The document summarizes a presentation on developing an enzymatic hydrolysis pre-treatment strategy to improve batch anaerobic digestion of wastewater from desiccated coconut processing plants. It discusses conducting experiments with enzymatic hydrolysis using lipase, initial pH adjustment, and varying inoculum to substrate ratios. The highest initial biogas production rates and biomethane yields were achieved with wastewater pre-treated with lipase and when the initial pH and inoculum to substrate ratio were optimized. While inhibition occurred initially due to lipids, digestion was recovered after 60 days, indicating the inhibition was temporary rather than permanent.

1. Development of an enzymatic hydrolysis pre-treatment
strategy to improve batch anaerobic digestion of wastewater
generated in desiccated coconut processing plants
Presenter: Mr. Kasun T. Samarasiri
B.Sc.Eng.(Hons) (Moratuwa-Sri Lanka)
M.Sc. Research Student
Department of Chemical and Process Engineering,
University of Moratuwa, Sri Lanka.
1
Supervisor: Prof. P.G. Rathnasiri
PhD (NTNU-Norway), M.Sc.(UMIST-Uk), B.Sc.Eng.(Hons) (Moratuwa-Sri Lanka)
Professor
Department of Chemical and Process Engineering,
University of Moratuwa, Sri Lanka.
Research Partner: Mr. Dave Fernando
Undergraduate Student
Department of Agriculture,
University of Peradeniya, Sri Lanka.
Presented on 3rd of July 2019
in 5th International Multidisciplinary Engineering Research Conference (MERCon 2019)
at University of Moratuwa, Sri Lanka

2. Outline of Presentation
1. Introduction
2. Research Gap and Research Problem
3. Objectives
4. Materials and Methods
5. Results and Discussion
6. Conclusions
2

3. What are the Most Commonly Used Strategies to Overcome Inhibition Caused by Lipids?
3
Strategies
used to
overcome
inhibition
Enzyme
Addition
Initial pH
Adjustment
in feed
Varying I/S
Ratio
Absorbent
Addition
Sludge
Recirculation
Dissolved Air
Flotation
In Anaerobic Digestion
2. RESEARCH GAP AND RESEARCH PROBLEM

4. In previous research studies effect of
1. enzymatic hydrolysis pre-treatment
2. initial pH adjustment
3. varying inoculum to substrate ratio
were conducted individually for different wastewaters
such as: Poultry slaughterhouse wastewater, dairy wastewater and olive
oil mill wastewater.
An alternative improved enzymatic pre-treatment strategy for the anaerobic
digestion of desiccated coconut wastewater via combined effect of the
above three strategies has not yet been conducted so far. 4
RESEARCH GAP

5. 5
Retardation of Treatment Efficiency of Anaerobic Bioreactors
due to the Presence of Lipids
RESEARCH PROBLEM
❑ Lipids attach around the hydrophobic cell walls: leads to mass transfer limitations
❑ Longer recovery times for lipid degradation: because lipids are high energy nutrients
than carbohydrates and proteins
❑ Reduction of the overall treatment efficiency: process inhibition in wastewater
treatment plant
Strategies Should be Developed to Overcome this Problem

6. 6
3. OBJECTIVES
❑ Detail characterization of wastewater and
identification of different constituents
containing lipids.
❑Develop new pre-treatment strategy to
overcome inhibition caused by lipids.

7. 7
4. MATERIALS AND METHODS
1. Enzymatic hydrolysis pre-treatment (by lipase)
2. Initial pH adjustment in feed (by NaOH)
3. Batch anaerobic digestion experiment (in 50 ml reactors
– 24 reactors)
Experiments Conducted

8. Conducted prior to the anaerobic digestion using lipase originated
from porcine pancreatic-Type II (Sigma-Aldrich) at controlled
temperature of 37°C and stirring speed of 100 rpm for 24 hours in
beakers which were placed inside water bath.
8
Enzymatic
hydrolysis
pre-treatment
experimental
setup
Enzymatic hydrolysis pre-treatment

9. 9
Enzymatic
Hydrolysis
Pre-treatment
Experimental Setup

10. Adding Sodium Hydroxide in between the enzymatic hydrolysis pre-
treatment and the anaerobic digestion.
The initial pH value of enzymatic pre-treated and enzymatic untreated
wastewater samples was adjusted to pH 7.0±0.2 using sodium hydroxide
pellets.
10
Initial pH adjustment in feed

11. ❑ Conducted using 50 ml syringes and
25 ml syringes under atmospheric
temperature (30°C - 32°C) and
pressure (1 atm) without mixing for
60 days.
❑ 24 identical anaerobic batch
reactors were performed.
11
Anaerobic Batch Reactor SetupAnaerobic Batch Reactor Setup

