DEPARTMENT OF ENVIRONMENTAL RESOURCES ENGINEERING

TREATMENT WETLAND PROJECT REPORT

ERE 675Ecological Engineering for Water Quality

Submitted By: Date Submitted:Nicole Ng December 10, 2015

______________________

Table of Contents

1 Introduction..............................................................................................................................1

2 Methods....................................................................................................................................4

2.1 Mesocosm wetlands..........................................................................................................4

2.2 Water quality measurements and chemical analysis.........................................................5

2.3 Microorganisms count.......................................................................................................6

2.4 Fecal coliform...................................................................................................................6

2.5 Data Analysis....................................................................................................................7

3 Results......................................................................................................................................8

3.1 Wetland water quality.......................................................................................................8

3.2 Microorganisms composition..........................................................................................11

3.3 Fecal coliform.................................................................................................................12

4 Discussion..............................................................................................................................14

5 Conclusions and Recommendations.......................................................................................18

6 References..............................................................................................................................19

i

1 Introduction

The accelerated industrialization throughout the world, specifically with regards to the

agricultural sector, resulted in increased adverse anthropogenic effects on natural ecosystems.

Agricultural runoff and wastewater results in a whole host of environmental adverse effects such

as degradation of aquatic ecosystems, loss of biodiversity, contamination of drinking water

resources, and global contamination by persistent organic pollutants (Cheng et al., 2002; Ongley

and Nations, 1996). Agricultural wastewater is typically characterized with higher concentration

of organic matter and nutrients than treated domestic municipal effluent (Ibekwe et al., 2003).

The excess nutrients may result in harmful algal bloom, which can lead to oxygen depletion from

cellular respiration, mass mortalities of aquatic organisms, and illness from consumption of toxic

algae. To combat agricultural wastewater pollutants, treatment systems are utilized to treat the

wastewater prior to discharging to freshwater resources and ecosystem so as to reduce adverse

environmental effects. Conventional methods of agricultural wastewater treatment include

centralized wastewater treatment facilities and anaerobic lagoons (manure lagoons) (Mustafa,

2013; Vanotti et al., 2007). Centralized wastewater treatment facilities are associated with high

costs of construction, operation, and maintenance. Due to this, it is typically unfeasible to

construct wastewater facilities in rural communities. Consequently, anaerobic lagoons are widely

used to treat and store manure due to their low costs and minimal maintenance relative to a

wastewater treatment facility. However, there are many environmental and health concerns

associated with using anaerobic lagoons, which includes: emission of ammonia, pathogens, and

water quality degradation (Aneja et al., 2000; Mallin, 2000; Sobsey et al., 2001). In recent years,

constructed wetlands have been gaining widespread attention as a low cost alternative to treating

polluted wastewater.

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Wetlands can act as sources and sinks for nutrients and carbon and has the ability to

transform nutrients, which is ideal in terms of treating polluted wastewater. Like any pollutant

removal processes, it is highly dependent on physical and biological processes. Many factors in

constructed wetlands can alter the performance in pollutant removal such as: vegetation cover,

media hydraulic conductivity, macrophytes species, water quality of influent, etc. Typically,

nutrients are one of the most worrisome pollutants due to the adverse ecological effects it can

cause. The conventional method of ammonia/ammonium removal is through nitrification-

denitrification. Nitrification (Eq. 1) is a two-step aerobic process which consists of nitritation,

transformation of ammonia/ammonium to nitrite facilitated by ammonia oxidizing bacteria

(AOB), and nitratation, transformation of nitrite to nitrate facilitated by nitrite oxidizing bacteria

(NOB). Denitrification is the transformation of nitrate to dinitrogen gas (Eq. 2).

NH4+ + 2O2 → NO3

- + 2H+ + H2O ( 1 )2NO3

- + 12H+→ N2 + 6H2O ( 2 )However, because both processes require high oxygen demand, this may increase costs

associated with operation in constructed wetlands. A new pathway for nitrogen removal

discovered in recent decades is anammox (anaerobic ammonium oxidation). Through this

process, ammonium is transformed into dinitrogen gas without the need to add COD to support

denitrification (Strous et al., 1997). Anammox in conjunction with nitritation greatly reduces the

oxygen demand for the ammonium-dinitrogen gas transformation than the traditional

nitrification-denitrification process.

