Biomass Conversion and Biorefinery

https://doi.org/10.1007/s13399-023-04043-w

ORIGINAL ARTICLE

Optimization of biomass and polyhydroxyalkanoate production

by Cupriavidus necator using response surface methodology
and genetic algorithm optimized artificial neural network
Pema Lhamo1 · Biswanath Mahanty1 · Shishir Kumar Behera2

Received: 12 January 2023 / Revised: 28 February 2023 / Accepted: 7 March 2023

© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023

Abstract
High polyhydroxyalkanoate (PHA) yield from selected substrates associated with minimal accumulation of residual biomass
can improve the process economy. In this study, different carbon (glucose, sucrose, glycerol, and acetic acid) and nitrogen
­(NH4Cl and urea) sources were screened for PHA production by Cupriavidus necator. The effects of incubation time, nitro-
gen, and phosphate concentration on biomass growth and PHA production were co-optimized through response surface
methodology (RSM) and genetic algorithm-optimized artificial neural network (GA-ANN). Sucrose and urea were found
to offer significantly better (p <0.001) biomass (1.468 ± 0.007 g ­l-1) and PHA (0.924 ± 0.02 g ­l-1) yield when compared
with other carbon and nitrogen sources. Though the performance of both the models remains similar for biomass (R2 =
0.97–0.98), GA-ANN (with six neurones in a hidden layer) seems exceptionally better in predicting PHA yield (R2 = 0.97)
when compared to the polynomial model (R2 = 0.92). The maximum PHA concentration of 2.69 g ­l-1 was predicted by the
ANN model at an incubation time of 62.80 h with 2.0 g l­ -1 of nitrogen and 4.0 g l­ -1 of phosphate concentration. The multi-
composite desirability using the GA-ANN model projected a better polymer-to-biomass ratio compared to the polynomial
model. The inclusion of a cost-benefit analysis framework may be warranted before recommending the optimal conditions
obtained through multivariate regression and GA-ANN models.

Keywords Polyhydroxyalkanoates · Central composite design · Multi-objective optimization · Composite desirability ·

Artificial neural networks

1 Introduction waste [1] warrant the exploration of biopolymers derived

from renewable resources as a sustainable and environment-
Plastics derived from fossil fuels have offered a comfort- friendly alternative [2]. Polyhydroxyalkanoates (PHAs),
able lifestyle and have driven the global economy for dec- biodegradable thermoplastic polymers accumulated by a
ades. However, serious environmental impacts associated diverse group of microorganisms, can be very similar to
with improper management and expensive recycling of the conventional petrogenic polypropylene in terms of molecu-
lar weight, stiffness, and melting point [3]. Though it has not
* Biswanath Mahanty been extensively commercialized, PHAs possess immense
bmahanty@karunya.edu potential in healthcare, packaging, tissue engineering, and
Pema Lhamo agriculture (Table S1). Being nontoxic and carbon neu-
pemalhamo20@karunya.edu.in tral, PHAs are promising biopolymers aligned with United
Shishir Kumar Behera Nations (UN) Sustainable Development Goals (SDGs) for
shishir.kb@vit.ac.in sustainable consumption and production (SDG 12) and
1
conservation and sustainable use of the oceans, seas, and
Department of Biotechnology, Karunya Institute marine resources (SDG 14) [4]. PHA-based products can be
of Technology and Sciences, Karunya Nagar, Coimbatore,
Tamil Nadu 641 114, India composted, mineralized, and reabsorbed by plants serving
2 as feedstock for PHA production, closing the loop of mate-
Industrial Ecology Research Group, School of Chemical
Engineering, Vellore Institute of Technology, Vellore, rial consumption cycles and fulfilling “circular economy”
Tamil Nadu 632 014, India model [5, 6].

