1. #adrdforum @adbioresources
PROFESSOR SANDRA ESTEVES
DIRECTOR, WALES CENTRE OF EXCELLENCE FOR ANAEROBIC DIGESTION, UNIVERSITY
OF SOUTH WALES
BIORESOURCE RECOVERY
FROM WASTES AND LOW
GRADE BIOMASS TO ORGANIC
ACIDS AND BIOPOLYMERS

2. Bioresource Recovery from Wastes
and Low Grade Biomass to
Organic Acids and Biopolymers
Prof. Sandra Esteves
sandra.esteves@southwales.ac.uk

3. Hydrogen Energy
Biohydrogen Systems
Advanced Nanomaterials
Bio Energy Systems
Anaerobic Digestion
Waste and Wastewater Treatment
Monitoring and Control
Environmental Analysis
Bioelectrochemical Devices
The
Hydrogen
Centre
Biochemicals and Bioplastics Production
Biogas Upgrading and Utilisation
Life Cycle Analysis

4. What about GREEN
Chemical and
Biopolymer Platforms?
Energy and material fluxes
The Fluxes in Today’s Society
are already Complex

5. Chemicals from Methane: Acetic Acid
Acetic Acid Production Route:
Price of Acetic Acid
Variable, but can be sold for $500-1300 per
metric tonne
Acetic Acid End-uses
Adhesives, coatings, inks, resins, dyes, paints and
pharmaceuticals. It can also be further converted into
other chemicals e.g. vinyl acetate, acetic anhydride,
cellulose acetate, terephthalic acid and polyvinyl chloride
Annual Global Production of Acetic Acid
10.7 million tonnes (34th highest production volume chemical)
CH4
2H2 + CO
CH3OHCH3COOH
Steam Reforming
+ H2O
Methane
Synthesis
Gas
Methanol
Acetic Acid
Methanol
Carbonylation
+ CO
CH4
Biomethane
Biohydrogen
Acetic Acid
2H2+ CO
CH3COOH CH3OH
Chemicals from Biomethane: Acetic Acid
Products from
anaerobic
fermentations

6. Chemicals from Methane: Urea
Urea Production Route:
CH4
2H2 + CO
NH3(NH2)2CO
Steam
Reforming
+ H2O
Methane Synthesis Gas
AmmoniaUrea
H2 + CO2
Water Gas
Shift Reaction
+ H2O
+ N2
Haber
Process
+ CO2
Hydrogen and
Carbon Dioxide
End-uses of Urea
91% of urea is used for the production of solid nitrogen-based
fertilisers. Non-fertiliser uses include the production of urea-
formaldehyde resins, melamine, animal feed and numerous
environmental applications
Annual Global Production of Urea
120 million tonnes (18th highest production volume chemical)
Chemicals from Biomethane: Urea
CH4
Biohydrogen
and carbon
dioxide
2H2+ CO
Products from
anaerobic
fermentations
H2+ CO2
Biomethane
NH3
Ammonia
(NH2)2CO
Price of Urea
$300-500 per metric tonne

7. Anaerobic Digestion Process
Rate limiting
Biogas

8. © University of South Wales
Acetate
Propionate
Eubacteria
Methanosaetaceae
Methanobacteriales
Methanomicrobiales
Methanosarcinaceae
Williams et al. 2013

9. © University of South Wales
Williams et al. 2013
Methanogens and VFA residuals

10. © University of South Wales
Propionate & Lithotrophic
Methanogens
Propionate
VFA(mg/l)
Williams et al. 2013

11. Diversity of Populations in Different Inocula
Phylum distribution
(%)*
Inoculum A Inoculum B
Methanosaeta 0 2
Methanosarcina 6 0
Actinobacteria 0 8
Firmicutes 55 11
Bacteroidetes 26 20
Planctomycetes 0 0
Proteobacteria 0 7
Spirochaetes 0 2
Synergistetes 1 7
Tenericutes 1 0
Verrucomicrobia 0 1
Chloroflexi 2 8
Unknown gene copies 8 33
Oliveira et al. To
be submitted
© University of South Wales

12. Integration of
Anaerobic Processes & PHA production
© University of South Wales

13. ~ 1/3 of the initial VS converted to VFAs
in a matter of a couple of days and the
rest can be produced in another
fermentation
Jobling-Purser et al., submitted
Experiments
Volatile Fatty Acids from Food
Wastes
© University of South Wales

14. Kumi et al., to be submitted
Volatile Fatty Acids from
Badmington Grass
© University of South Wales

15. VFAs in Percolate
MSW (Full Scale)
Oliveira et al. In preparation
Double solubilisation of organics to be
digested instead of composted and
available for biorefining products
© University of South Wales

