Biological Treatment of Recalcitrant Organic

Pollutants in RBC using Bioaugmentation

Suparna Mukherji

Professor, Centre for Environmental Science & Engineering,

IIT Bombay, Mumbai

Microbial Technology for Wastewater Management,

Department of Biotechnology, WBTU, Kolkata, India

23-25 January, 2015

Wastewater Treatment Plant

Physical, Chemical and Biological Unit Operations

Wastewater Treatment Flowsheet

Recalcitrant Organics in Wastewater

Recalcitrant organics found in domestic wastewater

Phenolic compounds

Hydrocarbons originating from oil

Surfactants

Pesticides, Pharmaceuticals, Personal care products

Not effectively removed during secondary treatment

Tertiary Treatment Options

Sorption on activated carbon

Advanced oxidation processes

A wide range of recalcitrant organics are found in industrial wastewaters

Petrochemical

Pharmaceuticals

Steel Mills

Coke ovens & Gasifiers

Xenobiotics in Industrial Wastewaters

Biomass Producer Gas

Gasifier

Wastewater

Wet Scrubber

Water IC Engines

Bacterial Degradation of Xenobiotics

Pure cultures with remarkable ability to degrade xenobiotics

have been isolated from contaminated environments

Bioaugmentation Adding microbes with enhanced degrading abilities

to achieve clean-up or treatment

For a target contaminant or contaminant matrix in wastewater select microbial strains with enhanced ability to degrade these contaminants

Research Questions

Can adequate removal be achieved ?

Will the externally added microbe survive in this scenario ?

Reactor operating conditions ?

Xenobiotics Degrading Bacteria

Bacteria that can use diesel as sole C & E Source

Exiguobacterium aurantiacum

Burkholderia cepacia

Oil

R2 = 0.80 R

2 = 0.97

0.00

0.05

0.10

0.15

0.20

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Initial abundance of n-alkanes in diesel (Area_initial)

Ma

xim

um

de

ca

y r

ate

of

n-a

lka

ne

s

(A/d

ay

)

AS1_C9-C26 ES1_C12-C26 ES1_C9-C11

Significance: Max decay

rate is essentially constant

irrespective of carbon

number in the range 0.07-

0.2 day-1 for C12 to C26

Mohanty, G. and S. Mukherji, International Biodeterioration and Biodegradation, 2008

Bioaugmentation Scenarios

Both strains applied in attached growth reactors

Rotating biological contactor

Burkholderia cepacia Oily wastewater

B. cepacia used along with an pre-acclimatized algal consortium

Exiguobacterium aurantiacum Gasifier

wastewater

E. aurantiacum along with pre-acclimatized activated sludge microbial consortia

Rotating Biological Contactor

Aeration and mixing achieved through slow rotation of the discs

Staging in RBC can improve quality of treated effluent

Typical Parameters: BOD Removal

Treatment level

Parameters Unit BOD

Removal

BOD removal &

nitrification

Nitrification

Hydraulic

loading

m3/m2.d 0.08 –

0.16

0.03 – 0.08 0.04 – 0.10

Organic

loading

g sBOD/m2.d 4 - 10 2.5 - 8 0.5 - 1

g BOD/m2.d 8-20 5 - 16 1-2

Max 1st

stage

organic load

g sBOD/m2.d 12-15 12-15

g BOD/m2.d 24-30 24-30

HRT h 0.7 -1.5 1.5 - 4 1.2 – 3

Eff. BOD mg/l 15 - 30 7 - 15 7 – 15

Graphical model for RBCs: Clark, 1978

Mass balance for substrate across RBC

Accumulation = Inflow-Outflow-sink due to biodegradation

Consumption both by attached & suspended growth

Contribution of suspended microorganisms to substrate

removal can be neglected, particularly for low retention time

Ignores the separate aerated & submerged sectors

Assumption for model

Complete mixing in the liquid volume

Organism decay neglected (decay rate < growth rate)

At steady state biomass in the reactor is constant

Mathematical Model:

VXY

XAY

QSQSdt

dSV

s

s,

fW

f

f0

Active biomass: product of wetted area of biodisc and the

attached active biomass per unit area of biodisc

Consumption of active substrate can be neglected since

attached biomass predominates degradation

V: Liquid volume in the reactor (m3)

Aw: Wetted Area (m2)

Xf’: Mass of attached active biomass in biofilm per unit disk area

X: Suspended biomass per unit volume

So & S: Substrate concentration in influent and effluent

Yf and Ys: Apparent yield of suspended and attached biomass in

biofilm

Modified Mass Balance Expression

(μm/Yf)Xf’: Max Amount of substrate removed per day

per unit surface area of disc (Area capacity constant; P)

