A kinetic model based on utilization of purple phototrophic bacteria
for nutrient recovery
Daniel Puyol*, Tim Huelsen, Edward Barry, Jurg Keller, Damien J. Batstone
Advanced Water Management Centre. The University of Queensland. Brisbane, QLD 4072, Australia
CRC for Water Sensitive Cities. PO Box 8000, Clayton, VIC 3800, Australia.
*
Corresponding author information: d.puyol@awmc.uq.edu.au
Abstract
In this work, the development of a kinetic model for wastewater treatment and nutrient recovery (N
and P) by purple phototrophic bacteria is presented. The model is based on chemical oxygen
demand (COD) and it is uptake-based, similarly to the IWA anaerobic digestion model 1 (ADM1).
Parameters estimation has been carried out for both autotrophic and heterotrophic growth modes,
in presence and absence of light as the energy source.
Keywords: Domestic wastewater, modelling, nutrient recovery, partition-release-recovery, purple
phototrophic bacteria.
Introduction
The development of alternative domestic wastewater treatment plants for energy and nutrient
recovery is still a key challenge (Batstone et al. 2014). Partition-Release-Recovery is a future
technology for domestic wastewater treatment. It enables recovery of energy, carbon and valuable
nutrients as N, P and K utilising biological agents to recover resources. A first step of concentration
of nutrients, mainly by assimilative growth (partition) is followed by the release of nutrients through
anaerobic digestion with net energy production (release). Subsequently, the nutrients are extracted
as an end product (recovery).
Partitioning allows the recovery of N and other nutrients, including P and K, through biological
assimilation, assisted by either selection of specialized microbes, and /or providing additional
carbon. However, current methods are still expensive and energy intensive. Our group has solved
the problem by proposing the use of purple phototrophic bacteria (PPB) that consumes low energy
infrared (IR) light and can accumulate C as PHA (Khatipov et al. 1998) and P as Poly-P (Hiraishi et al.
1991). In addition, no aeration is needed since this bacteria is facultative anaerobe. A recent study
has demonstrated that the process is robust and technically viable (Hulsen et al. 2014). PPB
organisms can growth heterotrophically using a wide range of organic compounds, but they are also
capable of growing autotrophically (Figure 1). Therefore, current models applied to traditional
secondary systems are imbalanced in C, N and P and need to be updated.
In this work, a new model applied for partitioning through PPB is presented. The model can predict
autotrophic and heterotrophic growth as well as assimilation of N and P. The model is currently
being validated against experiments.
PHOTOTROPHIC GROWTH OF PURPLE PHOTOBACTERIA
HETEROTROPHIC GROWTH
AUTOTROPHIC GROWTH
H2
(H2S, S2O3,Fe2+)
SUBSTRATE
CYCLIC
PHOSPHORYLATION
CO2
NADH
CALVIN
CYCLE
BIOMASS
H2
(Possible)
NH4+
H+
NAD+
ATP
NADHdh
ATPs
ADP
S2,SO42-,Fe3+)
E.T.S
H+ H+
NADH
H+
NITROGENASE
N2
(H+,
E.T.S
TCA
CYCLE
H+
NON CYCLIC
PHOSPHORYLATION
NADHdh
ATPs
CO2
CALVIN
CYCLE
NADH
H+
NAD+
ATP
H+
ADP
BIOMASS
Fd-ox
H+
RNf C
Fd-red
Figure 1: Schematic representation of the photoautotrophic and photoheterotrophic metabolism of
purple phototrophic bacteria.
Methods
Biomass was extracted from a lab-scale phototrophic anaerobic membrane bioreactor (PAnMBR,
Figure 2). Anaerobic batch experiments were performed using 100 mL serum bottles at ambient
temperature and illuminated with IR light using fluorescence lamps and UV-VIS absorbing foil.
Results from batch experiments have been used for obtaining model parameters. These include
Monod parameters from heterotrophic (soluble substrates) and autotrophic growth (bicarbonate),
as well as nutrient (N and P) and energy (light) limitation. The model is implemented in Aquasim
2.1d.
