1. Dr. Lance Schideman, Dr. Yuanhui Zhang, Dr. Micheal Plewa, John Scott
Young-Hwan Shin, Peng Zhang, Justin Pals
University of Illinois at Urbana - Champaign
1
Characterizing the fate and transport of bioactive
chemicals of emerging concern (CECs) from animal
manure during waste-to-energy processes

2. 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)

3. 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
INTRODUCTION
How CECs in the livestock manure can affect health of humans & ecosystems?
Interrupt transport of
CECs with Waste-to-
energy system

4. 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)

5. 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. 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. 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
0
20
40
60
80
100
120
E2%removal(Ce/C0)
Low spiking
HI spiking

8. Hydrothermal liquefaction (HTL) directly
converts wet biomass into crude oil
8
Gas Product
Post-HTL
WW
Oil
Product
Solid Residue
Demonstrated
HTL Feedstocks
Reactor
High T:200 – 350 oC
High Pressure : 80 – 120 atm
Municipal sludge
Manure
Algae
Crop residues
Woody materials
Eout : Ein > 3:1 at lab-scale (% solids =20%)
Eout : Ein > 10:1 w/ heat exchangers in
commercial applications

9. 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%
0%
10%
20%
30%
40%
50%
60%
70%
200 60 250 30 250 60 300 30 300 60 350 30 350 60
Bio-crude oil and solid residue
yield of LPAM biomass via HTL
oil solid residue

10. RESULTS & DISCUSSION- HTL destruction of bioactive CECs
10
0
20
40
60
80
100
120
%Removal
E2 removal E1 removal
 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

11. RESULTS & DISCUSSION- CHG destruction of bioactive CECs
11
 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
97.5
98.0
98.5
99.0
99.5
100.0
100.5
%Removal
E1 removal E2 removal

12. RESULTS & DISCUSSION- Destruction of Florfenicol by HTL
12
 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)

13. RESULTS & DISCUSSION- Florfenicol (FF) breakdown products
13
 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

14. RESULTS & DISCUSSION- Antibiotic resistance effects
14
 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 CHG
LB Negative control
LB Positive control
MABB effluent Negative control
MABB effluent Positive control
PHWW
(MABB Biomass)
PCWW
(PHWW
w/ MABB Biomass)
PCWW
(MABB Biomass)
PHWW
(CAS Biomass)
PCWW
(PHWW
w/ CAS Biomass)
PCWW
(CAS Biomass)
MeanNumberAntibioticResistanceE.coliWells
(ResistantJackpotWells/96-WellMicroplate)
0
10
20
30
40
50

15. 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/L
96,000 20,000 3,000 52,000
Net Energy
recovery
80% 79% 87% 35%

16. 16
Dynamic System Modeling with STELLA
A process
Internal Flows
External Input
Output
E2-Energy process model construction simulating CECs flow
Waste
Pretreatment
Excretion
Bioenergy Conversion
(HTL)
Adsorption and
Biological Treatment
CHG CHG
conversion
Biomass
Harvest
HTL destruction/ transformation
Biological degradation/uptake
Physical/chemical adsorption
Discharged in
treated WW

17.  > 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 eff
MABB eff_recycle 1
MABB eff_recycle 2
MABB eff_recycle 3
MABB eff_recycle 4
%RemovalofE2
0
20
40
60
80
100
120
E2concentration(ng/L)
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
% removal
% removal of each step
E2 concentration (ng/L)

18. 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
2

19. 19

20. 20

21.  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
0
500
1000
1500
2000
2500
3000
3500
concentration(μg/kg)
E2 in slurry E1 in slurry
E2: 27.26 ±0.58
E1: 25.95 ±0.59
RESULTS & DISCUSSIONS Occurrence of hormones

22. 22
Finishing
Farrowing
Gestation
Lagoon
ⒶⒷ
Ⓒ
INTRODUCTION
Sampling points of swine manure at SRC (Swine Research Center at UIUC)

23. Conventional Pig production cycle
INTRODUCTION
<Source: http://www.epa.gov/agriculture/ag101/porkglossary.html>
Pork glossary
a) 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
23
Gestation
- Manure slurry from Top & sludge
layer at finishing pit
(d)
(h)

24. MATERIALS & METHODS
24
Analytical methods
24
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

25. 0
50
100
150
200
250
300
350
400
450
500
0
1000
2000
3000
4000
5000
6000
7000
TP&TN(mg/L)
sCOD(mg/L)
Date
sCOD TN TP
 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