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Mixed microbial culture utilizing volatile fatty acids
(VFAs) can produce copolymer (HB: 45–70 mol%, HV:
30–55 mol%) with decreased glass transition, melting tem-
perature, crystallinity, and average molecular weight [7, 8].
Among others, Cupriavidus necator has been known to be
an efficient polymer producer accumulating PHAs up to
90% of its dry cell weight using simple sugars (glucose and
fructose) and VFAs [9, 10]. Having the versatility of both
autotrophic and heterotrophic growth, C. necator can utilize
a wide range of organic substrates [9]. The polymer yield,
composition, and material properties can differ based on
carbon and nitrogen sources [11].
Exorbitantly high cost of traditional substrates has led to
an exploration of agro-industrial wastes, such as lignocel-
lulosic materials, biodiesel waste, sucrose-rich molasses,
and vinasse from the sugar industry as cheap renewable raw
materials which can be used for PHA production with appro-
priate pre-treatment (Table S2) [12]. However, the nutrient
composition (i.e., C:N:P molar ratio) of the waste may not
be optimal as low C:N or C:P ratio can have a negative influ-
ence on the quantity and quality of the accumulated polymer
[13]. Therefore, it is crucial to select the appropriate sub-
strate feed and understand and optimize the defined media
composition (Table S3) for the overproduction of biopoly-
mers, before switching over to agro-industrial waste [14].
An increase in biopolymer production alone may not be
sufficient to improve the process economy as residual bio-
mass growth, PHA accumulation, and substrate consumption
are highly interdependent [15]. Several studies have reported
the optimization of culture medium for improvement in bio-
mass and PHA production [16]. Though the biopolymer yield
improves with the biomass concentration [17], the increased
cost of cell disruption and dissolution of residual biomass
might offset the benefit if the biopolymer recovery is not sub-
stantially improved. For such uncertain and competing objec-
tives, no single solution can be offered [18] and a trade-off
can be formalized using a composite desirability function for
those responses under multi-objective optimization [19].
Statistical experimental design-based response surface
methodology (RSM) has been widely explored to model
measured response(s) as a linear, quadratic, or cubic function
Table 1  Coded levels of different variables influencing PHA produc-
tion included in the CCD
Variables a Coded levels
−1.682 −1 0 1 1.682
Time (h) 4 21 47 72 90
Urea (g l­-1) 0.10 0.48 1.05 1.60 2.00
Phosphate (g ­l-1) 0.10 0.89 2.05 3.20 4.00
a
The initial concentration of carbon (sucrose) was maintained at 30 g
l­ -1 across the design
13
Biomass Conversion and Biorefinery
of independent variables [20, 21]. Though the RSM-based
model does not provide any mechanistic view of the process,
the prediction of the optimal factor setting and optimization
in a nonlinear system is quite effective [22]. On the other
hand, artificial neural network (ANN) is increasingly being
adopted to model complex nonlinear processes without the
need for explicit mathematical formalization, unlike RSM.
Though an efficient ANN model requires larger data set in
general, a smaller dataset statistically well distributed in
input dimension (e.g., in the case of RSM) can effectively
be utilized [23]. ANN has been adopted to model and opti-
mize poly-β-hydroxybutyrate (PHB) production in microbial
culture as a function of media composition [24]. The exten-
sion of RSM design into ANN modeling has been adopted
in different bioprocess systems, offering better predictability
[25, 26]. However, the choice of ANN topology (number of
neurons in the hidden layer, initialized weight, and bias) in
most of the relevant studies remains heuristic [27]. Genetic
algorithm can be adopted to fine-tune the ANN topology
and cross-validation–based data splitting for training, test-
ing, and validation subsets [28, 29]. Additionally, different
responses from the same process are separately mapped
using independent networks [30], which may not be con-
venient for a co-optimization problem.
This work aims at selecting appropriate carbon (glucose,
sucrose, glycerol, and acetic acid) and nitrogen ­(NH4Cl and
urea) sources for the growth of Cupriavidus necator and PHA
production using factorial design. The biopolymer production
with selected substrates using nitrogen and phosphate concen-
tration and incubation time as predictors was independently
modeled adopting central composite design (CCD)–based
RSM methodology and genetic algorithm optimized ANN
(GA-ANN). Subsequently, GA-driven co-optimization of the
process, i.e., maximized PHA accumulation while minimizing
biomass growth, was projected from a composite desirability
function in both the modeling frameworks. The optimal sub-
strate concentration evaluated from this study can be extended
to identify appropriate agro-industrial wastes for cost-effective
PHA production. To the author’s best knowledge, this is the
first report on the efficient utilization of sucrose by Cupri-
avidus necator for PHA production.
2 Material and methods
2.1 Microorganism and culture conditions
Cupriavidus necator KCTC 22469, purchased from Korean
Collection for Type Cultures, South Korea, was maintained
and subcultured in nutrient broth medium at 30°C. The
PHA production studies were carried out in mineral salt
medium (MSM) containing (g l­-1) ­NaH2PO4, 4; ­Na2HPO4,
4.6; ­K2SO4, 0.45; ­Mg2SO4, 0.39; ­CaCl2, 0.062; and 5 ml ­L-1
Biomass Conversion and Biorefinery

