemical
additions and other unknown variables that can influence the data. OCCI recommended conducting a
controlled study with a true control (manure untreated with Acti-Zyme) to that with Acti-Zyme treated
manure. This controlled study took place concurrently with the lagoon trial.
4.2 1 Trial Design
A separate controlled study was also initiated to compare the effects
of Acti-Zyme addition to data generated from manure with no
product added. Aged manure samples were used for the controlled
trial which were sampled from the dairy lagoon on May 15 (see
Figure 3).
This trial was implemented and housed at Olds College Composting
Technology Centre in an indoor facility. The controlled trial
replicated the conditions of the lagoon. The experiment used six 23L
clear plastic carboys. Two treatments were tested: a control
treatment where only manure (no Acti-Zyme) was added and a
treatment where Acti-Zyme was added weekly. Three replicates for a
total of six experimental units made up the controlled study trial (see
Figure 4).
At the beginning of the trial, each treated carboy received 10 L of
manure mixed with the equivalent of 3 g (one teaspoon)/500mL for a
total Acti-Zyme addition of 60 g per 10L of manure at the outset. This
was added on June 17 (Day 0) and was thoroughly mixed with the
manure in the carboy. The control or untreated carboys had 10L added
and mixed without the compound.
Weekly, 0.5L of manure was added to each carboy for 20 consecutive
weeks. The manure used was manure collected at the Dairy on May 15
and was stored in sealed buckets onsite. At each weekly 0.5L manure
addition of the treated carboys, there was 3 g of Acti-Zyme added. The
control group only received 0.5 L of manure.
A total of 20L of manure volume was realized in each carboy at the
conclusion of the trial. The controlled study had a representative
manure sample analyzed at the beginning of the trial, mid-way through the trial (at 2 months or Day 63),
and at the conclusion of the trial (end of 4 month period Day 133). The samples collected were tested by
Exova Laboratories (Calgary, AB) for full testing (see 4.1.4 for parameter details). Sampling was done by
Figure 3. Additional manure
collected for the controlled study on
May 15, 2014.
Figure 4. Preparation of the six
experimental units prior to the
addition of Acti-Zyme.
11
completely mixing the carboy prior to sample collection. Sampling was done on original samples
collected on May 15 and again on two carboys on August 19 (Day 63 of the experiment). Finally, a
sample was taken at the conclusion of the study on October 28 (Day 133 of the experiment) on the
same carboys sampled in August. To compare the results of unshaken carboys, two more samples were
taken from carboys which were not shaken (untouched)
during the entire trial.
A gas collection balloon was added to the mouth of the
vessels periodically to estimate the amount of gas produced
from each vessel. Temperature data was recorded daily
using a temperature logger (HOBOTM Onset 12) and all
vessels were housed in an enclosed building in the shade to
prevent excessive heating of the manure. Black plastic
covered the walls of the vessels to prevent light from shining
on the manure through the clear carboy walls (see Figure 5).
Table 3 shows the application and testing schedule during
the trial.
Plants were grown in a 20C± 5°C greenhouse where the day
length was 10 hours light: 14 hours dark (10L:14D). Plants
were checked daily and watered with distilled water as needed.
Table 3. Schedule of Acti-Zyme and Manure additions and Sampling for Testing Dates
Date Day Wk Activity Testing
May 15t Pre-Trial Collect Manure Sampling for Testing
June 9 Pre-Trial Confirm Rates and additions
June 17 Day 0 Add Compound to 10L - 60g to 10L manure in carboy
June 24 Day 7 2 Add 3 grams compound with 500g manure to treated group
July 1 Day 14 3 Add 3 grams compound with 500g manure to treated group
July 8 Day 21 4 Add 3 grams compound with 500g manure to treated group
July 15 Day 28 5 Add 3 grams compound with 500g manure to treated group
July 22 Day 35 6 Add 3 grams compound with 500g manure to treated group
July 29 Day 42 7 Add 3 grams compound with 500g manure to treated group
Aug 5 Day 49 8 Add 3 grams compound with 500g manure to treated group
Aug 12 Day 56 9 Add 3 grams compound with 500g manure to treated group
Aug 19 Day 63 10 Add 3 grams compound with 500g manure to treated group Sampling for Testing
Aug 26 Day 70 11 Add 3 grams compound with 500g manure to treated group
Sept 2 Day 77 12 Add 3 grams compound with 500g manure to treated group
Sept 9 Day 84 13 Add 3 grams compound with 500g manure to treated group
Sept 16 Day 91 14 Add 3 grams compound with 500g manure to treated group
Sept 23 Day 98 15 Add 3 grams compound with 500g manure to treated group
Sept 30 Day 105 16 Add 3 grams compound with 500g manure to treated group
Oct 7 Day 112 17 Add 3 grams compound with 500g manure to treated group
Oct 14 Day 119 18 Add 3 grams compound with 500g manure to treated group
Oct 21 Day 126 19 Add 3 grams compound with 500g manure to treated group
Oct 28 Day 133 20 Add 3 grams compound with 500g manure to treated group Sampling for Testing
Figure 5. Control carboys with logger
cables for temperature monitoring and
balloon gas production observation.