The objectives of this study were to (1) evaluate the effects of macrophytes species in the

VSBs, (2) evaluate the effects of anammox seeding sources in the FWSs, (3) examine differences

in performances between VSB2 and FWS1, (4) determine contribution of plant uptake for

2

nitrogen removal, and (5) identify relationships of nitrogen removal with microbial community.

This paper is mainly focused on objectives 1, 2, 3, and 5.

3

2 Methods

2.1 Mesocosm wetlands

Weekly batch operations were conducted under greenhouse conditions from September to

November of 2015 (hydraulic retention time (HRT) = 7 days). The experiment consisted of two

FWS and two vegetated submerged bed (WSB) wetlands, where each wetland was housed in

plywood containers (working dimensions: 0.42 m width × 0.45 m length × 0.53 m height) lined

with 45 mm ethylene propylene diene monomer (EMPD) black pond liner. The FWS wetlands

were dual lined to prevent macrophytes roots from penetrating the liners. The VSB wetlands

were layered with 40.5 cm of marble chips and approximately 0.5 L of Nucor electric furnace

slag in September 2014. VSB1 and VSB2 were vegetated with umbrella sedge (Cyperus

alternifolius) and narrow-leaf cattails (Typha angustifolia), respectively. The rooting substrate of

the FWS wetlands consisted of sand-loam from previous constructed wetlands and

approximately 1 L Nucor slag added in September 2014. FWS1 and FWS2 were vegetated with

Cyperus transplanted in May 2013 and December 2012, respectively. The vegetation was thinned

on 5/24/2013 and 9/18/2014 for both wetlands.

The influent for the weekly batch operation was 120 L of synthesized dairy wastewater

characterized by 64.2 g of dissolved NH4Cl, 0.75 L of dairy manure digester liquor, and 119 L of

tap water (140+ mg NH4+-N/L). Every seven days, the wetlands were drained at the bottom and

fed with 25 L of new influent, equivalent to a hydraulic loading rate of 18.90 mm/d and

ammonia loading rate of 2.90 ± 0.40 g/m2d. The dates of each operation is presented below in

Table 1. Seedings were added in each of the four wetlands on 9/16/2015. The seeding for VSB1,

VSB2, and FWS1 composed of 1 L of anaerobically digested dairy manure (ADDM), 0.5 L of

sewage, and 23.5 L of tap water. The seeding for FWS2 composed of 4 L of sludge digestive, 0.5

4

L of sewage, and 20.5 L of tap water. The ADDM was formed of 41 g of VS/L and 1.8 g N/L

Ammonium, while the sludge digestive was composed of 10 g Vs/L and 1.2 g N/L Ammonium.

The sludge digestive was obtained from Metropolitan Syracuse Wastewater Treatment Plant.

Table 1. Batch operation datesPeriod

sOperation

DatePRE 9/16/ - 9/23

1 9/23 - 9/302 9/30 - 10/73 10/7 - 10/144 10/14 - 10/215 10/21 - 10/286 10/28 - 11/47 11/4 - 11/11

2.2 Water quality measurements and chemical analysis

Field measurements and sample collections were conducted on a weekly basis. Water

quality parameters such as pH, temperature, oxidation-reduction potential (ORP), dissolved

oxygen (DO) were measured using respective probes. The probes were inserted a few

centimeters below water surface and stirred the water to ensure homogeneity and allow for

measurement stabilization (reading constant after 30 seconds). Five samples (four from effluent

prior to drainage and one from influent) were collected in high density polyethylene (HDPE)

bottles. These water samples were analyzed for NH3+, NO2

-, and NO3- in the laboratory using

QuikChem 8500 series flow injection analyzer, with the exception of the influent from the first

batch operation. Total inorganic nitrogen (TIN) was determined as the sum of NH4+-N, NO2

--N,

and NO3—N.