26.  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
8.8
3.8
3.3
2.64
2.38
0
1
2
3
4
5
6
7
8
9
10
UCSD
Centrate
UCSD
influent
Primary
effluent
Secondary
Eflluent
LPAM
CytotoxicityIndex(LC50)-1(10)3
Figure. 5 Comparison of Cytotoxicity index for LPAM & municipal
wastewater
Figure. 4 Cytotoxicity of LPAM organics via CHO cell assay

27.  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
INTRODUCTION
Removal of estrogenic hormones during biological processes
Proposed transformation pathway of estrogenic hormones
(Lee & Liu 2002; Hutchins et al., 2007)

28. 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
50
100
150
200
250
0 30 60 90 120 150 180 210
sCOD(mg/L)
Time (min)
CAS MABB
0
5
10
15
20
25
30
0 30 60 90 120 150 180
TP(mg/L)
Time (min)
MABB CAS
RESULTS & DISCUSSIONS
Profile of sCOD & TP removal in one cycle of MABB & CAS

29. 29
RESULTS & DISCUSSIONS
Profile 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
100
200
300
400
500
600
700
0 500 1000 1500 2000 2500
concentration(μg/L)
Time (min)
E2 E1 E3 Total EE2
0
100
200
300
400
500
600
700
0 500 1000 1500 2000 2500
concentration(μg/L)
Time (min)
E2 E1 E3 Total EE2
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)

30.  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
COD(mg/L)
Time (d)
COD inf
COD eff
Phase I-A Phase I-B Phase II Phase III
Shock loading
450 &
2370 mg/L
Low LPAM loading
Avg OLR: 42.3 mg/l/d
Mid LPAM loading
Avg OLR: 152 mg/l/d
Low LPAM loading
Avg OLR: 46.5 mg/l/d
68%
removal
58.4%
removal
80.9%
removal
Reactor broken
RESULTS & DISCUSSIONS
Long – term operation of Mixed algal-bacterial bioreactor

31. Phase I-B Phase II
% removal
NH4
+-N: 98.4
% removal
TN: 41.1
NH4
+- N: 94.8
% removal
TN: 43.9
NH4
+-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

32. 32
 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
0
20
40
60
80
100
120
E1%removal(Ce/C0)
Low spiking
HI spiking
0
20
40
60
80
100
120
E2%removal(Ce/C0)
Low spiking
HI spiking

33. RESULTS & DISCUSSIONS
33
 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
CHOCellMeanCytotoxicityIndexValue
(LC50
-1
)(10
3
)±SE
<----LessToxic------MoreToxic---->
0.1
1
10
100
1000
Sam
ple
4Sam
ple
5Sam
ple
12Sam
ple
13Sam
ple
14
Sam
ple
15
Sam
ple
16Sam
ple
24
Sam
ple
25
Sam
ple
26Sam
ple
27
LPAM
from
top manure pit
MABB effluent w/o GAC (top)
LPAM
from
bottom
manure pit
MABB effluent w/o GAC (bottom)
MABB effluent w/ GAC
CAS effluent w/o GAC
CAS effluent w/ GAC
Lagoon wastewater
Spiked LPAM
from
bottom
pit
MABB effluent w/ GAC (Spiked)
CAS effluent w/ GAC (Spiked)
YData
0
10
20
30
40
50

34. 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

35. 35
INTRODUCTION
Properties 17β-estradiol 17α-estradiol Estrone Estriol Florfenicol
Used abbreviation E2 EE2 E1 E3 FF
Class Steroid Steroid Steroid Steroid Antimicrobial
Cas registry number 50-28-2 57-63-6 53-16-7 50-27-1 73231-34-2
Molecular weight (g/mol) 272.3 296.4 270.4 288.4 358.21
Vapor pressure (Pa) 3 x 10-8
6 x 10-9
3 x 10-8
9 x 10-13
Negligible
Water 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 9.03
log Kow 3.1 - 4.0 3.67, 4.15 3.1 - 4.0 2.6 - 2.8 2.36
Molecular formula C18H24O2 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)

36. 36
Small batch reactor
• Optimization of Hydrothermal processes for CECs removal & Energy
• HTL/CHG/Combined HTL & CHG
• Efficiency of CECs removal & Energy recovery
• TotalVolume (ml): 40
• WorkingVolume (ml): 20
• Organic content (%): 25 ~ 30
Connector
Body: reactor
Gas inlet
Gas control valve
MATERIALS & METHODS

37. MATERIALS & METHODS
37
Analytical 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
38
 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. 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
CHG
1. 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

40. 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. 41

42. 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. 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 feminizationo Antibiotics in the drinking water
INTRODUCTION

44. MATERIALS & METHODS
44
Detailed Analytical methods: Sample prep
Flow diagram of the sample preparation method for the analysis of estrogenic hormones by ELISA and GC/MS
44