1

Dr. Lance Schideman, Dr. Yuanhui Zhang, Dr. Micheal Plewa, John ScottYoung-Hwan Shin, Peng Zhang, Justin Pals University of Illinois at Urbana - Champaign

Characterizing the fate and transport of bioactive chemicals of emerging concern (CECs) from animal manure during waste-to-energy processes

Problem/Opportunity Statement

2

Manure can be viewed as a problem… Excess nutrient runoff and spills can lead to eutrophication and hypoxia

Hormones can lead to endocrine disruption (e.g., fish feminization) Antibiotics can lead to antibiotic resistance (80% for livestock)

Manure can be viewed as an opportunity…

Big supply- 50 to 150 million dry tons/yr

Manure organics have a large energy content (1-2 Quadrillion BTU) Non-potable water reuse potential

Liquid portion of animal manure (LPAM) ~1 Billion tons/yr Nutrients can be used to grow additional bioenergy feedstocks Manure nutrients reduce cost & CO2 emissions for synthetic fertilizers

Antibacterial Drug Use (FDA, 2009)

Livestock animals Feeding with antibiotics

Storage of livestock manure (Pit or Lagoon)

Manure spreading in the field

River or Lake water Fish feminization Antibiotic resistant infection

Runoff/Drainage/flooding from soil to surface water

Antibiotics, Antibiotic resistant bacteria, and hormones

o Integrated manure management and bio-energy recovery system can interrupt transport

of CECs and thus reduce negative effects on the health of humans & ecosystems

INTRODUCTIONHow CECs in the livestock manure can affect health of humans & ecosystems?

Interrupt transport of CECs with Waste-to-energy system

Overall process diagram for integrated waste to energy system

4

a) swine manure storage, b) LPAM production, c) biomass production, and d) hydrothermal biomass conversion processes.

a)

b)

c)

d)

RESEARCH TOPICS & OBJECTIVES

5

Characterize CECs in the liquid portion of animal manure (LPAM)

Fate of CECs in biological & adsorptive water treatment processes

Mixed algal/bacterial bioreactor (MABB)

Conventional activated sludge (CAS)

With and without granular activated carbon (GAC) incorporated

Fate of CECs in hydrothermal biofuel conversion processes

Hydrothermal liquefaction (HTL) biomass to bio-crude oil

Catalytic hydrothermal gasification (CHG) biomass to syn-gas

HTL of biomass & CHG of HTL-wastewater

Dynamic process modeling describing the fate of bioactive CECs

6

Operating conditions for bioreactors

 Mixed Algal-

Bacterial Bioreactor (MABB)

Conventional Activated Sludge Bioreactor (CAS)

Reactor type Sequencing Batch Reactor

Sequencing Batch Reactor

Operating Volume (gal) 50 50

Light intensity (µmol photons/m2/s) 350 -

Temperature (˚C) 18 16

Aeration rate (L/min) 6 11

Organic Loading Rate (mg/L) 48.6 - 571 48.6 - 571

HRT (day) 1- 4 1- 4

SRT (day) 25 – 30 25 - 30

Fill volume ratio (VF/VT, %)

Estrogen Spike Conc(mg/L)

50

1.3 – 396

50

1.3 – 396

MATERIALS & METHODS- Capture of bioactive CECs

MABB

7

Estrogens were well captured in both bioreactors

84.3% - 99.9% removal MABB had slightly higher average

removal than CAS + 5.1 % removal

Reactors with GAC had slightly higher average removal

+ 4.2 % removal % Removal very similar during high

and low spiking events Used for STELLA modeling

RESULTS & DISCUSSION- Capture of bioactive CECs

MABB w/ GAC

MABB w/o GAC

CAS w/ GAC

CAS w/o GAC75

80

85

90

95

100

105Low spikingHI spiking

E2 %

rem

oval

(Ce/

C0)