Table 2  CCD for three variables Runa Time (h) Urea (g l­-1) Phosphate (g ­l-1) PHA (g ­l-1) Biomass (g l­-1)
with the observed and predicted
responses Observed Predicted Observed Predicted

1 21.43 1.61 0.89 0.47 0.27 0.48 0.46

2 4.00 1.05 2.05 0.05 0.08 0.47 0.44
3 21.43 0.48 0.89 0.71 0.71 0.48 0.54
4 47 1.05 2.05 1.64 1.55 0.83 0.85
5 47 0.10 2.05 0.57 0.68 0.77 0.72
6 47 1.05 2.05 1.64 1.55 0.83 0.85
7 47 1.05 2.05 1.61 1.55 0.87 0.85
8 47 1.05 2.05 1.63 1.55 0.85 0.85
9 47 1.05 2.05 1.42 1.55 0.84 0.85
10 21.43 0.48 3.20 1.1 0.05 0.51 0.54
11 72.56 0.48 3.20 0.88 0.77 0.8 0.81
12 72.56 1.61 0.89 0.46 0.28 0.98 0.98
13 47 1.05 4.00 1.34 1.41 0.82 0.82
14 47 2.00 2.05 1.05 1.28 0.83 0.86
15 47 1.05 2.05 1.43 1.55 0.87 0.85
16 72.56 0.48 0.89 0.98 0.72 1.07 1.06
17 21.43 1.61 3.20 1.25 1.20 0.48 0.79
18 90 1.05 2.05 0.38 0.69 1.1 1.11
19 72.56 1.61 3.20 2.1 1.92 1.08 1.06
20 47 1.05 0.10 0.32 0.59 0.76 0.75
a
Run no. 10 and run no. 17 are excluded from polynomial model for PHA and biomass, respectively

of trace element solution containing (g ­l-1) ­ZnSO4.7H2O,
0.1; ­MnCl2.4H2O, 0.03; ­H3BO3, 0.03; ­CoCl2.6H2O, 0.20;
­CuCl2.2H2O, 0.01; N ­ iCl2.6H2O, 0.02; and N
­ a2MoO4.2H2O,
0.03. The carbon and nitrogen sources (as per the experi-
mental design) were added to the MSM from a filtered steri-
lized stock solution.
PHA production. All pairwise multiple comparisons
between the groups were performed by adapting the
Holm-Sidak method. A significance level of α = 0.05
was used throughout the analysis. The statistical analysis
module of SigmaPlot (version 12.0) (Systat Software Inc.,
San Jose, California, USA) was used for the analysis.

2.2 Selection of carbon and nitrogen sources

2.3 Experimental design for modeling
The effects of various carbon (i.e., glucose, sucrose, glyc-
erol, and acetic acid) and nitrogen (i.e., urea and N ­ H4Cl) To model biomass growth and PHA production as a func-
sources on PHA production were evaluated using batch tion of incubation time, urea, and phosphate concentra-
studies. Each experimental set containing MSM (50 ml) tion, a CCD-based RSM was adopted. The initial concen-
supplemented with 30 g ­l-1 of carbon (filter sterilized) and tration of sucrose was maintained at 30 g l­-1 across the
0.5 g l­-1 of nitrogen, adjusted to pH 7.0, was inoculated design to offer different C/N ratios in the design space.
with overnight grown culture at a targeted biomass con- Five coded levels (−α, −1, 0, +1, +α) for each independ-
centration of 1.1 g ­l-1. The experimental sets were incu- ent variable and their corresponding values are shown
bated for 48 h at 30°C in a shaker incubator (110 rpm). in Table 1. The design contains 20 experimental runs
Following the measurement of biomass growth (λmax = including eight factorial points, six axial points, and six
600 nm), the culture was centrifuged at 4800 × g for 10 center-point replicates [31]. The CCD and associated
min. The culture supernatant and pellet were used for the response values are shown in Table 2. The experimental
measurement of residual substrate concentration and PHA results of CCD were fitted with a second-order polyno-
production, respectively. mial model (Eq. 1).
A two-way analysis of variance (ANOVA) was used 3 2 ∑
3 3
to detect the effects of carbon and nitrogen sources, (1)
∑ ∑ ∑
Y = 𝛽i0 + 𝛽i Xi + 𝛽ij Xi Xj + 𝛽ii Xi2
together with their interactions on biomass growth and i=1 i=1 j=i+1 i=1

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 Biomass Conversion and Biorefinery

Fig. 1  Effect of different carbon sources on a biomass, b PHA production, and nitrogen sources on c PHA production. Different letters indicate
the statistical difference between the groups (p < 0.050). Error bars denote the standard deviation of duplicate measurements

