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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

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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



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).

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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.

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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





Aug 5 Day 49 8 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


Sept 2 Day 77 12 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 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.

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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

(%

)


Day 6

Day 10

Day 15

00.5

11.5

22.5

33.5

4

Ave

Hei

ght

at D

ay 1

5 (

cm)


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



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



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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