Hydrothermal liquefaction (HTL) directly converts wet biomass into crude oil

8

Gas Product

Post-HTLWW

Oil Product

Solid Residue

Demonstrated HTL Feedstocks

Reactor

High T:200 – 350 oC High Pressure : 80 – 120 atm

Municipal sludgeManureAlgae

Crop residuesWoody materials

Eout : Ein > 3:1 at lab-scale (% solids =20%)Eout : Ein > 10:1 w/ heat exchangers in commercial applications

HTL successfully converted captured LPAM organics to bio-crude oil

• Biomass % solids = 20%, • HHV of biomass (dry) =

14,140 kJ/kg• Optimal operating condition

was 300 oC & 60 min reaction time• Oil HHV = 31,426 kJ/kg• Energy recovery = 80%

200 60 250 30 250 60 300 30 300 60 350 30 350 600%

10%

20%

30%

40%

50%

60%

70%

Bio-crude oil and solid residue yield of LPAM biomass via HTL

oil solid residue

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RESULTS & DISCUSSION- HTL destruction of bioactive CECs

250°C/60min

300°C/30min

300°C/60min

350°C/60min

400°C/60min0

20

40

60

80

100

120 E2 removal E1 removal

% R

emov

al

Most HTL operating conditions provide high % removal of hormones

300 ˚C / 60min showed more removal of hormones than 300 ˚C / 30min

Removal was more sensitive to Reaction time than temperature

% Removal of E1 and E2 under various Hydrothermal Liquefaction conditions

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RESULTS & DISCUSSION- CHG destruction of bioactive CECs All CHG conditions provided high % removal of E1 & E2 ( > 99% removal)

Higher than 450 ˚C, more than 99.87% of E1 and E2 removed

CHG Removal of CECs was more sensitive to temperature than retention time

% Removal of E1 and E2 during the CHG processes for different operating conditions

350°C/60min

400°C/60min

450°C/60min

500°C/60min

550°C/60min98.4

98.6

98.8

99.0

99.2

99.4

99.6

99.8

100.0

100.2 E1 removal E2 removal

% R

emov

al

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RESULTS & DISCUSSION- Destruction of Florfenicol by HTL

Detection limit of Florfenicol (FF) was 0.05 mg/L in high resolution GC/MS

99.9% of FF in DI water and LPAM were removed with HTL at 300 ˚C and 30 min

Removal of Florfenicol in the HTL process with DI water and LPAM

Florfenicol in LPAM (Post-HTL)

Florfenicol in DI water (Pre-HTL)

Florfenicol in LPAM (Pre-HTL)

Florfenicol in DI water (Post-HTL)

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RESULTS & DISCUSSION- Florfenicol (FF) breakdown products

4-MSB was the predominant FF breakdown product in the Post–HTL wastewater (5-30%)

4-MSAP and MPS were also detected at higher temperature

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RESULTS & DISCUSSION- Antibiotic resistance effects

Sensitive Antibiotic

Resistance Assay was

developed

Antibiotic Resistance

occurred when the

positive and negative

control varied

LPAM contributed to

antibiotic resistance

HTL & CHG processes

eliminated the capacity

of LPAM to induce

antibiotic resistance

Antibiotic Resistance fluctuation assay before & after HTL or CHGM

ean Num

ber Antibiotic R

esistance E

.coli W

ells (R

esistant Jackpot Wells/96-W

ell Microplate)

0

10

20

30

40

50

Comparison of different bio-energy processes

• HTL-CHG process was most favorable in terms of net energy yield, assuming heat exchange at 80% efficiency,

• Additional CHG(600C) after HTL can recovery the energy in PHWW at about 7% of total biomass

• Biomass not fully converted to energy products (oil and gas) in direct CHG process

• Significant decrease in oil yield (40% for HTL, 15% for direct CHG) and thus overall energy yield

HTL HTL-CHG Direct CHG

Reaction condition

300C 60min

HTL: 300C 60min

HTL: 300C 60min 400C 60

minCHG: 400C 60min

CHG: 600C 60min

Oil energy yield, kJ/g wet BM

2.45 2.45 2.45 0.98

Gas energy yield, kJ/g wet BM

n.a. 0.22 0.59 0.34

Net total energy

yield, kJ/g wet BM

2.26 2.24 2.46 1.00

Final Aq Product

COD, mg/L96,000 20,000 3,000 52,000

Net Energy recovery 80% 79% 87% 35%

16Dynamic System Modeling with STELLA

A process

Internal FlowsExternal InputOutput

E2-Energy process model construction simulating CECs flow

Waste Pretreatment

Excretion

Bioenergy Conversion(HTL)