16. Near Infrared Spectroscopy In
Bioreactor Performance Monitoring
Data Point
3.1
3.3
3.5
3.7
3.9
1 2 3 4 5 76 8
2.1
2.3
2.5
Volatile Solids
Total Solids
Bicarbonate Alkalinity
1500
2000
2500
3000
3500
4000
-400
-200
0
200
400
600
800
1000
1200
1400
1600
5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77
Volatile Fatty Acidsmg.L-1
mg.L-1
g.L-1
g.L-1
Data Point
3.1
3.3
3.5
3.7
3.9
1 2 3 4 5 76 8
2.1
2.3
2.5
2.1
2.3
2.5
2.1
2.3
2.5
2.1
2.3
2.5
Volatile Solids
Total Solids
Bicarbonate Alkalinity
1500
2000
2500
3000
3500
4000
-400
-200
0
200
400
600
800
1000
1200
1400
1600
5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77
-400
-200
0
200
400
600
800
1000
1200
1400
1600
5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77
Volatile Fatty Acidsmg.L-1
mg.L-1
g.L-1
g.L-1
Reed et al., 2011
© University of South Wales

17. © University of South Wales
VFA Production from Thermally
Hydrolysed Secondary Sludge
Kumi et al., to be submitted

18. Concentration of VFAs from
Sewage Sludges Pretreated Hydrolysates (Before Acidification)
Concentrate:
Nearly 20,000mg/l total VFAs, which was the aim for the application
Tao et al., submitted © University of South Wales

19. Modern human society depends on
the use of plastics
light weight, durable and versatile
and have been even cheap
Very short life span in many cases
Fossil fuel based plastics impose
adverse environmental impacts
non-biodegradable; persisting in the environment for a
long time causing severe damage to wildlife
Google images

20. Bioplastic Categories
Maize and/or potato starch
in blend with
polycaprolactones and
other biodegradable esters
PHA
PHA
Extraction
Biomass from crops Biomass from crops Biomass from crops and wastes
Starch, cellulose Sugars Sugars, oils, VFAs
Modification
Microbial
fermentation Microbial
fermentation
Starch and cellulose
materials
Lactic acid
Polyhydroxyalkanoates (PHA)Chemical
polymerisation
Poly(lactic acid)

21. ACADEMIC EXPERTISE FOR BUSINESS (A4B)
Collaborative Industrial Research Project
SuPERPHA – Systems and Product Engineering Research for
Polyhydroalkanoates (PHA)
July 2013 – Dec 2014 (£1.2M)
University of South Wales (lead)
Partners:
Swansea and Bangor Universities
Aber Instruments Ltd.
Axium Process Ltd.
Excelsior Technologies Ltd.
FRE-Energy Ltd.
Kautex-Textron Ltd.
Loowatt
NCH
Nextek Ltd.
Scitech Adhesives systems Ltd.
(Supported by BASF)
Thames Water
Waitrose
Welsh Water
© University of South Wales

22. © University of South Wales
 Polyhydroxyalkanoates (PHA) accumulate as intracellular carbon and energy
reserve naturally within a variety of gram positive and gram negative bacteria.
 General principle for PHA accumulation = Excess carbon + Nutrient deficiency.
 PHAs are thermoplastic polyesters with melting point 50-180ºC. UV stable, low
permeation of water and good barrier properties
 Properties can be tailored to resemble elastic rubber (long side chains) or hard
crystalline plastic (short side chains)
Polyhydroxyalkanoates
O
O
O
OO
O
OO
O
O O
O
O
O
O
Polyhydroxybutyrate
(PHB)
Brittle
PHBcoPHV
Hard/flexible
Medium chain length
Polyhydroxyalkanoate
(mclPHA)
Thermoplastic Elastomer

23. © University of South Wales
Cupriavidus necator
 Cupriavidus necator, industrial
PHA producer, has shown to
naturally produce PHB close to
85% of its dry weight.
 Gram negative, rod-shaped,
flagellate, chemo heterotrophic
(DSMZ).
 Species generally occurs in soil,
known for resistance to various
metals.
Xu et al., 2010 - TEM images of C. necator in
fermentation,(A) 24 h, (B) 62 h, (C) 70 h, and (D) 82 h.