5

2.3 Microorganisms count

Microorganisms were sampled three times during the course of the wetland operation

(10/15/15, 10/21/15, 10/28/15). Four 100 mL graduated beakers were obtained. The beakers

were used to obtain water (approximately 50 mL collected from the outflow stream) and

sediment (approximately 10 mL collected) samples from each of the FWS wetlands. Two

microscope were utilized for the identification of microorganisms: Fisher Scientific

Micromaster II Microscope equipped with a 10X eyepiece lens and objective lenses of 4X, 10X,

40X, and 100X and World Precision Instruments Precision Stereo Zoom Trinocular Microscope

III equipped with a 20X eyepiece lens and objective lenses up to 4.5X. Sediment samples were

diluted with deionized (DI) water at 20X dilution. Approximately 50 µL of water and diluted

sediment samples were transferred with a pipette onto VWR 76.2×25.4 mm, 1.2 mm thick micro

slides, which were covered with VWR 22×22 mm micro cover glass. In addition, approximately

100 µL of water and diluted sediment samples were also transferred onto a concavity slide.

Samples transferred onto the cover glass were analyzed with the Micromaster II Microscope,

while samples on the concavity slide were analyzed under the Trinocular Microscope. Flagellates

and ciliates were identified by their shape and motility.

2.4 Fecal coliform

Fecal coliform was collected once during the operation period on 10/22/15. Three samples

with volumes of 15, 45, and 100 mL were collected from each of the wetland. Fecal coliform

procedure was adopted from Standard Method 9222D. Samples were brought to the laboratory to

be filtered using a membrane filter through vacuum filtration. The filters were removed and

placed in petri dishes. The filters were treated with at least 2 mL of M-FC medium. The petri

dishes were then incubated in the oven for 24 ± 2 hours at 44.5 ± 0.2oC. After the incubation

6

period, the filters were viewed through the microscope for fecal coliform colonies (indicated by

various shades of blue).

2.5 Data Analysis

Concentration and mass removal efficiency, and areal mass and volumetric removal rate

were calculated for ammonia, NO2-+NO3

-, and TIN using Eq. 4-7, respectively.

ℜc=C i−Ce

Ce×100 ( 3 )

ℜm=Ci V i−C e V e

Ci V i×100 ( 4 )

MRRa=C iV i−C e V e

A s τ ( 5 )

MRRv=C iV i−C e V e

τ ( 6 )

where REc is the concentration removal efficiency (%), Ci and Ce are the influent and effluent

concentrations respectively (g N/L), REm is the mass removal efficiency (%), Vi and Ve are the

influent and effluent volumes respectively (L), MRRa is the areal mass removal rate (g N/m2d),

As is the surface area (m2), τ is the hydraulic retention time (7 days), and MRRv is the volumetric

mass removal rate (g N/m3d).

7

3 Results

3.1 Wetland water quality

All analysis data from herein excludes operation period PRE (9/13 – 9/26) due to four

different influents were added to each of the CWs and NH3+, NO2

-, and NO3- were not analyzed

for the first set of influent. Temperature, pH, DO, ORP, and water volume for influent and

effluent wastewater (WW) during the course of the operation period is presented in Figures 1-5,

respectively. The operational conditions of the four CWs are summarized in Table 2.

0 1 2 3 4 5 6 7 815

17

19

21

23

25

27

29

Operation Period

Effllu

ent T

empe

ratu

re (o

C)

Figure 1. Influent and effluent (WW) temperature

0 1 2 3 4 5 6 7 86.006.206.406.606.807.007.207.407.607.808.00

Operation Period

Efflue

nt p

H

Figure 2. Influent and effluent WW pH

0 1 2 3 4 5 6 7 80

2

4

6

8

10

12

Operation Period

Efflue

nt D

O (m

g/L)

Figure 3. Influent and effluent WW DO levels

0 1 2 3 4 5 6 7 8-80

-60

-40

-20

0

20

40

60

Operation Period

Efflue

nt O

RP (m

V)

8

Figure 4. Influent and effluent WW ORP

0 1 2 3 4 5 6 7 80

5

10

15

20

25

30

Operation Period

Efflue

nt w

ater

vol

ume

(L)