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4.3 Greenhouse Study
To determine the phytotoxicity and nutrient effects on plants,
three rates of manure from three sources were tested on a
variety of crops. The manure sources were: lagoon manure
which was collected on October 6, 2014 after agitation,
treated manure from the control study, and untreated manure
also from the controlled study. Each manure was mixed
thoroughly and applied to soil and soilless media planted with
either barley (CDC Meredith) or corn (Dekalb C26-28RIB
Complete ®) in one of three application rates in a blocked
design. Five seeds were planted per replicate. Three
replicates were used for each rate of manure application.
Manure moisture was determined prior to application.
In addition, fine curled peppercress seed (Stokes, Thorold, AB) was also tested in soilless media at three
rates for all manure samples described above. Germination was monitored and recorded at day 6, 10
and 15 for all plants. Individual plant heights were recorded on day 15 post planting. Figure 6 shows the
trays used. Figure 7 shows the experiment layout and colour coding of the trials. Manure was added at
the appropriate designated rate to each row. Blue staked rows were untreated manure additions, red
stakes denoted lagoon manure additions and orange staked rows denoted treated manure additions.
Media Type
Rate A Rate B Rate C
Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3
Control Untreated
Manure Untreated
Manure Untreated
Manure Untreated
Manure Untreated
Manure Untreated
Manure Untreated
Manure Untreated
Manure Untreated
Manure
Control Lagoon Manure
Lagoon Manure
Lagoon Manure
Lagoon Manure
Lagoon Manure
Lagoon Manure
Lagoon Manure
Lagoon Manure
Lagoon Manure
Control Treated Manure
Treated Manure
Treated Manure
Treated Manure
Treated Manure
Treated Manure
Treated Manure
Treated Manure
Treated Manure
Figure 7. A depiction of the experimental layout of the greenhouse trials.
Rate of manure application was determined by following rates typically used in industry. Application
rates of 8,000 to 12,000 imperial gallons/acre were suggested by the client and three rates were tested
in the greenhouse study based on this suggestion. Table 4 below describes the rates and calculations
used.
Table 4. Rates used in agricultural applications and the resulting volumes used in the greenhouse
study.
Rate Imperial Gallons/Acre mL/Tray Well
A 4,000 10
B 8,000 20
C 12,000 30
Figure 6. Barley at day 15 post planting
showing germination and height differences
when given various rates of one of three
manure treatments.
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5.0 RESULTS
5.1 Lagoon Study
Figure 8 below shows the average temperature and rainfall during the lagoon trial. Data was sourced
from Alberta Agriculture website from the nearest weather station which was Olds College.
http://agriculture.alberta.ca/acis/alberta-weather-data-viewer.jsp
Figure 8. Summary of precipitation and average temperature during the lagoon trial.
Large precipitation events would be expected to impact the concentration of nutrients in the lagoon and
may cause turbulent mixing and Increased suspended solids. Large precipitation events were
experienced in mid-May, mid-June and mid-August.
Table 5 summarizes the analytical data collected from the lagoon over the course of the trial. The first
sampling events were intended to provide baseline data for the water quality parameters of interest.