[Q(S0-S1)]/AW: Actual Amount of substrate removed per

unit surface area per day (Removal coefficient ; R)

'

fW

f

f0 XA

YQSQS

dt

dSV

SK

S

g

mf

SK

SXA

YQSQS

dt

dSV

g

'

fW

f

m0

Monod’s Equation

Mathematical Model

At steady state, dS/dt=0,

The plot of 1/S & 1/R, will have slope Kg/P & intercept 1/P

Model can be used for data obtained from RBC operating

at steady state at various HRT

Yf calculated by kg of sludge produced/ kg of soluble

BOD5

Mass/area of attached growth determined by scrapping

dry wt of biomass scrapping from known area disc

P

1

S

1

P

K

R

1 g

Mathematical Model

Graphical Determination

(1/S) L/mg

1/R

m2- day/mg

1/P

(Kg/P)

Challenges in Treating Oily WW

Complex composition of oil & presence of other

toxic substances

Difficulty in using suspended growth systems

EPS production adversely affects sludge settling

Long start-up and acclimation period

Large HRT requirement

Oily Wastewater Treatment Algal-Bacterial System

Algae specifically blue-green algae/ cyanobacteria can facilitate hydrocarbon degradation

demonstrated through batch studies

Both a direct and indirect role has been reported

Direct role:

Heterotrophic growth on hydrocarbons in addition to autotrophic growth

Indirect role:

In natural environment oil degrading bacteria are found to adhere on to surfaces of macroalgae and cyanobacterial mats

Schematic of RBC Reactor

Diameter

Shaft

Length of Trough

Discs

Disc

Width of Trough

Spacing between discs: 14-15 mm; 35% Submergence

Discs rotated at 10 rpm using electric motor and reduction gear system

Working Volume 4 L

Organisms used for inoculation

Burkholderia cepacia: isolated from Arabian Sea Sediments

Sessile fresh water algal culture obtained from rock surfaces

Media optimized for growth of both algae and bacteria

Three stage Rotating Biological Contactor (RBC) fed with 0.6% diesel oil as sole substrate

Oily Wastewater Treatment Algal-Bacterial System

Working Volume = 4L; 27 discs with effective surface area 0.83 m2; 10 rpm

Operated in flow through mode performance

evaluated at pseudo-steady state

Acclimatization of Algae to Diesel

In the Absence of Diesel In the Presence of Diesel 0.5% (v/v)

Loss of culture diversity in response to stress

Experimental Set-up

6

Illumination: 1100 lux; Light-Dark Cycle 18:6 hrs

Biofilm Development

0.2% diesel in feed; Inoculation: algae:bacteria VSS ratio 3.5:1

Biofilm development within 17 days in Batch mode

Composition of Synthetic Wastewater

No soluble substrate/ easily degradable substances added

COD in influent is entirely due to oil; COD in effluent is due to oil and suspended biosolids

Due to high oil conc. in influent COD estimation procedure was modified

Influent and Effluent Characteristics

All Concentrations are expressed in mg/l except pH

As HRT decreases the OLR and HLR increases, until excessive OLR causes extensive

biofilm growth and sloughing at 12 hrs

Stage-wise Removal

TPH Removal

(OLR: 23.9-38.2 g/m2-day)

0

20

40

60

80

100

120

24 hr 21 hr 18 hr 15 hr

HRT (hrs)

Sta

ge

-wis

e T

PH

Re

mo

va

l b

y G

C (

%)

Ist Stage IInd Stage IIIrd Stage

0

20

40

60

80

100

120

24 hr 21 hr 18 hr 15 hr

HRT (hrs)S

tag

e-w

ise

CO

D R

em

ov

al (%

)

Ist Stage IInd Stage IIIrd Stage

Maximum removal of oil/TPH/COD occurred in the first stage

Mukherji S. and A. Chavan, Chemical Engg. J., Vol. 200-202, 459-470, 2012.