Figure 5. Influent and effluent water volume

Table 2. Average influent and effluent water quality parameters ab

Influent Effluent

VSB1 VSB2 FWS1 FWS2

Temperature (oC) 21.1 ± 3.5 21.7 ± 3.2 22.0 ± 2.8 21.0 ± 3.2 20.7 ± 3.3DO (mg/L) 8.8 ± 0.6 1.7 ± 0.7* 0.5 ± 0.4 1.2 ± 0.4 1.5 ± 0.3pH 7.6 ± 0.1 7.0 ± 0.2 7.0 ± 0.1 7.2 ± 0.3 6.9 ± 0.4ORP (mV) -56 ± 6.9 -22 ± 8.3 -17 ± 2.5 -30 ± 19.0 -13 ± 26.3a mean ± standard deviationb data does not include operation period PRE (9/16 – 9/23)* Only six observations; DO was not measured on 10/7/15 due to no remaining water in VSB1

The average concentration and mass removal efficiencies for NH3+ and TIN for all

wetlands is shown in Figure 6. The average MRRa and MRRv for NH3+ and TIN for all wetlands

is presented in Figures 7 and 8, respectively. The MRRa and MRRv are similar between NH3+ and

TIN due to low influent NO2- and NO3

- concentrations. NH3+ MRRa, concentration removal

efficiency, mass removal efficiency, and MRRv during the course of the operation period is

presented in Figures 9-12, respectively. Figure 13 shows the average and standard deviation of

the concentration and mass removal efficiency of NO2-+NO3

-.

9

VSB1 VSB2 FWS1 FWS20.00

10.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

CRE - NH3 MRE - NH3 CRE - TIN MRE - TIN

Rem

oval

Effi

cienc

y (%

)

Figure 6. Average concentration and mass removal efficiencies of NH3+ and TIN. CRE refers to

the concentration removal efficiency and MRS refers to the mass removal efficiency. Errors bars depict standard deviation

VSB1 VSB2 FWS1 FWS20

0.20.40.60.8

11.21.41.61.8

2

MRRa - NH3 MRRa - TIN

Area

l mas

s rem

oval

rate

(g

N/m

2d)

Figure 7. Average areal mass removal rates for NH3

+ and TIN. Error bars depict standard deviation

VSB1 VSB2 FWS1 FWS20

2

4

6

8

10

12

14

16

MRRv - NH3 MRRv - TIN

Volu

met

ric m

ass r

emov

al ra

te (g

N/

m3d

)

Figure 8. Average volumetric mass removal rates for NH3

+ and TIN. Error bars depict standard deviation

0 1 2 3 4 5 6 7 802468

101214161820

Operation Period

NH3+

vol

umet

ric m

ass

rem

oval

rate

(g N

/m3d

)

Figure 9. NH3+ MRRv for all wetlands

during operation

0 1 2 3 4 5 6 7 8-10.00

0.0010.0020.0030.0040.0050.0060.0070.00

Operation Period

NH3+

conc

. rem

oval

ef

-fic

ienc

y (%

)

Figure 10. NH3+ concentration removal

efficiency during operation

10

0 1 2 3 4 5 6 7 80.00

10.0020.0030.0040.0050.0060.0070.0080.00

Operation Period

NH3+

mas

s rem

oval

ef

-fic

ienc

y (%

)

Figure 11. NH3+ mass removal efficiency

during operation

0 1 2 3 4 5 6 7 80

0.5

1

1.5

2

2.5

3

Operation Period

NH3+

are

al m

ass r

emov

al

rate

(g N

/m2d

)

Figure 12. NH3+ MRRa for all wetlands

during operation

VSB1 VSB2 FWS1 FWS2-600

-500

-400

-300

-200

-100

0

100

CRE MRE

NO2-

+ N

O3-

rem

oval

effi

cienc

y (%

)

Figure 13. Concentration and mass removal efficiency of NO2-+NO3

-

3.2 Microorganisms composition

During the course of microorganisms sampling, there were much difficulty identifying

ciliates and flagellates due to inexperience with identifying samples under the microscope, and

poor camera quality. Typically, 1-2 flagellates were found for each 100 µL water sample, which

is equivalent to 1-2×103 flagellates/mL. Ciliates were found during the course of the

identification period, however due to poor camera quality, it was difficult to count and measure.