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Biomass Conversion and Biorefinery

Table 3  Two-way ANOVA Source of variation PHA Biomass

of carbon and nitrogen source
selection for biomass and PHA DF SS MS F P DF SS MS F P
production
C 3 1.686 0.562 493.960 <0.001 3 0.044 0.015 46.882 <0.001
N 1 0.006 0.006 5.498 0.047 1 0.001 0.001 4.194 0.075
C×N 3 0.027 0.009 7.824 0.009 3 0.002 0.001 1.966 0.198
Residual 8 0.009 0.001 8 0.002 0.001
Total 15 1.728 0.115 15 0.049 0.003

where Y represents any of the responses (PHA, biomass)
and β0, βi, βij, and βii are the regression coefficients for inter-
cept, linear effects, two-way interactions, and square terms,
respectively.
2.4 Quadratic model selection and statistical
analysis
The response obtained from the different batch experimen-
tal runs was fitted to a quadratic model and checked for
the significance of linear and nonlinear model terms in
ANOVA. Following the removal of the outlier (based on
studentized residual), a stepwise regression was performed
while adding or removing the predictors at each step (criti-
cal p values set at 0.05 and 0.10, respectively) until the
final model is achieved. The accuracy of the final model
was determined by the coefficients of determination (R2),
and adjusted R2 (adj-R2) values, while the significance of
the terms was evaluated using ANOVA. The model selec-
tion and ANOVA analysis were performed using the step-
wiselm function in Matlab® ver. 9.5.
2.5 Artificial neural network model
optimized using genetic algorithm. The L-M backpropagation
algorithm (trainlm) was used to train the network with a manual
sample division into training, validation, and test sets.
2.5.1 Genetic algorithm–based optimization of ANN
architecture
The number of HL neuron initialized weight and bias values
were optimized by a genetic algorithm [32]. For a given
number of predictors (­ Ni), response (­ No), and HL neurons
­(Nh), the initialized population consists of a real-valued vec-
tor ai ∈ [−1, 1] of length (l) as in Eq. (2).
(2)
( ) ( ) ( )
l = Nh + Ni × Nh + Nh × No + Nh + No
The first Nh genes are to be decoded in binary reflecting
if a hidden neuron is included or not. The second and third
term in the RHS of the equation represents the number of
weights from IL to the HL and from the HL to the OL. The
fourth term accounts for the number of bias values to the
HL and OL neurons.
The decoding of HL neuron selection [33] is based on Eq. (3).
{
1 if ai > 0
yi =
0 otherwise (3)

ANN model was developed to predict biomass growth and PHA

where ­yi refers to the ith hidden neuron which is utilized or not.
production from the experimental data set. The adopted ANN
All bias and weights pertinent to an unused HL neuron are also
architecture comprises one input layer (IL) containing three pre-
disregarded from architecture. No transformation for the weight
dictors, one hidden layer (HL), and one output layer (OL) with
and bias values was required. A population size of 500 and a
two responses, i.e., biomass and PHA. However, the number
maximum generation of 200 were adopted in GA optimization,
of neurons in HL and the initialized weight and bias (bj) were
retaining other parameters at their default settings in Matlab®

Table 4  ANOVA for the Model terms PHA Biomass

regression model of PHA and
biomass production SS DF Mean SS F p value SS DF Mean SS F p value

Total 5.898 18 0.328 0.686 18 0.038

Model 5.416 8 0.677 14.050 <0.001 0.674 8 0.084 70.925 <0.001
Linear 1.721 3 0.574 11.902 0.001 0.590 3 0.197 165.590 <0.001
Nonlinear 3.696 5 0.739 15.339 <0.001 0.084 5 0.017 14.124 <0.001
Residual 0.482 10 0.048 0.012 10 0.001
Lack of fit 0.426 5 0.085 7.600 0.022 0.010 5 0.002 6.481 0.031
Pure error 0.056 5 0.011 0.002 5 0.000

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 Biomass Conversion and Biorefinery

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 Biomass Conversion and Biorefinery