12. 12
Anaerobic
Batch Reactor
Experimental Setup

13. 13
a (no lipase) b (0.01 (w/v) % lipase) c (0.1 (w/v) % lipase)
A (Scenario 1. pH
adjusted)
Aa(I), Aa(II),
Aa(III), Aa(IV)
Ab(I), Ab(II),
Ab(III), Ab(IV)
Ac(I), Ac(II),
Ac(III), Ac(IV)
B (Scenario 2. pH
not adjusted)
Ba(I), Ba(II),
Ba(III), Ba(IV)
Bb(I), Bb(II),
Bb(III), Bb(IV)
Bc(I), Bc(II),
Bc(III), Bc(IV)
(I) 1:4 ratio – 80% wastewater + 20% Inoculum in volume basis (v/v)%
(O&G added from wastewater = 0.150g, sCOD in mixture = 5292.46 mg/l)
(II) 2:3 ratio – 60% wastewater + 40% Inoculum in volume basis (v/v)%
(O&G added from wastewater = 0.112g, sCOD in mixture = 5323.11 mg/l)
(III) 3:2 ratio – 40% wastewater + 60% Inoculum in volume basis (v/v)%
(O&G added from wastewater = 0.075g, sCOD in mixture = 5353.77 mg/l)
(IV) 4:1 ratio – 20% wastewater + 80% Inoculum in volume basis (v/v)%
(O&G added from wastewater = 0.037g, sCOD in mixture = 5384.42 mg/l)
a. Wastewater pre-treated without lipase
b. Wastewater pre-treated with 0.01 (w/v)% lipase
c. Wastewater pre-treated with 0.1 (w/v)% lipase
Introduction to Different Pre-Treatment Scenarios

14. ❑ pH – YSI 1200 laboratory pH
meter.
❑ Daily biogas production – Daily
volume displacement in head
space
❑ Methane composition –
syringe protocol developped by
Paul Harris, 2010
❑ Chemical oxygen demand
(COD) – ASTM D 1252-00
❑ Total solid (TS) – APHA method
1684
❑Total volatile solid (TVS) – APHA
method 1684
❑ Volatile suspended solid (VSS)
– APHA method 1684
❑ Bio-methane yield – Calculated
❑ Oil and grease content – APHA
method 10056
❑ Fatty acid content – Gas
Chromatography
14
Measured Parameters

15. Characteristics of Desiccated Coconut Wastewater (DCWW)
Parameter Range
pH value 4.86 ± 0.25
Total Chemical Oxygen
Demand (tCOD) mg/l
6462.32 ± 608.22
mg/l
Soluble Chemical Oxygen
Demand (sCOD) mg/l
5261.81 ± 246.33
mg/l
Total Solid Content (TS) g/l 5.49 ± 0.042 g/l
Total Volatile Solid Content
(TVS) g/l
3.25 ± 0.124 g/l
Oil and Grease Content
(O&G) mg/l
3.748 g/l
Experimentally Determined Physicochemical
Characteristics of Wastewater
Experimentally Determined Fatty Acid Composition of Oil and
Grease in DC Wastewater (Analyzed by the Gas Chromatograph)
15

16. Rod shaped Methanosaeta (acetotrophic) which produce methane from
acetate were observed in the inoculum. (Images were obtained from
microscope)
Parameter Value
Soluble Chemical Oxygen
Demand (mg/l)
5415.07 ±
44.24
Total Solid (g/l) 78.86 ± 2.56
Volatile Solid (g/l) 66.6 ± 2.38
Volatile Suspended Solid (g/l) 67.23 ± 0.89
Physicochemical characteristics of
anaerobic inoculum obtained from a
dairy wastewater treatment plant
16
Microscopic image of methanogens in the inoculum
Results from Characterization of Inoculum

17. 17
Cumulative Biogas Production During First 10 Days
Maximum Biogas Production was Observed in
Enzyme Added, pH Adjusted and I:S Ratio (v/v)%
2:3 Category “Ac(II)” Samples

18. 18
40% WW and 60% IN in Volume Basis
20% WW and 80% IN in Volume BasisInitial
Biogas
Production
is higher
Initial Biogas Production Rates During First 10 Days
I:S (v/v)% 1:4 I:S (v/v)% 2:3
I:S (v/v)% 3:2
I:S (v/v)% 1:4
At 0.1 (w/v)% lipase
concentration,
Most of the pH
adjusted reactors,
achieved the highest
initial biogas
production rate.

19. 19
Relationship Between: IBPR with pH & I/S Ratio
Initial Biogas Production Rate got higher when
Initial pH of Wastewater got higher (from 0.5 – 2.0 (I/S) Ratio in Volume Basis)
Initial pH
adjustment
enhance IBPR

20. 20
Relationship Between: IBPR with Enzyme addition & I/S Ratio
Initial Biogas Production Rate got higher when
Amount of enzyme added was higher (from 0.5 – 2.0 (I/S) Ratio in Volume Basis)
Enzyme addition
enhance IBPR

21. 21
Bio-methane Yield after Complete Degradation in 60 Days
Bio-methane yield of reactors under the
category (I) were higher because they
contained higher organic loading
In category ‘II’ reactors were higher in
enzyme added ‘c’ and pH adjusted ‘A’
reactor – Ac(II)