The baseline analysis of the influent (north site) shows approximate values for BOD at 12,000mg/L,
nitrite at 7.5ug/g, nitrate at <600ug/g, variable ammonium levels (6920-18500ug/g), total nitrogen at
1.6% dry weight, available phosphorus at 3200ug/g, total phosphorus at 5000mg/kg, TDS of 12,500mg/L,
TSS of 21,000mg/L, a 10.6 % total solids by wet weight and a pH of 7.3. Fecal coliform counts were
elevated. Following the first few weeks of baseline testing, the Acti-Zyme addition began regularly in the
manure scraping operation. In addition, there were two events where the lagoon was charged with
additional Acti-Zyme- June 2 and August 19. This is a highly dynamic and complex biological system
which makes it very difficult to make conclusive observations. Stratification within the lagoon, aerobic
and anaerobic biological activity, weather influences (precipitation events tend to dilute nutrient
concentrations, evaporative losses tend to concentrate nutrients) and processing influences (addition of
wash water, agitation of the lagoon etc) tends to make the findings highly variable. In examination of
the sampling results at the north site post Acti-Zyme addition, there may be a slight reduction in BOD
-5
0
5
10
15
20
25
0
5
10
15
20
25
30
35
15-May-14 15-Jun-14 15-Jul-14 15-Aug-14 15-Sep-14 15-Oct-14
Tem
per
atu
re (
°C)
Pre
cip
itat
ion
(m
m)
Precip. Air Temp. Ave. (°C)
14
over time. Total nitrogen and phosphorus levels appear to remain somewhat consistent at the influent,
but are observed in slightly higher concentrations at the south site (opposite the influent) (total nitrogen
as high as 2.55% and total phosphorus as high as 9170mg/kg). Dissolved and suspended solids are highly
variable, likely due to precipitation events, lagoon agitation and other factors. The pH remains quite
consistent over time. It is difficult to make any firm conclusions about the impact of Acti-Zyme addition
on water quality parameters because of the complexity of the system and the many variables affecting
the results.
15
Table 5. Data Summarized from the Lagoon Study
Lagoon Sampling Date
Units
15-May 27-May 2-Jun 9-Jun 24-Jun 8-Jul 22-Jul 5-Aug 19-Aug 2-Sep 16-Sep 3-Oct
6-Oct 4-Nov
Sample Notes
Agitation of
Lagoon Baselin
e Sample
Baseline Sample
Baseline Sample
Agitation of Lagoon
Final Sample
% Change
From May 15 to
Oct 3
Agitation of Lagoon
Final Sample
Post Trial Sample
Additional Acti-Zyme Addition
Yes Yes
Day of Trial
0 7 22 36 50 64 78 92 106 123
126 155
Biochemical Oxygen Demand - North Site
mg/L 11800 13100 12200 14500 6310
9210
9450 8340
- 29% Decrease
Biochemical Oxygen Demand - South Site
mg/L
7680
8460 12500
11000 6650
Nitrite - North Site ug/g
7.3 7.8 21 0.4 7.2 <0.2 4 3 5.4 <1 <1 0.4 -94.5%
Decrease
Nitrite - South Site ug/g
4 4 <1 <1 0.6
1 9.8
Ammonium - North Site ug/g
18500 7920 6920 14900 7610 8470 19000 11000 9650 9890 16200 16800 -9.1%
Decrease
Ammonium - South Site ug/g
15600 2640 14300 9790 10300
9840 27300
Nitrate - North Site ug/g
<90 <60 <600 <70 <600 <60 <600 <600 <30 <600 <70 <600
Nitrate -South Site ug/g
<1000 <10 <60 <70 <30
<30 <100
Total Nitrogen - North Site % Dry Wt
1.49 1.69 1.63 1.32 1.62 1.69 2.52 1.59 2.02 1.87 2.89 2.06 +38%
Increase
Total Nitrogen - South Site % Dry Wt
2.18 2.55 2.22 2.33 1.66
1.67 2.33
Available Phosphorus (phosphate) - North Site
ug/g 3800 3200 2900 360 2800 300 3300 3100 2900 3100 5700 200
-95% Decrease
Available Phosphorus (phosphate)- South Site
ug/g 5700 5900 7000 7000 340
390 2200
Total Phosphorus - North Site mg/kg
5200 4770 4830 640 4910 4220 6980 5230 4270 5050 8700 5440 +4%
Decrease
Total Phosphorus - South Site mg/kg
8360 7970 9170 9010 5340
5300 5650
Fecal Coliforms - North Site MPN/g
>1100 >1100 >110000 >110000 46000
>11000
24000 110000
Fecal Coliforms - South Site MPN/g
230
2300 9300
46000 >11000
Total Dissolved Solids - North Site mg/L
12300 9920 15200 6100
12400
11600 9260 -25%
Decrease
Total Dissolved Solids - South Site mg/L
12500
12600 13000
11200 7600
Total Suspended Solids - North Site mg/L