Parameter estimation has been performed in Aquasim 2.1d. The modified Aquasim has been used to
determine the two-parameter uncertainty surface for specific uptake rate (kM) and saturation
constant (KS) and simulate the substrate consumption of the batch tests. 95% Confidence intervals
have been calculated by minimization of the residual sum of squares. The method for determining
parameter estimation is described in Batstone et al. (2003).
Biomass yield were calculated accounting for the initial and final biomass concentration based on
substrate consumption. Biomass concentration was further transformed into COD and then yields
are expressed in COD terms.
Biomass decay rate and hydrolysis first order parameters were calculated by non-linear regression
using Aquasim 2.1d. Data to fit to first order equations was VSS (decay rate) and sum of volatile fatty
acids (hydrolysis).
Figure 2: Lab-scale PAnMBR during steady-state operation. The characteristic dark-red colour of the
phototrophic bacteria is readily observable.
Results and discussion
Model mechanisms
The key separate growth mechanisms that need to be included are heterotrophic (both in presence
and absence of light) and autotrophic growth. Hydrolysis is also required in a domestic sewage feed
situation. Environmental conditions play a relevant role in controlling the kinetics of PPB.
Temperature and pH affect the behaviour of the PPB biomass. Therefore, thermodynamics and pH
limitation need to be included in a later development of the model. Possible inhibitory effects
contemplated are related with wastewater constituents like free ammonia (which is pH-dependent)
and heavy metals (mainly Cu and Zn), which can be addressed by using simple non-competitive
inhibitory functions. Finally, competitive substrate inhibition can occur between the various soluble
substrates since PPB are very versatile. In a first approach, no differentiation between substrates is
made, but a comparative study on competitive kinetics of substrate depletion can identify how these
substrates behave differently.
The model is represented in a Petersen matrix as shown in Table 1. The model is based on COD and
uptake parameters as the IWA-ADM1. However, it also incorporates a simplification of particulate
matter degradation (Xc) through a simple hydrolytic process and it is based on a single biomass
group (YPB). The model includes the heterotrophic growth (YPB,h) which is represented by organic
soluble substrate consumption (Ss), and hydrogen (Sh2) and bicarbonate (SIC) production. The
heterotrophic growth is also divided into photoheterotrophic mode (with light as the energy source,
YPB,ph), and chemoheterotrophic mode (with organic matter as the energy source, YPB,ch). The
autotrophic growth (YPB,a) includes use of bicarbonate as C source and hydrogen and reduced
sulphur species (SIS) as electron donors. However, we observed in previous experiments a net
increase of the total COD in the system that this model is not able to explain (Huelsen et al. 2014),
which is being investigated and will be addressed in an extension to the model. This hybrid model
includes nutrient assimilation (SIN, and SIP) and limitation of the process by nutrients (N and P
concentration, IIN, IIP) and energy in the phototrophic mode (IR light intensity, Ie) by using switch
functions, and also an inhibition factor due to free ammonia (IFA). All the processes are Monod-based
with the exception of hydrolysis and biomass decay, which follow first order kinetics.
Table 1: Petersen matrix of the proposed model for nutrient recovery by purple phototrophic
bacteria
SIN
SIP
SI
XPB
Xc
XI
Rate
fh2,XC
fIN,xc
fIP,xc
fsi,xc
0
-1
fxi,xc
khydXC
-fSS,ph
fIC,Ss
0
fh2,Ss
fN,B/YPB,
fP,B/YPB,
0
YPB,ph
0
0
h
h
kM(Ss/KSs+Ss)IFAII
NIIPIe
fN,B/YPB,
fP,B/YPB,
0
YPB,ch
0
0
h
h
kM(Ss/KSs+Ss)IFAII
NIIP
fN,B/YPB,
fP,B/YPB,
0
YPB,a
0
0
a
a
kMic(SIC/KSIC+SIC)
IFAIINIIPIe
-1
1
0
kdec,XPBXPB
0
-fh2,IC
0
0
0
0
0
Soluble inert (mg
COD/L)
0
-fIS,IC
Inorganic phosphorous
(mg P_PO4/L)
Decay of XPB
-1
fh2,Ss
Inorganic nitrogen (mg
N_NH4/L)
0
0
H2 (mg H2-H/L)
Autotrophic
uptake
fIC,SS
Inorganic Sulfur (mg S/L)
-fSS,ch
Particulate inert (mg
COD/L)
Sh2
fIS,XC
Composite biomass (mg
COD/L)
SIS
fIC,XC
Phototrophic biomass
(mg COD/L)
SIC
fss,xc
Inorganic carbon (mg
C_HCO3/L)
Photoheter
otrophic
uptake
Chemohete
rotrophic
uptake
Soluble substrate (mg
COD/L)
Process
Hydrolysis
Ss
Components
Parameters estimation
Parameters estimation has been performed by using data from the batch experiments. An example
of Monod parameters determination is shown in Figure 3. Triplicate experiments are used for the
estimation of the specific uptake rate (kM) and the saturation constant (KS) values. Table 2 shows a
summary of parameter values and standard deviations in light conditions (phototrophic growth
mode). The biomass yield had been previously calculated by accounting for the increment of VSS
with respect to the COD consumed. The activity of the biomass in dark conditions (chemotrophic
growth mode) using simple substrates was very low so parameters estimation was not possible. A
further experiment using complex substrates will be carried out to explore fermentation capacity of
PPB in absence of light.