◂Fig. 2  Three-dimensional surface plots (left panels) and two-dimen- where subscript i ∈ {P, X} indicates either PHA (P) or bio-
sional contour plots (right panels) showing the binary effect of a, d mass (X) as the response. The Yi, Li, and Ui are modeled
urea concentration-time, b, e urea-phosphate concentrations, and c, f
phosphate concentration-time on microbial PHA production using the
and minimum and maximum acceptable responses attainable
reduced quadratic model. Variables not projected in surface/contour by changing the predictors. The shape of the desirability
plots are on hold at their respective center-point reference level, i.e., function depends on the value of “s.” The overall (global)
phosphate 2.05 g l­-1; urea 1.05 g ­l-1; and time 47 h desirability D is the weighted geometric mean of all the indi-
vidual desirability functions as in Eq. (7).
2022b. The average normalized square error (ANSE) for both the ��
responses (i.e., biomass and PHA) on the entire dataset was set as w
(7)
∑
D = wi di i
an objective function for minimization [32], as shown in Eq. (4).
n � n �
where wi is the weight factor for the responses indicating
their importance to the overall desirability [35].
⎛∑ �2 ∑ �2 ⎞
⎜ yX,i − ŷ X yP,i − ŷ P ⎟
i=1 i=1
ANSE = ⎜ n
�2
+ n ⎟∕2
�2 ⎟ (4)
2.7 Analytical methods
⎜ ∑� ∑�
⎜ tX,i − ̂tX tP,i − ̂tP ⎟
⎝ i=1 i=1 ⎠

where yX,i, yP,i are predicted and tX,i, tP,i are target responses of 2.7.1 Estimation of biomass and substrate concentration
the ith sample for biomass and PHA, respectively. The
ŷ X , ŷ P , ̂tX , ̂tP are sample averaged predicted and target The biomass concentration in experimental sets at the end
responses for biomass and PHA, respectively. of the incubation period was estimated from optical density
measurements of resuspended biomass (λmax = 600 nm) and
2.6 Multi‑objective optimization a calibration constructed in a priori between dry cell weight
and optical density measurement. The estimation of residual
The individual desirability [34] for PHA production (maximi- sucrose, urea, and phosphate concentration in the culture
zation) and biomass growth (minimization) in the multi-objec- supernatant was measured using the dinitrosalicylic acid
tive optimization are shown in Eqs. (5) and (6), respectively. (DNS) method [36], diacetyl monoxime [37], and ammo-
nium molybdate-ascorbic acid method [38], respectively.
⎧
⎪ 0 if YP < LP
⎪ � �S 2.7.2 PHA extraction and quantitation
dP = ⎨ YP −LP
UP −LP
if LP ≤ YP ≤ UP (5)
⎪
⎪ 1 if YP > UP
⎩ The biomass collected after PHA accumulation was centri-
fuged at 4800 × g for 10 min and dried at 60°C in a hot air
oven. The dried pellet obtained was suspended in chloroform
⎧ 1 if YX < LX
⎪� �S (25 ml), sonicated for 45 min at 50°C, and left to dissolve for
dX = ⎨ UX −YX
UX −LX
if LX ≤ YX ≤ UX (6) 5 days at 30°C in a shaker at 120 rpm [39]. The solution was
⎪
0 if Y > U then filtered to remove all undissolved material.
⎩ X X

Fig. 3  Optimized ANN archi-

tecture a performance during
training and b comparison of
the experimental and predicted
responses

13
 Biomass Conversion and Biorefinery

Table 5  Composite desirability for PHA and biomass production adopting different response weights
Model Weightage Variables Projected response Desirability D
-1 -1 -1 -1
PHA X T (h) N (g ­l ) P (g ­l ) PHA (g ­l ) X (g ­l ) P/X PHA X

Polynomial model 1.00 0.00 61.87 2.00 4.00 2.40 1.10 2.18 1.00 0.14 1.00
0.75 0.25 55.42 1.72 4.00 2.24 1.02 2.19 0.93 0.21 0.64
0.50 0.50 35.74 1.26 2.42 1.51 0.79 1.91 0.63 0.39 0.49
0.25 0.75 20.32 0.88 0.99 0.73 0.52 1.40 0.30 0.59 0.51
GA-ANN model 1.00 0.00 62.80 2.00 4.00 2.69 1.01 2.69 1.00 0.18 1.00
0.75 0.25 47.00 2.00 3.76 2.52 0.77 3.27 0.93 0.37 0.74
0.50 0.50 38.98 2.00 3.61 2.19 0.65 3.37 0.81 0.47 0.62
0.25 0.75 28.31 2.00 3.30 1.41 0.48 2.94 0.52 0.60 0.58

The extracted PHAs in the chloroform layer were quantified
using a crotonic acid assay [40]. Briefly, 100 μl of the organic layer
was air-dried to which 1 ml of concentrated sulfuric acid was added
and kept in a water bath at 100°C for 10 min. The solution was then
cooled and mixed properly and spectrophotometrically measured
(λmax = 235 nm) against a sulfuric acid blank. Crotonic acid assay
of PHB purchased from Sigma-Aldrich was used as standard.
1
H proton NMR analysis was used to determine the com-
position of PHA produced by C. necator. The PHA samples
were dissolved in deuterated chloroform ­(CDCl3), and the
spectra was recorded against tetramethyl silane as an inter-
nal standard using a Bruker BioSpin GmbH 300 Mhz NMR
spectrometer. The chemical shifts were reported in ppm.