22. 22
6. CONCLUSION AND RECOMMENDATIONS
❑Lipase pre-treatment enhanced the initial biogas production rate
❑Initial pH adjustment in feed enhanced initial biogas production
rate
❑Inhibition caused by lipids was not a permanent inhibition but it
reduced the rate of biogas generation in anaerobic digestion
process.
❑Cumulative biogas production after 60 days is not affected by the
combined pre-treatment
❑VS reduction % after 60 days is not affected by the combined pre-
treatment
❑Biomethane yield increased when I/S ratio was decreasing

23. References
23

24. 24
Desiccated Coconut
Wastewater
How does Wastewater Generate in Desiccated Coconut (DC) Industry?
Raw Coconut De-husking De-shelling Paring
Splitting
Washing and
Inspection
Pasteurization/
Sterilization
Size Reduction
Drying
Screening,
Grading and
Packing
Testa Washing
Water
Coconut Water
Washing Water
Condensate Loss
}19%
76%
5%

25. COD variations of anaerobic reactor in a DC
WWTP (22/23 May 2016)
COD removal efficiency of anaerobic reactor in
a DC WWTP (22/23 May 2016)
25
COD
Removal
Efficiency
is Low
COD is
above
6500
mg/l
At a certain threshold point of tCOD above 6,500 mg/l,
tCOD removal efficiency reduced from 92% to 28% due to INHIBITION.
(UASB anaerobic reactor)

26. According to Plant Performance and Test Analysis Conducted,
the Oil and Grease Content Varied from 2 g/l to 11 g/l
26
Oil and Grease
Content in the
effluent is
above 1 g/l
But according to the literature, it was beyond the average maximum
inhibitory concentrations 1 g/l of other anaerobic reactors

27. 27
Pre-Treatment Strategy Pre-Treatment Using Wastewater References
Enzymatic pre-treatment Lipase produced by solid-state
fermentation of the fungus P. restrictum
Effluents from poultry
slaughterhouses
(Valladão et al., 2007)
Porcine pancreatic lipase Effluents from poultry
slaughterhouses
(Dors et al., 2013).
Porcine pancreatic lipase Dairy wastewater (Mendes et al., 2006)
Enzyme produced through solid-state
fermentation of penicillium sp. fungus
Dairy wastewater (Rosa et al., 2009)
Enzymatic extract preparation from
pseudomonas aeruginosa KM110
Dairy wastewater (Qumsari et al., 2012)
Porcine pancreas lipase Swine slaughterhouse waste (Ning et al., 2016)
Initial pH adjustment Sodium hydroxide Slaughterhouse waste (Affes et al., 2013)
Sodium hydroxide, Potassium hydroxide
and Calcium hydroxide
Dairy wastewater (Rani et al., 2012)
Varying substrate to
inoculum ratio
Different substrate to inoculum ratios Oily (Triolein) effluent (Cirne et al., 2006)
What are the Different Treatment Strategies Used in
Previous Research Studies?

28. 28
Degradation of long term bio-
degradable substances
Biogas Production Rate in Category “I” Reactors
Degradation of easily
biodegradable substances

29. 29
Degradation of long term bio-
degradable substances
Biogas Production Rate in Category “II” Reactors
Degradation of easily
biodegradable substances

30. 30
Degradation of long term bio-
degradable substances
Biogas Production Rate in Category “III” Reactors
Degradation of easily
biodegradable substances

31. 31
Degradation of long term bio-
degradable substances
Biogas Production Rate in Category “IV” Reactors
Degradation of easily
biodegradable substances

32. 32
Lag Phase : About 6 Days/ Complete Gas Production : 24 Days
Lag phase in pH adjusted Reactors was lesser than pH not adjusted Reactors
Inhibition is recovered
after 60 days
Cumulative Biogas Production of Wastewater in Category “I” Reactors
This longer lag
phase is the
reason for the
temporary
inhibition.

33. 33
Lag Phase : No Lag/ Complete Gas Production : 45 Days
Lag phase in pH adjusted Reactors was lesser than pH not adjusted Reactors
Inhibition is recovered
after 60 days
Cumulative Biogas Production of Wastewater in Category “II” Reactors
amount of long
term bio-
degradable
material was higher
than “I” because of
addition of higher
inoculum content

34. 34
Lag Phase : No Lag/ Complete Gas Production : 45 Days
Lag phase in pH adjusted Reactors was lesser than pH not adjusted Reactors
Inhibition is recovered
after 60 days
Cumulative Biogas Production of Wastewater in Category “III” Reactors
amount of long
term bio-
degradable
material was higher
than ‘II’ because of
addition of higher
inoculum content

35. 35
Lag Phase : No Lag/ Complete Gas Production : 45 Days
Lag phase in pH adjusted Reactors was lesser than pH not adjusted Reactors
Inhibition is recovered
after 60 days
Cumulative Biogas Production of Wastewater in Category “IV” Reactors
Amount of long
term bio-
degradable
material was higher
than ‘III’ because of
addition of higher
inoculum content