17900 24500 4700 23400
N/A
16800 N/A
Total Suspended Solids - South Site mg/L
6540
33500 N/A
4500
% Solids - North Site % Wet Wt
5.7 8.0 10.6 7.6 11.3 9.7 3.1 8.8 7.3 8.8 3.6 4.3
% Solids - South Site % Wet Wt
4.6 4.1 4.2 3.7 7.4
7.8 2.0
pH - North Site
7.42 7.37 7.27 7.45 7.48
7.32
7.48 7.21 -3%
Decrease
pH - South Site
7.35
7.41 7.25
7.07 8.04
* calculated from June 2 baseline data
16
5.2 Controlled Study
The manure used in this study was recorded at 6% at the outset of the trial. At the conclusion, when
used for greenhouse trial testing, both control and treated manure was 4% suggesting some evaporative
loss in the carboy systems over the four months affecting nutrient concentration levels recorded in the
controlled study. BOD was reduced in both the treated and control manure samples at day 63 and again
at the end of 133 days. Elevated ammonium levels and Increases in total nitrogen were seen in both the
treatment and control manure samples with the treated manure yielding higher values as the trial
progressed. Available phosphorous (phosphate) levels Increased in both treatments as the trial
progressed. A reduction in dissolved solids and an Increase in suspended solids was noted in both
treatments over time. Gas production observed was low and did not produce significant, measurable
data and no differences in temperature or odour was noted between the treatments in this study.
Table 6. Data Summarized from the Controlled Study.
Analyte Carboy Sample Units 15-May Baseline
19-Aug-14 (Day 63)
% Change 28-Oct-14 (Day 133)
% Change
Available Nitrite
Baseline
ug/g
7.3
Control 5 -32% Dec
22.8 +312% Inc
Treated 3 -59% Dec
29 +397% Inc
Untouched Control
39.1 +535 % Inc
Untouched Treated
19.9 +272% Inc
Available Ammonium (Dry Basis)
Baseline
ug/g
18500
Control 9240 -50% Dec
20900 +13% Inc
Treated 22000 +18% Inc
22500 +22% Inc
Untouched Control
23600 +28% Inc
Untouched Treated
25400 +37% Inc
Available Nitrate (Dry Basis)
Baseline
ug/g
90
Control 50 -45% Dec
100 +12% Inc
Treated 50 -45% Dec
100 +12% Inc
Untouched Control
100 +12% Inc
Untouched Treated
100 +12% Inc
Total Nitrogen
Baseline
% Dry Wt
1.49
Control 1.76 +18% Inc
2.37 +58% Inc
Treated 1.82 +22% Inc
2.31 +55% Inc
Untouched Control
2.11 +42% Inc
Untouched Treated
2.20 +47% Inc
Available Phosphorus (phosphate)
Baseline
ug/g
3800
Control 4100 +8% Inc
6500 +71% Inc
Treated 4600 +21% Inc
6300 +66% Inc
Untouched Control
5700 +50% Inc
Untouched Treated
5800 +53% Inc
Inc = Increase; Dec = Decrease
17
Analyte Carboy Sample
Units 15-May Baseline
19-Aug-14 (Day 63)
% Change 28-Oct-14 (Day 133)
% Change
Total Phosphorus
Baseline
mg/kg
5200
Control 5500 +6% Inc
7660 +47.5% Inc
Treated 6320 +21.5%
Inc 7080 +36% Inc
Untouched Control
6470 +24.5% Inc
Untouched Treated
6130 +18% Inc
Fecal Coliforms
Baseline
MPN/g
>1100
Control <0.3 Sig Dec
7 Sig Dec
Treated 0.4 Sig. Dec
<3 Sig. Dec
Untouched Control
<3 Sig. Dec
Untouched Treated
<3 Sig. Dec
Total Dissolved Solids
Baseline
mg/L Dried at 180°C
12300
Control 13200 +7.5% Inc
11300 -8% Dec
Treated 11000 -10.5%
Dec 9000 -26.5% Dec
Untouched Control
10200 -17% Dec
Untouched Treated
9200 -25% Dec
Total Suspended Solids
Baseline
mg/L
17900
Control N/A
24400 +31% Inc
Treated N/A
33000 +84.5% Inc
Untouched Control
39600 +121% Inc
Untouched Treated
34300 +91% Inc
% Solids
Baseline
%
5.7
Control 5.1 -11% Dec
5.4 -5% Dec
Treated 5.5 -4% Dec
5.7 No Change
Untouched Control
5.4 -5% Dec
Untouched Treated
5.3 -7% Dec
Biochemical Oxygen Demand
Baseline
mg/L
11800
Control 7640 -35% Dec
7100 -40% Dec
Treated 9300 -21% Dec
4520 -61.5% Dec
Untouched Control
6210 -47% Dec
Untouched Treated
4900 -58.5% Dec
pH
Baseline
7.42
Control 7.58 +2% Inc
7.4 -0.3% Dec
Treated 7.7 +4% Inc
7.67 +3% Inc
Untouched Control
7.47 +0.66% Inc
Untouched Treated
7.63 +2.8% Inc
Inc = Increase; Dec = Decrease
18
5.3 Greenhouse Study
Figure 9 shows the view of the trays and plants in the growth trial study at 15 days post planting. A
summary of manure moisture is shown in Table 7.