Decay rate constants (kdec,xPB) were calculated to be 0.08 ± 0.04 and 0.16 ± 0.02 d-1 in light and dark
conditions, respectively. These values are not statistically different, so that the decay rate is not
necessary to be split based on metabolic growing conditions. Hydrolysis constant (khyd) was
calculated to be 0.21 ± 0.03 d-1. These lower values indicate that losing PPB biomass can be easily
avoided by using sludge retention times of 3 d or lower, which also lead to the optimization of the
carbon, nitrogen and phosphorus assimilation.
Figure 3: Fitting of experimental data (symbols) for acetate uptake for estimating Monod
parameters. Error bars are standard deviations from triplicates. Lines are model fittings.
Table 2: Summary of parameters estimation for PPB in light conditions.
Compound
kM
St dev
mg COD/mg
VSS d
Acetate
4.2
Propionate
2.3
Butyrate
2.95
Ethanol
2.4
Ammonium*
Phosphate**
-
0.2
0.1
0.07
0.3
-
mg IC /mg VSS
d
Bicarbonate 0.07
0.02
St dev
χ2
HETEROTROPHY
mg COD/L
KS
21
1.5
0.5
3.4
0.023
0.081
2
142
1.8
339
0.6
175
3.8
1548
0.048
0.299
0.005
0.0002
AUTOTROPHY
mg IC/L
undergoing undergoing
478
Ym
St dev
Y***
mg VSS/mg COD
mg
COD/mg
COD
1.42
1.34
1.30
1.30
-
0.71
0.67
0.65
0.65
-
0.04
0.12
0.04
0.03
-
mg VSS/mg IC
1.7
0.5
mg
COD/mg IC
3.4
* In mg NH4-N/L. ** In mg PO4-P/L ***1 g VSS = 2.00 ± 0.29 g TCOD
Implications and future model additions
Model parameters calculated in this work will be used for simulation of PPB process for domestic
wastewater treatment. Partitioning is the first and most important process in the nutrient recovery
process by the partition/release/recovery concept. Modelling this process is of utmost importance for
a correct implementation of this technology in pilot plant and full-scale. The model is currently being
validated by using data from a lab-scale PAnMBR working in continuous mode for more than 200 d.
Once validated, the model will be implemented in Matlab/Simulink.
Future upgrades of the model will include pH prediction as well as pH inhibition. Temperature
dependency of PPB is also being assessed. Gas phase will be added to the model as well, so that
bicarbonate concentration will be dependent on CO2 concentration in the gas phase through standard
gas-liquid transfer. Bioaccumulation of P as Poly-P is going to be addressed as a side process nonrelated with biomass growth. Assimilation of K will be also added in the model. In addition, possible
competitive inhibition between substrates (mainly acetate, propionate, butyrate and ethanol) could
be implemented in the model by spreading the substrate consumption and including competitive
inhibitory functions.
Conclusions
A model has been developed to predict the purple phototrophic bacteria behaviour on domestic
wastewater treatment. The model is implemented in Aquasim 2.1d. Parameters estimation has been
performed with strong statistical support. These parameters are being used for scaling-up and
controlling the process. The model, once validated, will be implemented in Matlab/Simulink.
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