11

Below is a photo of a ciliate that was identified and taken with a cell phone camera (Figure 14).

There were also typically 1-2 flagellates identified in the sediment sample at 20X dilution.

However, it is not conclusive because there were much more debris in the sediment sample than

the water sample, where the debris could have been mistaken for a microorganism.

Figure 14. Photo of a ciliate taken at 100X magnification taken with camera phone

3.3 Fecal coliform

Only one fecal coliform sampling was conducted. During this sampling, there was only one

result of fecal coliform that was identified in the FWS2 45 mL sample. However, this is not

conclusive because no other samples had a blue tint, an indication of fecal coliform. Below are

photos of fecal coliform samples (Figures 14 and 15). Due to very little indication of fecal

coliform colonies in the samples, no other fecal coliform identification was conducted.

12

Figure 15. Fecal coliform sample Figure 16. Fecal coliform sample

13

4 Discussion

Comparing the VSB wetlands, VSB1 had relatively lower effluent temperature, higher DO

concentration, neutral pH, and lower ORP than VSB2. For nitrogen removal performance, VSB1

significantly outperformed VSB2 in mass removal (64.0% to 39.8% - NH3+, 60.1% to 38.6% -

TIN), while had modest performance in concentration removal (20.8% to 13.8% - NH3+, 20.7% -

13.8% -TIN), MRRa (1.9 to 1.2 g N/m2d NH3+and TIN), and MRRv

(14.1 to 8.9 g N/m3d NH3+

and TIN). Both VSB1 and VSB2 had ORP values below 0 mV, indicating that these wetlands

were operating under anoxic or anaerobic conditions. DO levels in VSB1 appears to be slightly

higher than VSB2, which appears to be nearly depleted by the end of every weekly batch cycle.

Figure 13 shows that both VSB1 and VSB2 had negative removal concentration efficiency for

NO2-+NO3

- but approximately 30% NH3+ concentration removal efficiency. This indicates that

NH3+

is being transformed through nitrification. The positive mass removal efficiency of NO2-

+NO3- in VSB1 could be an indication that denitrification occurred, removing NO2

-+NO3- out of

the system. There is a large variation between the concentration and mass removal efficiency for

VSB1. In Figure 5, VSB1 consistently utilizes more water than any other wetland, thus

increasing the concentration of the effluent. Overall, VSB1 outperforms VSB2 in nitrogen

removal, however it has a higher water demand than VSB2, while VSB2 has the lowest overall

nitrogen removal rate with the exception of MRRv. This may indicate that Cyperus requires high

water demand. Typically, Typha has a very high water demand and Cyperus, found in drier sites,

has a higher capacity to tolerate water stress, needing less water. The water loss could be due to

the “oasis” effect where evapotranspiration can be higher than calculated potential

evapotranspiration in small-scale experimental systems (Kantawanichkul et al., 2009).

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FWS1 and FWS2 were seeded with ADDM and sludge digestive, respectively. Both

wetlands had similar nitrogen removal efficiency, with FWS2 slightly outperforming FWS1 in

all removal rates analysis. Figure 4 shows that after operation period 4, ORP values of VSB2

effluent were all above 0. This indicates that during this period, VSB2 was operating under

aerobic conditions. During this operating period 4, all nitrogen removal rates were increased,

with FWS2 generally having the highest rates, with the exception of NH3+ concentration

removal. One thing to note is that the greenhouse was heated on 10/14/15. Figures 17-18 shows

temperature and DO levels in FWS1 over 24-hour period. Figure 19 shows the temperature and

DO level in FWS2 during the heating in the greenhouse over 24-hour period. Without heating on

in the greenhouse, the greenhouse is subjected to diurnal air temperature, with temperature

dropping off in the late evening. DO levels begin at supersaturated condition before being

depleted approximately 12 hours after the wetland was replenished with fresh wastewater

influent. With the heat on, temperature remained relatively constant in FWS2 and DO took twice

as long to be depleted in FWS2. This can potentially indicate that during heating on, there is

more oxygen to be able to devote to nitrification-denitrification, increasing nitrogen removal

rates during this time. It should be noted that during this period, the influent the lowest

temperature.