Adsorption and Biological Treatment

CHG CHG conversion

BiomassHarvest

HTL destruction/ transformation

Biological degradation/uptake

Physical/chemical adsorption

Discharged in treated WW

Concentrated

biosolid

Dilute

d liq

uid

> 99.9% of hormones can be removed in the integrated treatment system

RESULTS & DISCUSSION- Modeled System CEC removal

Bottom slurry

Screened slurry

Bag filtraion

LPAM (MF)

MABB effMABB eff_recycle 1

MABB eff_recycle 2

MABB eff_recycle 3

MABB eff_recycle 4

% R

emoval of E

2 0

20

40

60

80

100

120E

2 concentration (ng/L)

0

2000

4000

6000

8000

10000

12000

14000

16000

18000% removal % removal of each step E2 concentration (ng/L)

Conclusions

LPAM: Liquid Portion of Animal Manure containing nutrients and dissolved

organics (including bioactive CECs) can become a valuable resource

>98% of CECs in LPAM can be captured w/ an adsorptive-biological reactor

Using algae and GAC enhance the uptake of organics

>99.9% CEC removal possible with optimized multi-step system

> 85% of the energy content of LPAM can be harvested by hydrothermal

biofuel conversion processes

Antibiotics are broken down sufficiently by hydrothermal processes to

eliminate the development of antibiotic resistance

Effluent LPAM is cleaned up significantly for improved surface water quality

or potential water reuse applications

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19

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Range of E2 & E1 in swine manure slurry (UIUC) was lower than previous studies (Fine et al., 2003;

Hanselman et al., 2003; Irwin et al., 2001; Raman et al., 2004; Shappell et al., 2007; Sim et al., 2011; Singh et al., 2013).

Spiked concentrations of E1 & E2 were in the range of practical concentration of hormones.

E1 & E2: 1.3 µg/L (low spiking) < real LPAM << 396 µg/L (High spiking) << 3214 µg/kg

gSlurryfSlurry

sSlurrybSlurry

fSlurry pit(Lit)

fLagoon(Lit)

bSlurry(Lit)

sSlurry(Lit)0

500

1000

1500

2000

2500

3000

E2 in slurry E1 in slurry

conc

entr

ation

(μg/

kg )

E2: 27.26 ±0.58 E1: 25.95 ±0.59

RESULTS & DISCUSSIONS Occurrence of hormones

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Fini

shin

g

Farr

owin

g

Ges

tatio

n

Lagoon

Ⓐ Ⓑ

Ⓒ

INTRODUCTION

Sampling points of swine manure at SRC (Swine Research Center at UIUC)

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Conventional Pig production cycle

INTRODUCTION

<Source: http://www.epa.gov/agriculture/ag101/porkglossary.html>

Pork glossarya) Breeding: producing offspring

b) Gestation: period when sow is pregnant

c) Farrowing: period from birth to weaning

d) Weaning: removal of piglets from their mother

e) Piglet: young pig

f) Finishing: growing piglets to market weight

g) Heat: estrous period of sow

h) Slaughter: killing pigs

i) Boar: Castrated male pigs

(a)

(b)

(c)

(d)

(e)

(f) (g)

- Fresh solid, urine, slurry, & LPAM

Finishing

Reproduction cycle of sows Life cycle of growing pigs

Breeding

* Sampling points

Gestation

- Manure slurry from Top & sludge

layer at finishing pit

(d)

(h)

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MATERIALS & METHODSAnalytical methods

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Swine manure samples Centrifugation Extraction

Evaporation & concentrationELISA or GC/MS analysis at ng/L level

Figure. 2 Flow diagram of the samples preparation and analytical methods of estrogenic hormones