Table 7. Summary of Manure Treatments and Moisture Contents.
Figure 9. Photos of growth trial plants at day 15 post planting.
Manure Treatment Percent Dry Matter (%) Percent Moisture (%)
Lagoon 2% 98%
Treated 4% 96%
Untreated (Control) 4% 96%
Corn in Soilless Media
Corn in Soil
Barley in Soilless Media Barley in Soil
Peppercress in Soilless Media
19
Corn
Germination results show that corn took time to germinate in the 15-day trial with higher and rates of germination and earlier emergence of plants noted in the
soil versus the soilless media. Overall, increasing rates of manure decreased germination rates with the exception of lagoon manure in soilless media. Heights of
plants, which is an indication of their vigor, had mixed results likely due to the various rates of germination and emergence. Notable is the heights in treated
manure at Rate A in soilless media.
Figure 10. Percent germination and average plant height of corn grown in soilless media with various application rates of manure treatments over a 15-day
growth trial.
Figure 11. Percent germination and average plant height of corn grown in soil with various application rates of manure treatments over a 15-day growth trial.
0%
20%
40%
60%
80%
100%
Per
cen
t G
erm
inat
ion
(%
)
Corn in Soilless Media
Day 6
Day 10
Day 15
00.5
11.5
22.5
33.5
4
Ave
Hei
ght
at D
ay 1
5 (
cm)
Corn in Soilless Media
0%20%40%60%80%
100%
Per
cen
t G
erm
inat
ion
(%
)
Corn in Soil
Day 6
Day 10
Day 15
0123456
Ave
Hei
ght
at D
ay 1
5 (
cm)
Corn in Soil
20
Barley
Barley took less time to emerge than the corn. Results show almost all seeds germinated in the soil and the most variability and reduced germination was noted
in the untreated manure in the soilless media. Height data is more uniform with most additions of manure outperforming the control.
Figure 12. Percent germination and average plant height of barley grown in soilless media with various application rates of manure treatments over a 15-day
growth trial.
Figure 13. Percent germination and average plant height of barley grown in soil with various application rates of manure treatments over a 15-day growth trial.
0%
20%
40%
60%
80%
100%
Per
cen
t G
erm
inat
ion
(%
)
Barley in Soilless Media
Day 6
Day 10
Day 15
02468
101214
Ave
Hei
ght
at D
ay 1
5 (
cm)
Barley in Soilless Media
0%
20%
40%
60%
80%
100%
Per
cen
t G
erm
inat
ion
(%
)
Barley in Soil
Day 6
Day 10
Day 15
02468
101214
Ave
Hei
ght
at D
ay 1
5 (
cm)
Barley in Soil
21
Peppercress
Lagoon manure impacted early germination and emergence of peppercress in all three application rates.
As a result, plant height was also highest in the lagoon manure treatment and as rates of manure
increased, plants were taller.
Figure 14. Percent germination and average plant height of peppercress grown in soilless media with
various application rates of manure treatments over a 15-day growth trial.