2.7.3 Polymer characterization 3 Results and discussion

The IR spectrum of air-dried biopolymer was analyzed with
Bruker VERTEX 80v (Bruker, Germany) Fourier transformation
infrared (FTIR) spectrophotometer. The analysis was performed
using a sample-laden KBr disk, and the spectrum was recorded in
the range 4000–400 c­ m−1 with a resolution of 2 ­cm−1 measured
in transmittance mode.
3.1 Selection of carbon and nitrogen sources
The effect of different carbon and nitrogen sources on
biomass growth and PHA production is shown in Fig. 1.
It is evident that biomass accumulation across the carbon
sources (except between glucose and glycerol) is significantly

Fig. 4  Fourier transform infra-

red (FTIR) spectrum of biopoly-
mer extracted from Cupriavidus
necator

13
Biomass Conversion and Biorefinery
different (p <0.001), where it varied between 0.499 ± 0.006 g
­l-1 (acetic acid) and 1.468 ± 0.036 g l­ -1 (sucrose) after 48 h of
incubation (Table S4). The amount of accumulated polymer
from different carbon sources followed in the order of sucrose
> glucose > glycerol > acetic acid, irrespective of the iden-
tity of nitrogen sources. The difference in the mean biomass
accumulation between urea and N ­ H4Cl was not statistically
significant (p = 0.075) and can be attributed to random sam-
pling variability (Table 3). Though carbon (p <0.001) and
nitrogen (p = 0.047) sources had a significant influence on
PHA production, multiple comparison tests revealed that
Fig. 5  1H NMR spectra of extracted PHA from Cupriavidus necator
the difference between urea and ­NH4Cl was only discernible
when glucose was used as the carbon source.
The choice of an “ideal substrate” for biomass growth
and PHA production significantly varies from one microor-
ganism to another. For example, glucose has been shown to
produce higher biomass and PHA yield for Bacillus megate-
rium, when compared to the other substrates (e.g., glycerol,
sucrose, acetate) [41]. However, in the case of Ralstonia
eutropha, such substrate selection makes only a nominal
difference [42]. Moreover, the preferred substrate for bio-
mass growth may not necessarily be the best choice for
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 Biomass Conversion and Biorefinery

Table 6  PHA concentration and intracellular PHA content in pre- and post-optimization scenarios reported in the literature
Microbial strain Feedstock Optimized parameters PHA conc. PHA content Ref.
(g ­l-1) (% CDW)
Before After Before After

Thermus thermophilus Sodium gluconate Time, C/N ratio, and phosphate concentration 0.33 0.47 25.78 41.23 [53]
Cupriavidus necator Glucose Agitation speed, glucose, and urea concentra- 6.80 11.83 75.98 92.13 [63]
tion
Burkholderia cepacia Waste glycerol Aeration rate, agitation speed, and cultivation 1.18 3.60 51.98 63.94 [14]
period.
Ralstonia eutropha Fructose Carbon sources 1.40 6.75 43.08 50.37 [42]
Cupriavidus necator Crude glycerin Crude glycerin and nitrogen concentration 0.11 2.81 73.33 64.75 [46]
Bacillus cereus Molasses Molasses, ammonium sulfate, and initial pH 1.22 1.42 35.16 40.23 [64]
Burkholderia sp. Kraft lignin Incubation period, pH, and carbon concentra- 0.02 0.04 23.96 32.26 [65]
tion
Cupriavidus necator Crude glycerol Glycerol, ­(NH4)2SO4, and trace element con- 22.94 24.98 50.01 54.01 [66]
centration
Azohydromonas lata Sucrose Sucrose, urea, and trace elements concentration 3.85 5.95 47.53 48.57 [62]
Halomonas daqingensis Algal biodiesel waste residues Algal biodiesel waste residues, NaCl, and pH 0.21 0.24 68.71 65.19 [67]
Ralstonia eutropha Glucose Glucose, ammonium chloride, furfural, 2.10 2.10 60.00 61.76 [68]
hydroxymethylfurfural, and acetate
Bacillus aryabhattai Sweet sorghum juice Total sugar and urea concentration 2.30 2.43 35.49 39.07 [69]
*
Optimization refers to conditions for increasing PHA, if otherwise not stated