11/5/20132/18/2014

2/26/20143/20/2014

5/21/20147/24/2014

0

1000

2000

3000

4000

5000

6000

7000

0

50

100

150

200

250

300

350

400

450

500sCOD TN TP

Date

sCOD

(mg/

L)

TP &

TN

(mg/

L)

The water quality parameters of LPAM showed seasonal variation based on temperature

sCOD, TN & TP of 6 different LPAM increased with temperature of manure storage

sCOD of LPAM was adjusted to 4,000 mg/L to make bioreactor feedstock

RESULTS & DISCUSSIONS Water quality analysis

Chinese Hamster Ovary (CHO) cell assay (Hsie AW, 1975; Wagner et al., 1998) used

to investigate cytotoxicity of LPAM

Organics in LPAM has a cytotoxicity index of 2.38 which is less toxic than raw

municipal wastewater (8.8), primary effluent (3.8) and secondary wastewater effluent

(2.64)

RESULTS & DISCUSSIONS

0

1

2

3

4

5

6

7

8

9

108.8

3.83.3

2.64 2.38095238095238

Cyto

toxi

city

Inde

x (L

C50)

-1(1

0)3

Figure. 5 Comparison of Cytotoxicity index for LPAM & municipal wastewater

Figure. 4 Cytotoxicity of LPAM organics via CHO cell assay

Sorption onto biomass (E2 >90%) is dominant

Desorption of E2 from the biomass is insignificant (Andaluri et al., 2012)

Biotransformation could be occurred in the bioreactors by microorganisms

INTRODUCTIONRemoval of estrogenic hormones during biological processes

Proposed transformation pathway of estrogenic hormones (Lee & Liu 2002; Hutchins et al., 2007)

28

MABB shows similar sCOD & higher TP removal than CAS with lower aeration

Aeration rate: CAS (11 LPM) & MABB (7 LPM)

60 ~ 65% of sCOD was removed within 3 hours of operation in each reactor

TP removal ratio of each reactor was MABB ( 16.9%) & CAS (4.47%) in 3 hours

0 30 60 90 120 150 180 2100

50

100

150

200

250

CAS MABB

Time (min)

sCO

D (m

g/L)

0 30 60 90 120 150 1800

5

10

15

20

25

30

MABB CAS

Time (min)

TP (m

g/L)

RESULTS & DISCUSSIONSProfile of sCOD & TP removal in one cycle of MABB & CAS

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RESULTS & DISCUSSIONSProfile of hormones removal in one cycle of MABB operation

Within 11 hours, 88 ~ 96 % of total hormones were removed in both of reactors

GAC addition accelerate & increase the removal of hormones in the MABB

% removal of total hormones in 1 cycle was 98.2 ~ 99.4%

0 500 1000 1500 2000 25000

100

200

300

400

500

600

700

E2 E1 E3 Total EE2

Time (min)

conc

entr

ation

(μg/

L)

0 500 1000 1500 2000 25000

100

200

300

400

500

600

700

E2 E1 E3 Total EE2

Time (min)

conc

entr

ation

(μg/

L)

Figure. 7 Removal of estrogenic hormones in mixed algal-bacterial bioreactor without Granular Activated Carbon (GAC)

Figure. 6 Removal of estrogenic hormones in mixed algal-bacterial bioreactor with Granular Activated Carbon (GAC)

sCOD removal from LPAM ranged from 58.4% to 80.9% with increasing organic

loading from 42.3 to 152 mg/L/day

High Shock loading w/ GAC shows fast recovery (450 mg/L << 2370 mg/L)

≈≈

0

200

400

600

800

1000

1200

1400

1600

1800

50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210

CO

D (m

g/L

)

Time (d)