TCOD Removal

(OLR: 21.7-34.7 g/m2-day)

Biomass Estimation During HRT Study

Total Biomass (Algae + Bacteria) Algal Biomass

0

10

20

30

40

50

60

70

24 hr 21 hr 18 hr 15 hr

HRT (hrs)

VS

S (

mg

/cm

2)

Ist Stage IInd Stage IIIrd Stage

0.0

2.0

4.0

6.0

8.0

10.0

12.0

24 hr 21 hr 18 hr 15 hr

HRT (hrs)C

hlo

rop

hy

ll-a

(m

g/c

m2)

Ist Stage IInd Stage IIIrd Stage

At 15 Hrs HRT, high OLR resulted in significantly higher VSS is the first

stage, primarily due to increase in bacterial biomass; algal biomass was

low in all stages

n-Alkane Removal During HRT Study

• Greater than 98% removal of n-

alkanes, C9-C11 was achieved in

the Ist stage at all HRTs studied

• Greater than 86% removal of all

n-alkanes in the IInd stage

(cumulative) achieved at all HRTs

studied except at 15 h HRT

• Almost complete removal of all n-

alkanes occurred at HRT of 21 h

min5 10 15 20 25 30

pA

48

50

52

54

56

58

60

FID2 A, (D:\HPCHEM\1\DATA\ANAL\HT210021.D)

4.1

34

4.4

57

5.9

70

9.1

99

9.4

03

10

.09

0 1

0.1

54

10

.28

0 10

.69

3 1

1.0

31

11

.43

3 1

1.5

64

11

.64

3 1

1.7

59

11

.84

9 1

1.9

41

12

.23

8

13

.12

1 1

3.2

85

13

.44

1 1

3.6

58

13

.96

3 1

4.1

13

14

.82

1 1

4.9

78

15

.12

6 1

5.2

65

15

.66

3 1

5.8

22

15

.94

0 1

6.1

09 1

6.7

54

16

.95

4 1

7.0

55

17

.20

1 1

7.5

31

17

.81

9 1

7.9

28

18

.29

6 1

8.5

98

18

.70

5 1

8.8

00

18

.88

3 1

9.0

15

19

.47

5 1

9.6

17

20

.03

4 2

0.1

64

20

.31

2 2

0.4

24

20

.51

6 2

0.6

62

21

.02

5 2

1.1

75

21

.30

8 2

1.4

00

21

.98

4 2

2.1

64

22

.29

4 2

2.4

10

22

.59

3 2

2.7

15

23

.22

4

23

.84

0

25

.84

7

29

.35

7

34

.15

7

min5 10 15 20 25 30

pA

38

39

40

41

42

43

44

FID2 A, (D:\GCBACK~1\HPCHEM\1\DATA\ANAL\HT240002.D)

3.7

87

3.8

84 4

.212

4.5

41

4.6

85

5.0

78

5.1

93 5

.698

17

.48

8

19

.12

4 1

9.2

57

20

.39

8 2

0.7

40

21

.21

3

22

.14

4

23

.15

7

min5 10 15 20 25 30

pA

50

51

52

53

54

55

56

FID2 A, (D:\HPCHEM\1\DATA\ANAL\HT150003.D)

4.2

54

5.6

67

13

.63

8

23

.27

7

Ist Stage

IInd Stage

IIIrd Stage

Effect of N:P Ratio HRT: 21 hrs

All Concentrations are in mg/l except pH

The N:P mole ratio is higher than is typically used for hydrocarbon degradation

The N and P levels added are lower than typically used for algal-bacterial systems

Chavan, A. and S. Mukherji, J of Hazardous Materials, Vol. 154, 63-72, 2008.

Cultures in Reactor Isolation of Cultures

1. Cycloheximide Test

2. Phycobiliprotein estimation

3. Test for Nitrogen Fixation

4. Microscopy to observe the morphology

5. Test for tolerance to various hydrocarbons

Phormidium;

Oscillatoria; Chrococcus

B. cepacia

Yeast

Sorption of Oil on Algal-Bacterial Biomass

After continuous operation of reactor for more than 1 year, 82 g of dry biomass was collected from RBC discs

Soxhlet extraction performed

0.582 g oil/g biomass over and above controls

Oil associated with biomass consisted of UCM hump: accumulation of HMW structurally complex fraction

Bulk of oil representing aliphatic fraction is biodegraded

Diesel Oil

Oil extracted from Biomass

Unlike soluble substrate, oil is not uniformly distributed in the aqueous phase within each stage to the RBC due to rotation of the discs

Attached bacteria degrades the adsorbed substrate

Two mass balances required, cannot be decoupled

Aqueous phase

Solid phase

Diesel

High molecular weight structurally complex fraction: volatilization and biodegradation negligible

Aliphatic fraction: Sorbs on to biomass and inert NAPL on the discs; bacteria in the biofilm degrades the aliphatic fraction

Thickness and porosity of the biofilm is dependent upon the consumption of aliphatic fraction of oil and also adsorption of inert fraction of oil on to the biofilm

System Conceptualization NAPL Utilization in Algal-Bacterial RBC

An artificially designed consortia of oil degrading

Burkholderia cepacia and oil tolerant algae can be

utilized for degrading petroleum hydrocarbon

containing WW

The benefits of algal-bacterial association include: No requirement for soluble carbon source

Stable pH due to generation of alkalinity by algae

Moderate DO levels inspite of high TPH loading

Good settleability of sludge

Clark’s model could not be applied

Algal-Bacterial System for Oily WW

Treatment: Conclusions

Algal-Bacterial System for Oily WW Treatment: Conclusions

The system can operate well over the HRT range 18-24 hrs.