0%
20%
40%
60%
80%
100%
Per
cen
t G
erm
inat
atio
n (
%)
Cress Seed in Soilless Media
Day 6
Day 10
Day 15
0
0.5
1
1.5
2
2.5
3
3.5
Control Lagoon -Rate A
Lagoon -Rate B
Lagoon -Rate C
Treated -Rate A
Treated -Rate B
Treated -Rate C
Untreated- Rate A
Untreated- Rate B
Untreated- Rate C
Ave
Hei
ght
at D
ay 1
5 (
Cm
)
Cress Seed in Soilless Media
22
6.0 CONCLUSIONS
The project was designed to evaluate the changes in manure nutrient profile, physical parameters
Including various water quality tests, and plant growth trials when Acti-Zyme was added to the dairy
manure system. Additionally, a bench-scale controlled study comparing manure samples with the Acti-
Zyme to a control (no Acti-Zyme added) was also completed. Finally, growth trials of various plants
investigated the toxicity or nutrient value of various manure treatments.
The lagoon study investigated the use of Acti-Zyme in an applied agricultural setting. Although used
extensively in many lagoon systems across Canada, this was the first objective look at manure nutrient
and biological activity over a four month period. As in all applied research and in situ applied research in
particular, various factors have an effect on outcome. In this case, rainfall events, fluctuations in
temperature, stratification within the lagoon, aerobic and anaerobic biological activity, and discharge of
wash water are potential influencing factors in the lagoon study. Overall the farmer noted that the
lagoon pumped out well at the conclusion of the study and did not negatively affect manure agitation or
field application and noted more odour from the lagoon after a rain event. When comparing the
agitated baseline sample from May 15 to the agitated final sample on Oct 3, BOD decreased 29%; nitrite
decreased 94.5%; ammonia decreased 9.1%; Total Nitrogen increased 38%; Available Phosphate
decreased 95%; and Total Dissolved Solids decreased 25%. Conclusions are limited by the fact that this is
a comparison made between two data points in a highly dynamic environment.
The controlled study was of value as it allowed for the control and elimination of various influencing
factors that the lagoon trial could not control, and also it provided a comparison of treatments which
the lagoon study could not. Here there was the opportunity to compare results side by side over four
months which showed trends of reducing BOD and shifts in dissolved solids to suspended solids,
however these trends were noted in both the treated and untreated manure samples.
Finally, the growth trials in the greenhouse provided a preliminary look at the impact of manure on plant
germination and growth. In some cases, germination was delayed which resulted in smaller plants at
day 15 post planting. The general observation is that rate of application does affect the trial, perhaps
due to moisture content and the coverage the manure had on seeds germinating which may have
impeded emergence. More testing is needed to address optimal application rates for various plants; its
effects on plants; and ultimately, their yield and feed quality. Overall, initial trial results show that
manure, regardless of treatment or rate, was equal to or better than the control, suggesting manures
did not negatively impact germination and growth.
7.0 RECOMMENDATIONS
Considerations for future research in an open dairy lagoon scenario Include investigating the dose
response and impact of various factors such as wash water discharge and rain events, and influences of
agitation, stratification and oxygen levels at various depths. Replicated experimental units are needed
to compare results and provide firm conclusions. Further greenhouse and field studies applying the
manure treated with Acti-Zyme can confirm the relationship between the bioactive compound and
23
agricultural field applications. These trials may examine such things as: (1) Water to solids ratio, (2)
sampling at more than three sites immediately after product application and at opposite side of lagoon
to establish a baseline for each scenario, (3) examine crop production following application of manure
and assess factors such as protein, gluten, crop growth rate, and (4) controlled greenhouse studies
applying various dose rates in various applications.
24
8.0 REFERENCES
Alberta Agriculture, 2014. Available from: http://agriculture.alberta.ca/acis/alberta-weather-data-
viewer.jsp
Murdoch Tome, 1991. Streamkeeper's Field Guide: Watershed Inventory and Stream Monitoring
Methods. Available from: http://www.dnr.mo.gov/env/esp/waterquality-parameters.htm
25
9.0 APPENDIX
26
Appendix A – Extractable Nitrate, Phosphate and Potassium in Soil
27
28
Appendix B –Extractable Ammonium and Nitrate
29
30
Appendix C - Biochemical Oxygen Demand
31
Appendix D – Total Dissolved Solids
32
33
Appendix E – Total Suspended Solids
34
Appendix F - Moisture
35
Appendix G – Exova Lagoon Study Data Results
36
37
38
39
40
41
42
43
44
45
46
47
48
Appendix H - Exova Controlled Study Data Results
49
50
51
52
53
54
55
56
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