COD infCOD eff

Phase I-A Phase I-B Phase II Phase III

Shock loading450 & 2370 mg/L

Low LPAM loadingAvg OLR: 42.3 mg/l/d

Mid LPAM loadingAvg OLR: 152 mg/l/d

Low LPAM loadingAvg OLR: 46.5 mg/l/d

68% removal

58.4% removal

80.9% removal

Reactor broken

RESULTS & DISCUSSIONSLong – term operation of Mixed algal-bacterial bioreactor

Phase I-B Phase II

% removal NH4

+-N: 98.4 % removal TN: 41.1NH4

+- N: 94.8

% removal TN: 43.9NH4

+-N: 85.8

Total Nitrogen (415 ~ 833mg/L) of LPAM feedstock was removed up to 44% in

the effluent of MABB

NH4+-N (113.5 mg/L ) of LPAM feedstock were removed up to 98.4% in the

effluent of MABB

Long – term operation of Mixed algal-bacterial bioreactor RESULTS & DISCUSSIONS

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Low & high spiking in bioreactors were tested for STELLA modeling

Similar % removal of hormones except E1 in MABB w/o GAC

% removal of E1 in algal pre-treatment and HTL ranged from 43.63 to 76.20% (Pham et al., 2012)

RESULTS & DISCUSSIONS

MABB w/ GAC

MABB w/o GAC

CAS w/ GAC

CAS w/o GAC82

84

86

88

90

92

94

96

98

100

102Low spikingHI spiking

E1 %

rem

oval

(Ce/

C0)

MABB w/ GAC

MABB w/o GAC

CAS w/ GAC

CAS w/o GAC75

80

85

90

95

100

105Low spikingHI spiking

E2 %

rem

oval

(Ce/

C0)

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RESULTS & DISCUSSIONS

Cytotoxicity was decreased in the effluent of MABB

After the spiking of CAS, cytotoxicity was increased in the effluent of reactors

Antibiotics in the spiked feedstock could kill most of the nitrifying bacteria in CAS

Cytotoxicity was increased after the addition of GAC to each reactor

250 300 350 Wastewater Treatment Groups

CH

O C

ell Mean C

ytotoxicity Index Value

(LC50

-1)(10

3) ±SE

<---- Less Toxic ------ More Toxic ---->

0.1

1

10

100

1000

Figure. 12 CHO cell Cytotoxicity Index Values (CTI) of the analyzed samples

a. Sample 4 & 12: LPAM from top/bottom of finishing pit

b. Sample 13 & 14: Effluent from MABB with/without GAC

c. Sample 15 & 16: Effluent from CAS with/without GAC

d. Sample 24: Lagoon wastewater

e. Sample 25: LPAM from bottom of finishing pit

Y D

ata

0

10

20

30

40

50

1. Algal-bacterial bioreactor captured >65% of LPAM organics in one cycle and MABB is more

energy effective process than CAS with lower aeration & free light energy

2. Algal-bacterial bioreactor captured > 98.4% of NH4+-N and > 44% of TN from LPAM in one

cycle, and the removal increased with the addition of GAC

3. Granular Activated Carbon (GAC) can protect and stabilize the reactor from shock loading

4. Algal-bacterial bioreactor removed > 98.2% of estrogenic hormones from LPAM

5. GAC can accelerate & increase the removal of hormones in MABB & CAS

6. Activation of conjugated hormones might increase hormone concentrations in CAS without

GAC

7. Low & high CECs spiking in bioreactors were tested for STELLA modeling, and GAC

contribute to increase the removal of E1 & E2 in each MABB & CAS reactor

CONCLUSIONS

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INTRODUCTION

Properties 17β-estradiol 17α-estradiol Estrone Estriol FlorfenicolUsed abbreviation E2 EE2 E1 E3 FFClass Steroid Steroid Steroid Steroid AntimicrobialCas registry number 50-28-2 57-63-6 53-16-7 50-27-1 73231-34-2Molecular weight (g/mol) 272.3 296.4 270.4 288.4 358.21Vapor pressure (Pa) 3 x 10-8 6 x 10-9 3 x 10-8 9 x 10-13 NegligibleWater solubility (20°C, ppm) 3.9 - 13.3 4.8 0.8 - 12.4 3.2 - 13.3 over 400mg/L at pH > 5.5

pKa 10.5 - 10.7 10.21 10.3 - 10.8 10.4 9.03log Kow 3.1 - 4.0 3.67, 4.15 3.1 - 4.0 2.6 - 2.8 2.36