TPH and COD removal rate is proportional to the loading

rate, but beyond a certain value complete failure is

observed

Maximum removal of TPH and COD occurred in the first

stage

Optimum N:P ratio is 28.5:1 to 38:1. At higher ratios algal

dominance increased but reactor performance deteriorated

Technology is feasible for oily WW treatment

Challenges in Treating Gasifier WW

Complex composition with recalcitrant organics

across various group types

High acute toxicity –Fish based assays

High chronic toxicity, carcinogenicity and mutagenicity associated with PAHs, benzene, phenolics

High concentration of ammonium nitrogen

Cannot be discharged directly into municipal sewers

The toxic organics can inhibit biological treatment

Can cause nitrification inhibition and lead to eutrophication

Biomass Gasifier WW

Characteristics CESE, IIT Bombay,

1 KWe, 3 Hrs Run

SPRERI,Anand,

6 Hrs Run, 10 KWe

HPS, Patna,

6 Hrs Run, 45 KWe

pH 7.76 ± 0.05 8.33 ± 0.05 7.79

Alkalinity 370 ± 12.0 1340 ± 40.0 3100 ± 90.0

TSS 884 ± 22.5 876 ± 32.5 903 ± 42.5

COD 1808 ± 79.3 7805 ± 79.3 4800 ± 70

BOD 320 ± 15 2245 ± 75 1080 ± 100

TOC 480 ± 5.2 1280 ± 24.7 1020 ± 24

NH+

4-N 530 ± 32 510 ± 32 850 ± 40

Organic Nitrogen 21 ± 1.5 150 ± 4.5 78 ± 4.5

Phenolics 120 ± 10 1040 ± 85 580 ± 25

Thiocyanate N.D N.D N.D

Cyanide N.D N.D N.D

All values are in mg/L except pH

Organics in Synthetic WW

Phenol o-Cresol Benzene Naphthalene

Pyrene Fluoranthene Phenanthrene

Pyridine Quinoline

OH

CH3

OHOH

CH3

OH

1-Napthol

Properties of Components

Compound Chemical

Formula

MW

(g/mole)

Tb

(0C)

Cs

(mg/L)

KH

(atm-m3/mol)

Log KOW

Phenol C6H6O 94.1 181.7 83000 1.53X10-6 1.46

o-Cresol C7H8O 108.1 201.8 24000 1.9X10-6 1.94

1-Naphthol C10H7 OH 144.7 286 866 4.6X10-8 2.85

Pyridine C5H5N 79.1 115.2 76000 1.2X10-5 0.65

Quinoline C9H7N 129.1 109 6100 1.5X10-6 2.03

Benzene C6H6 78.11 80.1 1800 4.8X10-3 2.13

Naphthalene C10H8 128.1 218 32 4.5 x 10-3 3.3

Phenanthrene C14H10 178.2 340 1.20 2.56x10-5 4.45

Fluoranthene C16H10 202.2 375 0.20-0.26 6.5x10-6 4.90

Pyrene C16H10 202.2 393 0.13 1.14x10-5 4.88

Synthetic Wastewater Composition

All components were added to MSM to formulate synthetic wastewater COD range 1350-8100

Components Abbreviation WW1

(mg/L)

WW2

(mg/L)

WW3

(mg/L)

WW4

(mg/L)

Phenol PHEL 250 400 600 1600

o-cresol CRES - 150 450 1000

1-Naphthol 1-NAPH - 120 400 1000

Pyridine PYRI 280 200 250 800

Quinoline QUIN 280 280 250 900

Benzene BENZ 200 360 480 800

Naphthalene NAPH 60 60 60 200

Phenanthrene PHEN 0.5 0.5 0.5 0.5

Fluoranthene FLAN 0.2 0.2 0.2 0.2

Pyrene PYR 0.12 0.12 0.12 0.12

0

20

40

60

80

100

0 50 100 150 200 250 300

-dS

i/dt

(mg

/L-d

ay)

Concentration, Si (mg/L)