24. © University of South Wales
This presentation outlines the investigations related to three
main important factors :
1. Optimal feeding of VFA for maximum PHA production;
2. The establishment of a real-time tool for determination of
the optimum polymer harvesting time;
3. The utilisation of nutrients from digestates for bacterial
growth and PHA production; and
4. Evaluation of the effects of sodium chloride on bacterial
growth and PHA accumulation.
Various control strategies for
maximum PHA production

25. © University of South Wales
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 5 10 15 20 25 30 35 40 45 50
PHA(g/l)
Time(h)
1% Acetic Acid
2% Acetic Acid
3% Acetic Acid
4% Acetic Acid
5% Acetic Acid
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 10 20 30 40 50 60 70 80
PHB(g/L)
Time (h)
1%Butyric Acid
2%Butyric Acid
3%Butyric Acid
4%Butyric Acid
5%Butyric Acid
Batch fermentations with single VFA
addition of 1 – 5 % v/v acetic acid and
1 – 5 % v/v butyric acid
When VFA was supplied as a single
feed, it was found that concentrations
higher than 3% v/v VFA led to substrate
inhibition.
Only 18% acetic acid and 12% of
butyric acid was converted into PHA,
resulting in less than 65% (w/w) of PHA
content in the microbial cells.
VFA supplied as a single feed
Kedia et al., 2015

26. © University of South Wales
Monitoring Real Time PHA Accumulation
Online capacitance (pF/cm) profile and ex-situ measured PHA yield in
medium fed with acetic acid as the carbon source or without excess carbon
source.
0
1
2
3
4
5
0
0.25
0.5
0.75
1
1.25
0 10 20 30 40 50
PHA(g/l)
Capacitance(pF/cm)
Time (h)
Capacitance- Acetic Acid Capacitance- without excess carbon
PHA (g/l)- Acetic Acid PHA (g/l) - without excess carbon

27. © University of South Wales
Online capacitance (pF/cm) profile and ex-situ measured PHA yield in
medium fed with butyric acid as the carbon source.
Monitoring Real Time PHA Accumulation

28. © University of South Wales
PHA Concentration / Yield from
Digestates and NM
In D2, PHA concentration was
increased by almost 3x when
compared to D1 and NM.
The cells were almost 90%
packed with PHA in D2.
0
3
6
9
12
15
0 10 20 30 40 50 60
NM D1 D2
Time (h)
PHA(g/l)
PHA Yields and % CDW:
NM - 0.21 g PHA/ g VFA (28 h);
78 % CDW
D1 - 0.14 g PHA/ g VFA (48 h);
84% CDW
D2 - 0.48 g PHA/ g VFA (43 h);
90% CDW

29. © University of South Wales
Effect of NaCl concentration on
bacterial growth
0
1
2
3
4
5
6
7
8
0 10 20 30 40 50
3.5 g/l NaCl 6.5 g/l NaCl 9 g/l NaCl
12 g/l NaCl 15 g/l NaCl No salt
Time
CDW(g/l)
At 24 h, max CDW was demonstrated
by 9 g/l NaCl concentration
9 g/l NaCl = CDW 6.8 g/l
3.5 g/l NaCl = CDW 6 g/l
6.5 g/l NaCl = CDW 6.1 g/l
Control = CDW 6.4 g/l
For fermentations with NaCl
concentrations of 12 g/l and 15 g/l the
CDW was 69 - 70% lower than compared
to the control at 24 h, indicating an
inhibitory effect at higher salt
concentrations demonstrated by the
lower cell growth of C. necator cells.
CDW profile for NaCl concentration
fermentations and control

30. AD integration with Biopolymers
• Digestate Nutrient Management
• Biopolymer PHA digests well – high CH4 yield –
contributing to increasing C:N ratio in digesters and
increase in digestate quality
© University of South Wales

31. Recycling
Bioplastics
Through AD
Processes
© University of South Wales

32. Anaerobic Biodegradability of
Polymers
-100
0
100
200
300
400
500
600
0 10 20 30 40 50 60 70
MethaneyieldmlCH4/gVSadded
Days
© University of South Wales

33. © University of South Wales
The sole responsibility for the content of this document lies with the authors. It does not necessarily reflect the funders opinion. Neither the
authors or the funders are responsible for any use that may be made of the information contained therein.
Acknowledgments
Dr. Tim Patterson, Dr. Gopal Kedia, Dr. Pearl Passanha, Phil Kumi, Ben Joblin-Purser, Dr.
Des Devlin, Dr. James Reed, Dr. Julie Williams, Dr. Gregg Williams, Dr. Christian Laycock,
Prof. Richard Dinsdale, Prof. Alan Guwy, Dr. Robert Lovitt and team (Swansea
University) and Dr. Robert Elias and team (Bangor University)