PHA production. For example, glucose and sucrose though
resulted in equivalent biomass growth in Burkholderia
sacchari, the PHA production was at least one-fold higher
with the former substrate than that in the latter one [43].
In this study, the low biomass growth and PHA production
from acetic acid (when compared to carbohydrates) may
be attributed to a change in intracellular pH and metabolic
disturbance from the dissociation of the acid at a high con-
centration [44]. A reasonably good growth (6.843 ± 0.213 g
­l-1) and PHA production (4.440 ± 0.118 g ­l-1) were observed
with C. necator when grown at moderate (1 g ­l-1) acetate
concentration [45].
3.2 Optimization of PHA production using
a quadratic model
The biomass and PHA concentration across all the twenty
experimental runs (Table 2) varied between 0.47–1.10 g
­l-1 and 0.05–2.10 g ­l-1, respectively. The maximum bio-
mass and PHA production were observed in the experi-
mental run no. 18 (urea: 1.05 g l­-1, phosphate: 2.05 g
volatile fatty acids) has been shown to accumulate about
1.66 g l­-1 and 1.02 g l­-1 PHA [47, 48]. Under optimal
culture conditions, PHAs accumulation by B. megaterium
strain Ti3 has also been very low (0.61 g l­ -1) after 48 h
of incubation [49]. Exception to studies in fed-batch and
continuous reactor, a high PHA yield is rather difficult in
lab-scale batch experiments [50].
The ANOVA analysis of the full quadratic model used
to fit the biomass and PHA accumulation data from CCD
experiments suggested a significant lack of fit for both the
responses (Table S5). Observations with unusually high
studentized residuals, i.e., run no. 10 (for PHA) and run
no. 17 (for biomass), were dropped from step-wise regres-
sion analysis. Reduced quadratic model equations (in coded
form) after the elimination of insignificant terms (p >
0.05), for PHA and biomass, are shown in Eqs. (8) and (9),
respectively.
YPHA = 1.553 + 0.182X1 + 0.179X2 + 0.245X3
+ 0.177X1 X3 + 0.397X2 X3 − 0.413X21 (8)
− 0.202X22 − 0.195X23

­l -1) and 19 (urea: 1.61 g l­-1, phosphate: 3.20 g l­-1) after

90 h and 72 h, respectively. Though biomass produc-
tion was very similar, PHA accumulation was found to
be significantly different between the two experimental
conditions. A similar PHA concentration (2.81 g l­ -1) has
been reported from C. necator utilizing crude glycerol
and ammonium sulfate [46]. C. necator culture utilizing
either defined (glucose) or complex media (waste-derived
YX = 0.848 + 0.199X1 + 0.041X2 + 0.020X3
− 0.063X1 X3 + 0.0814X2 X3 − 0.025X21 (9)
− 0.019X22 − 0.023X23
The fitness of the polynomial models for PHA produc-
tion (R2 = 0.92, adj-R2 = 0.85) and biomass growth (R2 =
0.98, adj-R2 = 0.97) suggested a good agreement between