Phenol Pyridine Quinoline

Benzene Naphthalene

0

0.1

0.2

0.3

0 0.2 0.4 0.6

-dS

i/dt

(mg

/L-d

ay)

Concentration, Si (mg/L)

Phenanthrene Fluoranthene Pyrene

In the synthetic gasifier wastewater the components present

at similar concentration were degraded at comparable rates

Jeswani, H. and S. Mukherji, International Biodeterioration and Biodegradation, 2013

Batch Studies: E. aurantiacum

Batch Biokinetics: E. aurantiacum Degradation of a Synthetic Gasifier Wastewater (WW1)

0.00

0.01

0.02

0.03

0.04

0 500 1000 1500 2000

µ (

hr

-1)

S (mg/L)

Components Concentration

(mg/L)

Phenol 250

Pyridine 280

Quinoline 280

Benzene 200

Naphthalene 60

Phenanthrene 0.5

Fluoranthene 0.2

Pyrene 0.12

COD of wastewater 1460 mg/L

SK

S

s

m

m= 1.08 day-1

Ks= 516 mg/L

Jeswani, H. and S. Mukherji, International Biodeterioration and Biodegradation, 2013

Batch Studies: E. aurantiacum

0

0.2

0.4

0.6

0.8

0 25 50 75 100

T ime (hrs )

Ab

so

rba

nc

e a

t 6

00

nm

C ontrol(Indigenous culture) 100% B G WW

50% B G WW 25% B G Ww

Wood based gasifier wastewater from SPRERI 7680 mg/L COD, Final COD 5760, 960,

160, 0 mg/L for controls, 100%, 50% and 25% dilutions

Bioaugmentation in RBC

Cultures Source

Bacterial cultures

Exiguobacterium aurantiacum Soil contaminated with diesel oil

obtained from a tanker refueling

station

Activated sludge (AS) consortia Mahananda dairy, Mumbai

Activated sludge process

Media used for the study was Mineral Salt Media (MSM)

AS consortia was maintained on 500 mg/l of sodium acetate

E. aurantiacum was maintained on 150 mg/L pyrene in MSM

E. aurantiacum: Characteristics

Colony Morphology on PPYG Medium

circular, up to 2.5 mm diameter, butyrous

easily emulsified, orange and opaque

Gram Staining Characteristics

variable depending on substrate

Gram –ve for colonies grown on NB

Gram +ve for colonies grown on pyrene and phenol

Grows over pH range 4-11

Shape: Rod shaped in log growth phase (2.5 m x 1 m)

Catalase positive; Oxidase negative

Forms acid with glycerol, sucrose, fructose

Sensitive to Vancomycin

Reactor Studies

Start Up and Acclimatization

• 1.5 litres of activated sludge and 500 ml of E. aurantiacum and 2 L of synthetic wastewater

• Development of bacterial biofilm was observed after 17-21 days of inoculation

Transient Phase

• Flow Initiated

• 7-9 days

Sampling Phase

• Samples were analysed in duplicate during the pseudo-steady state condition

Reactor was operated at a HRT of 24 hrs and various parameters analysed for each

stage of the RBC

Additional abiotic studies in flow through mode in absence of biofilm

Biochemical tests

5 ml of phosphate

buffer+ biofilm

4x2 cm2 acrylic sheet on

mid disc of each stage

0.5x0.5 cm2

Serial

dilutions

Streaking and

spreading on plates

Nutrient broth

24 Hrs , colonies

observed

NB 100 mg/L

Pyrene

Colonies also streaked

on PPYG plates

Catalase, oxidase, sugar

tests, acid formation

and nitrate reduction

tests performed

Abundance of each

colony type determined

Analysis of Specific Components

HPLC, Jasco, Japan, equipped with Diode Array Detector

(DAD) and Fluorescence Detector (FLD)

Column: Restek Pinnacle II PAH column; pore size 110 Å, particle size as 4 μm, ID of 4.6 mm, length 250 mm, proprietary C18 bonding

Mobile Phase: 1 ml/min; Methanol-Water Gradient

0

20

40

60

80

100

0 2 4 6 8 10

%M

eth

an

ol

Time (min)

Time

(min)

Excit.

λ (nm)

Emis.