Molecular formulaC18H24O2

C20H24O2 C18H22O2 C18H22O3 C12H14Cl2FNO4S 

Structure    

Characteristics of emerging contaminants

Transformation pathways of estrogenic hormones• Transformation and breakdown of hormones

under hydrothermal processes

• To understand removal of hormones in

hydrothermal processes, we need to know the

change of each concentration of hormones

Proposed transformation pathway of estrogenic hormones (Lee & Liu 2002; Hutchins et al., 2007)

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Small batch reactor

• Optimization of Hydrothermal processes for CECs removal & Energy

• HTL/CHG/Combined HTL & CHG• Efficiency of CECs removal & Energy recovery• Total Volume (ml): 40 • Working Volume (ml): 20• Organic content (%): 25 ~ 30

ConnectorBody: reactor

Gas inlet

Gas control valve

MATERIALS & METHODS

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MATERIALS & METHODSAnalytical methods

37

PHWW/PCWW samples Centrifugation Extraction

Evaporation & concentrationGC/MS analysis ng/L level at ISTC

Figure. 2 Flow diagram of the samples preparation and analytical methods of estrognic hormones

38

RESULTS & DISCUSSIONS

Cytotoxicity: 250 ˚C and 300 ˚C < 350˚C

Toxicity in PHWW is proportional to temperature

Higher energy yield shows less cytotoxicity

Modification of HTL conditions may affect the cytotoxicity of PHWW

Figure 2. CHO cell cytotoxicity index values for each PHWW sample

250 300 350 250 ˚C 300 ˚C 350 ˚C

More toxic

39

CONCLUSIONS

1. Highest Bio-crude oil yields of HTL at 300˚C /60 min

2. Hormones removal is more sensitive to Reaction time than temperature

3. 300 ˚C /60 min is effective operating condition for HTL to provide simultaneous

bioenergy production and removal of hormones, COD and cytotoxicity

4. Chrome, Arsenic, Zinc, Cadmium, and Lead were removed up to 99.6% removal

after HTL

HTL

CHG1. Highest Bio-crude oil yields of HTL at 300˚C /60 min

2. Removal of CECs is sensitive to temperature in CHG

3. Ra-Ni was the most effective catalyst to remove estrogenic hormones

4. Amount of catalyst doesn’t affect the removal of hormones

5. Ru & Ru/NaOH shows the highest COD removal

Acknowledgement

Project team

• Dr. Lance Schideman, Ph.D., P.E.

• Dr. Yuanhui Zhang, Ph.D.

• Dr. Michael Plewa

• Peng Zhang

Illinois Sustainable Technology Center

Agricultural & Biological Engineering

Crop Science

Agricultural & Biological Engineering

• Funding: United States Department of Agriculture (USDA)/NIFA/Grant 11332987

41

42

Antibiotics

(FDA, 2009; Mellon M, 2001)

Antibiotics resistant bacteria develop from exposure to low-levels of antibiotics

& life threatening infections (Wise et al., 1998: Schuh et al., 2011)

30 ~ 90% drugs are excreted in urine & feces (Sarmah et al., 2006, Berge et al., 2006)

Spending for antibiotics infections was increased 10 times from 1998 to 2009 (Infectious Disease Society of America)

80%

Antibiotics usage for livestock

1834 tons

14,266 tons

INTRODUCTION

43

Estrogenic compounds

Major source: farm animals & humans (Shore et al., 1993; Raman et al., 2004)

Annual excretion from farm animal: 41 tons in the USA (Lange et al., 2002)

Commonly detected compounds: estrone (E1) & 17b-estradiol (E2) (Nichols et al., 1997)

Adverse effects to reproductive system with E2 (10~ ng/L) (Routledge et al., 1998; Schuh et al., 2011)

Reduced reproductive abilities & Feminization of aquatic species

o Male fish feminization

o Antibiotics in the drinking water

INTRODUCTION

44

MATERIALS & METHODSDetailed Analytical methods: Sample prep

Flow diagram of the sample preparation method for the analysis of estrogenic hormones by ELISA and GC/MS

44