15

0

5

10

15

20

25

0

2

4

6

8

10

TemperatureDO

Wat

er te

mpe

ratu

re, o

C

DO

, mg/

L

Figure 17. Temperature and DO levels in VSB1 over 24-hour period

0

5

10

15

20

25

0

2

4

6

8

10

TemperatureDOW

ater

tem

pera

ture

, oC

DO

, mg/

L

Figure 18. Temperature and DO levels in VSB1 over 24-hour period

0

5

10

15

20

25

0

2

4

6

8

10

TemperatureDO

Wat

er te

mpe

ratu

re, o

C

DO

, mg/

L

Figure 19. Temperature and DO levels in FWS2 over 24-hour period

Comparing VSB2 and FWS1, FWS1 outperformed VSB2 in nitrogen removal rates, with the

exception of MRRv. VSB2 ORP values remained consistently below zero, operating at anaerobic

16

conditions during the entire operation period, where FWS1 operated in aerobic conditions in the

latter periods of the operation duration. In Figure 13, FWS1 had lower NO2-+NO3

- nitrogen

removal than VSB2. This could possibly indicate several points. First is that with VSB2 having

lower NH3+ removal rates, not as much NH3

+ was transformed to nitrite as compared to FWS1.

Second, it is possible that nitrification did occur, followed by denitrification transforming NO2-

and NO3- to dinitrogen gas.

Lack of fecal coliform in the effluent indicates that fecal coliform was reduced at a high

rate. Studies have shown that FWS constructed wetlands could reduce fecal coliform by 52% and

98% in subsurface flow constructed wetlands. (Cameron et al., 2003; Thurston et al., 2001).

Since fecal coliform in the influent wastewater was not analyzed, it is not conclusive to

determine that fecal coliform was indeed reduced.

Our results showed that on average there are 1-2×103/mL in the water samples, with

variable results in the sediment samples in the FWS constructed wetlands. In other studies, it was

concluded that there were 687/mL ciliates and 3.8×103/mL nanoflagellates (Puigagut et al., 2012;

Tao et al., 2007). Typically, protozoa are used as indicators of treatment efficiency in constructed

wetlands. By analyzing on just nitrogen removal rates, FW2 had the highest which could be a

possible indication that FWS2 may have the highest microbial composition. This is further

complemented by Figure 13, where FWS1 and FWS2 have lower NO2-+NO3

- removal

efficiencies, indicative that nitritation and nitratation indeed occurred and that AOB and NOB

exists in these constructed wetlands.

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5 Conclusions and Recommendations

Overall, VSB1 had the highest average nitrogen removal rates but FWS2 performed well

midway through the operation duration. The negative removal efficiencies of NO2-+NO3

- indicate

that the main removal pathway of NH3+ is through nitrification. It is not conclusive whether

denitrification or anammox had actually occurred. One recommendation for this project is to

verify the occurrence of anammox by detecting accumulation of hydrazine following addition of

hydroxylamine (Tao et al., 2011). Another recommendation is that influent be tested for fecal

coliform to be able to determine if fecal coliform was actually reduced. One other

recommendation is to perform in situ measurements of denitrification as a proxy to separate the

difference between anammox and bacterial denitrification (Xue et al., 1999). For applied

recommendations, VSB constructed wetlands should be implemented in areas where there is

plenty of precipitation since the Cyperus has a high water demand. A follow-up project should

look at the effects of sludge digestive seedings in VSB constructed wetlands on nitrogen removal

performance, since FWS2 had higher nitrogen removal rate than FWS1.

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Sobsey, M.D., Khatib, L.A., Hill, V.R., Alocilja, E., Pillai, S., 2001. Pathogens in animal wastes and the impacts of waste management practices on their survival, transport and fate. White Pap. Anim. Agric. Environ. MidWest Plan Serv. MWPS Iowa State Univ. Ames IA.

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Vanotti, M.B., Szogi, A.A., Hunt, P.G., Millner, P.D., Humenik, F.J., 2007. Development of environmentally superior treatment system to replace anaerobic swine lagoons in the USA. Bioresour. Technol. 98, 3184–3194. doi:10.1016/j.biortech.2006.07.009

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