λ (nm)

Gain Atten

0 254 350 1 16

9 240 420 10,20,30 16

FLD Time Programming

HPLC Chromatogram: Synthetic WW4

PH

EL 1-

NA

PH

NA

PH

PH

EN

FLA

N

PY

R C

RE

SO

L

PH

EL

BEN

Z

NA

PH

PY

RI

QU

IN

FLD

DAD at 254 nm

Time (Min)

Res

po

ns

e (

µV

) R

es

po

ns

e (

µV

)

Time (Min)

Cmpd RT

(min)

PHEL 3.0

CRES 3.3

1-NAPH 3.5

PYRI 3.8

QUIN 4.2

BENZ 4.4

NAPH 6.6

PHEN 10.6

FLAN 12.0

PYR 13.8

Flow Through Mode Operation Stage wise COD Removal at Pseudo Steady State

0

500

1000

1500

2000

0 5 10 15 20 25 30 35 40

Time (days)

CO

D (

mg

/l)

Influent COD Effluent COD from 1st stage

Effluent COD from 2nd stage Effluent COD from 3rd stage

HRT: 26.7 hrs

a b

a) Chains of bacteria in stage 1 b) Chains of bacteria in stage II c) Mesh of polymers

visible in stage 1 d) Coral reef formation in stage III

c d

Scanning Electron Microscopy

Biofilm in the three RBC stages

Confocal Scanning Microscopy Stage 1 Active Biofilm

The 20 panels show scan at every 30µm from 600µm from bottom (B)

to top of the biofilm

600µm from

bottom

(B)

0µm at top

(T)

Biofilm Thickness: CSLM

First Stage: 1.1 mm

No stain visible in top 100 m & bottom 600m

Second Stage: 0.5 mm

No stain visible in top 50 m & bottom 200 m

Third Stage: 0.1 mm

No stain visible in top 20 m & bottom 40 m

0

200

400

600

800

1000

1200

1400

1600

1800

25 45 65 85 105 125

CO

D (

mg

/L)

Time (Days)

Influent 1st_Stage_Effluent

2nd_stage_effluent 3rd_Stage_Effluent

Performance at Variable HRT

2 day 1.5 day 1 day 0.67 day 0.5 day

(OLR: 3.3-13.9 g/m2-day; HLR=0.002-0.01 m3/m2-day)

Wastewater of COD 1400 mg/L (WW1)

Biokinetics: Clark’s Model

y = 30.083x + 0.0059

R2 = 0.984

0

0.02

0.04

0.06

0.08

0 0.001 0.002 0.003

1/S 1

1/R

Parameters Stage 1 Stage 2 Stage 3

P 243.9 169.5 46.08

Ks (mg/L) 5076 5568 1431

Y 1.1 0.93 1.04

Xa (g/m2) 309 275 57.6

µmax (d-1) 0.974 1.15 1.04 Plot for Stage 1

P

1

S

1

P

K

R

1 s

Jeswani, H. and S. Mukherji, Bioresource Technology, 20132

Parameters Concentration* (mg/L)

WW1 WW2 WW3 WW4

Infl. Effl. Infl. Effl. Infl. Effl. Influ Effl.

COD 1357

+30

124

+10

2100

+250

258

+5

3210

+10

300

+40

8100

+30

1307

+25

BOD 630

+35

40

+16.5

1156

+35

128

+10

630

+35

130

+14.5

3220

+35

510 +30

BOD/COD 0.46 0.32 0.55 0.50 0.51 0.43 0.40 0.39

TOC 540

+4.5

95

+5.5

830

+5

132

+5.0

1320

+40

202

+15

3890

+40

890 +30

Phenolics 250 ND 410

+12.5

240

+10

600

+10.5

75

+10

1600

+45

510 +50

Performance for various Wastewaters: HRT= 1 day

(OLR: 6.2-38.6 g/m2-day) Jeswani, H. and S. Mukherji, Chemical Engg Journal, 2015

Parameters

Concentration* (mg/L)

WW1 WW2 WW3 WW4

Infl. Effl. Infl. Effl. Infl. Effl. Influ Effl.

pH 7.4

+0.02

6.3

+0.04

7.0

+0.02

6.3

+0.04

6.9

+0.02

4.8

+0.04

6.7

+0.02

4.8

+0.02

Alkalinity 270

+15.5

230

+16.5

312

+15.5

232

+16.5

392

+12.5

286

+12

560

+15.5

365

+20.5

TSS 28 +2 32

+3.5

28

+2

46

+3.5

28

+2

52

+3.6

28 +2 69

+2

NH+4-N 282

+10.5

160

+4.5

282

+10.5

180

+4.5

282

+10.5

165

+6.5

282

+10.5

210

+30

NO3-N 3.8

+0.25

4.6

+0.02

3.6

+0.25

4.2

+0.02

3.6

+0.25

4.1

+0.02

3.6

+0.25

3.7

+0.25

Performance for various Wastewaters: HRT= 1 day

COD Reduction at Steady State

0

400

800

1200

1600

0 2 4 6 8 10 12 14

CO

D (

mg

/L)

Time (Days)

WW1: 6.2 g/m2/d

0

500

1000

1500

2000

2500

3000

0 2 4 6 8 10 12 14

CO

D (

mg

/L)

Time (Days)

0

1000

2000

3000

4000

0 2 4 6 8 10 12 14

CO

D (

mg

/L)

Time (Days)

0

2000

4000

6000

8000

10000

0 2 4 6 8 10 12 14

CO

D (

mg

/L)

Time (Days)

WW4: 38.6 g/m2/d WW3: 15.7 g/m2/d

WW2: 9.8 g/m2/d

Effect of Influent WW on Performance

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

0 1 2 3 4 5 6 7 8

Lo

g (

CO

D)

Stage Number

WW1 WW2 WW3 WW4

Stage wise Performance

Concentration of Individual Compounds

Compound WW1 WW2 WW3 WW4

C in C out C in C out C in C out C in C out

PHEL 250 13.1 250 18.8 600.0 72.3 1600 411.7

CRES 150 21.7 450.0 60.6 1000 115.7

1-NAPH 120 19.4 400.0 30.8 1000 240.5

PYRI 280 ND 200 23.4 250.0 36.7 800 108.7

QUIN 280 ND 280 25.2 250.0 41.8 900 172.6

BENZ 200 ND 360 65.5 480.0 63.0 800 101.7

NAPH 60 7.4 60 5.9 60.0 8.0 200 38.1

PHEN 0.5 0.1 0.5 0.074 0.5 0.06 0.5 0.10

FLAN 0.2 0.006 0.2 0.031 0.2 0.03 0.2 0.051

PYR 0.12 0.005 0.12 0.025 0.12 0.015 0.12 0.029

% Removal of Components at 24 h HRT Wastewater: WW1

RBC with Biofilm RBC without Biofilm

% Removal of Components at 24 h HRT Wastewater: WW2

RBC with Biofilm RBC without Biofilm

% Removal of Components at 24 h HRT Wastewater: WW3

RBC with Biofilm RBC without Biofilm

% Removal of Components at 24 h HRT Wastewater: WW4

0 25 50 75 100

PHEL

CRES

1-NAPH

PYRI

QUIN

BENZ

NAPH

PHEN

FLAN

PYR

% Removal

Stage3_WW4 Stage2_WW4 Stage1_WW4

RBC with Biofilm RBC without Biofilm

0 25 50 75 100

PHEL

CRES

1-NAPH

PYRI

QUIN

BENZ

NAPH

PHEN

FLAN

PYR

% Removal

Stage3_WW4 Stage2_WW4 Stage1_WW4

Removal in Presence of Biofilm

Significantly higher removal in presence of the biofilm

%Removal decreases as organic strength increases

Complete removal of pyridine, quinoline and benzene in 3-stage RBC from WW1

% Removal is lowest for pyrene, the component having the lowest concentration

%Removal of phenol is lowest in WW4 toxic effect due to high concentration

For most components removal in the first stage is 50% or higher but pyrene and fluoranthene removal is much less than 50% for these wastewaters

The second and third stages also play a significant role in removal of the components

Individual Compound Removal Effect of WW Strength on % Residual

0

20

40

60

80

100

WW1 WW2 WW3 WW4

Ph

en

ol

Resid

ual

(%)

0

20

40

60

80

100

WW2 WW3 WW4

o C

reso

l R

esid

ual

(%)

0

20

40

60

80

100

WW1 WW2 WW3 WW4Q

uin

olin

e

Resid

ual

0

20

40

60

80

100

WW1 WW2 WW3 WW4

Pyri

din

e

Resid

ual

(%)

1st Stage 2nd Stage 3rd Stage

Phenol & Quinoline

Increasing strength

has adverse effect on

removal

Pyridine & o-Cresol

Increasing strength

has no adverse effect

on removal

0

20

40

60

80

100

WW1 WW2 WW3 WW4Nap

hth

ale

ne R

esid

ual

0

20

40

60

80

100

WW1 WW2 WW3 WW4

Ph

en

an

thre

ne

Resid

ual

(%)

0

20

40

60

80

100

WW1 WW2 WW3 WW4Flu

ora

nth

en

e R

esid

ual

1st Stage 2nd Stage 3rd Stage

0

20

40

60

80

100

WW1 WW2 WW3 WW4Pyre

ne R

esid

ual

(%)

1st Stage 2nd Stage 3rd Stage

Individual Compound Removal Effect of WW Strength on % Residual

Although the

Concentration of

PAHs is constant

in these WW

%removal differs

Abiotic Losses in RBC Absence of Biofilm

Contribution of abiotic losses to COD removal is low

Cumulative abiotic loss increases in the subsequent stages

The %abiotic loss is least in WW4 and increases as the

organic strength is decreased

%Abiotic loss is highest for naphthalene and benzene,

intermediate for phenol, cresol, pyridine and quinoline and

least for the high MW PAHs

Benzene and naphthalene are the most volatile components, hence, their volatilization is highest

Biofilm may give rise to additional abiotic loss due to

sorption of hydrophobic components on the biofilm

Relative

Abundance

Stage

No. Mean SE

WW1

1st 0.79 0.081

2nd 0.82 0.119

3rd 0.58 0.032

WW2

1st 0.80 0.078

2nd 0.82 0.081

3rd 0.65 0.034

WW3

1st 0.76 0.022

2nd 0.73 0.091

3rd 0.58 0.032

WW4

1st 0.65 0.017

2nd 0.62 0.038

3rd 0.67 0.047

Viable Bacteria in the Biofilm

SEM Micrographs Biofilm : WW3

a b

c d

(a) Stage 1 (b) and (c) Stage 2 (d) Stage3

Sorption on the Biofilm

Estimated after running with WW4 for an extended

period; Biofilm collected and dried; Dry biomass

in stage 1, 2 and 3 were 6.5, 4.82 and 4 g/m2

Soxhlet extracted with DCM, Solvent exchange

COD in stage 1, 2 and 3 were 926, 672 and 432

mg/g

Sorption on Biofilm

Components

Mass adsorbed on the biofilm

(mg/g)

Distribution Coefficient

KD (L/g)

1st stage 2nd stage 3rd stage 1st stage 2nd stage 3rd stage

Phenol 240.8 192.2 119.8 0.29 0.36 0.29

o-Cresol 43.8 25.6 - 0.07 0.06

1- Naphthol 35.4 25.1 14.6 0.06 0.06 0.06

Pyridine 13.5 10.6 - 0.03 0.05

Quinoline 32.3 26.4 21.4 0.06 0.08 0.12

Benzene 20.8 18.8 16.1 0.06 0.09 0.16

Naphthalene 42.9 28 11.6 0.34 0.45 0.30

Phenanthrene 0.161 0.1 0.06 0.62 0.67 0.60

Fluoranthene 0.137 0.07 0.05 1.14 0.93 0.98

Pyrene 0.049 0.03 0.014 0.54 0.67 0.48

Conclusions

This system comprising of E. aurantiacum and mixed

microbial consortium is capable of achieving good removal of

organics from gasifier wastewater

Removal in presence of the biofilm is significantly higher than abiotic losses

WW3 and WW4 with OLR exceeding 15 g.m2.day-1 does not meet discharge standards

Log(COD) versus Stage Number plots can indicate the number of stages necessary for effectively treating the wastewater

WW4 having the highest organic strength shows lower

%removal High concentration of organics has adverse

effect on activity of the microorganisms

Conclusions

E. aurantiacum is the most predominant organism in the

biofilm

Viable counts in the biofilm increases with increase in organic strength

Abundance of E. aurantiacum is higher in the first two stages compared to the third stage

For WW4 abundance of E.aurantiacum in the first two stages is relatively lower than for other wastewaters

Decrease in COD correlates well with removal of the

hydrophilic and hydrophobic aromatic hydrocarbons

Further post-treatment is recommended due to the toxic

nature of the constituents

Ph.D. students

Gita Mohanty

Anal Chavan

Hansa Jeswani

R. Surve, CESE for fabricating & help in maintaining reactor

SEM & CSLM was conducted in SAIF-CRNTS, IIT Bombay

Funds provided by DBT, New Delhi

Acknowledgements

References

Chavan, A. and S. Mukherji, J of Hazardous Materials, Vol.

154, 63-72, 2008.

Jeswani, H. and S. Mukherji, 2012 Bioresource Technology,

111; 12-20.

Jeswani, H. and S. Mukherji, 2013 International

Biodeterioration and Biodegradation, 80, 1-9.

Jeswani, H. and S. Mukherji, 2015 Chemical Engineering

Journal, 259: 303-312.

Mohanty G. and S. Mukherji, 2008, International

Biodeterioration and Biodegradation, 61(3): 240-250, 2008.

Mukherji, S. and A. Chavan, 2012 Chemical Engineering

Journal, 200-202, 459-470.