13
Biomass Conversion and Biorefinery
experimental and predicted values. The model fitness and its
coefficient (Table 4) were evaluated using the significance of
linear and nonlinear terms and their lack of fit assessment (α
= 0.01). All the linear, interactions (except between time and
phosphate), and square terms were significant (p < 0.05) in
the PHA accumulation model (Eq. 8).
The coefficient (in absolute values) for the linear term
of phosphate on PHA production (Eq. 8) is about 1.5 times
higher than those of urea and incubation time, suggesting
its strong influence [51]. Negative coefficients of quadratic
terms for urea and phosphate suggest an inhibitory effect of
those variables on PHA accumulation at a higher level. This
is in agreement with the nutrient-limiting condition required
for PHA production [46, 52].
The interaction effect of the predictors on biomass
growth remains three to four times smaller than in PHA
production. The negative coefficient of the square terms
suggested that the impact of changing incubation time,
nitrogen, or phosphate concentration would get diminished
(low sensitivity) at their higher level. The significant influ-
ence of time, nitrogen, and phosphate concentration on
cell growth and PHA accumulation is in line with other
studies [53].
The quadratic model equations (Eqs. 8 and 9) represent the
relationship between independent and dependent variables.
The importance of individual predictors and their optimal set-
tings can be estimated in order to maximize polymer produc-
tion or minimize biomass growth. Though the coefficient of
the model equations are very specific to the problem under
investigation, the relative importance of the predictors and
their nature of influence (synergistic/antagonistic) can be
extended to studies for cost-effective PHA production [54].
The response surface and contour plots for PHA accu-
mulation, drawn as a function of two independent variables
(holding the other at respective center-point, phosphate 2.05
g ­l-1, time 47 h, and urea 1.05 g l­-1), are shown in Fig. 2.
At the lower end of incubation time and urea concentra-
tion, the PHA accumulation seems to be considerably low,
which increases until reaching maxima and then declines
with further increase of the variables (Fig. 2a). An optimum
combination of independent variables for PHA accumulation
can be traced to the clear peak in the response surface within
the design boundary. According to Eq. (8), a maximum PHA
production (2.40 g ­l-1) can be expected after 61.87 h of incu-
bation in the experimental run starting with 2 g ­l-1 of urea
and 4 g ­l-1 phosphate.
3.3 Artificial neural network–based model
and optimization
The GA-based optimization projected six neurones in the
hidden layer (Fig. S1) while minimizing composite normal-
ized error for both PHA and biomass in the dataset. The
optimal initialized weight and biases for the optimal ANN
architecture are shown in Table S6. The training of the
optimized network converges at 4 epochs (Fig. 3a), as net-
work performance (MSE) gets stabilized. The performance
of training (R2X: 0.98, R2P: 0.98), validation (R2X:0.98,
R2P:0.96), testing (R2X:0.91, R2P:0.90), and entire data set
remains satisfactory (Fig. 3b). The ANN model suggested a
maximum PHA yield of 2.69 g l­-1 at 62.80 h of incubation
with initial nitrogen and phosphate concentrations 2.0 g ­l-1
and 4.0 g ­l-1, respectively. Though the maximum PHA yield
predicted by the ANN model is higher than that of the poly-
nomial model, the optimal condition (i.e., time, nitrogen,
and phosphate concentration) for maximum PHA recovery
in both the models had been very similar.
3.4 Multi‑objective optimization using polynomial
model and ANN
Though PHA accumulation in microbial culture is closely
linked with the biomass growth, the relative proportion of
polymer to biomass can vary depending on culture condi-
tions, initial biomass or substrate, or choice of harvesting
time. The economic benefit from improvement in PHA pro-
duction must be greater than the inflated budget for pre-
treatment and downstream processing of increased amount
of biomass [16]. In this context, composite desirability can
be adopted to co-optimize such contrasting objectives, giv-
ing appropriate weight to each of them [19].
Considering the biomass and PHA production as the
objectives, the individual and composite desirability func-
tions are used to find the optimal combination of process
variables. The individual desirability of biomass decreases
(data not shown) with an increase in either incubation time,
urea, or phosphate concentration. However, the desirability
of PHAs initially increases with time or substrate concentra-
tions, reaches a maxima, and then declines. Although opti-
mizing individual responses provide a better result for each
response, the optimal parameter settings are different from
that of the composite desirability [55]. As polymer produc-
tion is often growth-associated, high composite desirability
in media optimization can be expected when the target is
set to maximize either or both of these responses [56]. High
desirability can be attained in optimization if PHA produc-
tion remains the only objective [57].
The assessment of composite desirability for PHA and
biomass production, while changing their relative impor-
tance in polynomial or ANN models, is shown in Table 5.
The first scenario focusing on the maximization of poly-
mer accumulation suggests the production of 2.40 g ­l-1 of
PHA along with 1.10 g l­ -1 biomass in the polynomial model.
However, ANN suggests a little higher PHA accumulation
(2.69 g l­-1) with similar biomass production (1.01 g l­-1) on
similar culture conditions as the polynomial model. This
13

suggested that a higher yield of PHA with low biomass can
be achieved, thereby decreasing the cost of downstream
processing. Significantly lower polymer production (1.02-
2.11 g ­l-1) with a higher proportion of biomass (2.26-3.41
g ­l-1) has been reported in other studies, utilizing either
defined or complex media under optimized conditions [47,
48]. Increasing the importance for biomass minimization to
0.25, 0.50, or 0.75 projected a drop in biomass accumulation
as well as in PHA production, for both the model variants.
However, a relative decrease in PHA to that of the biomass
was found to be lower in the ANN model than that of the
polynomial regression. The polymer-to-biomass ratio con-
tinued to increase for the ANN model. The culture condi-
tions projected for reduced priority of biomass (weightage 0)
are somewhat similar for both model variants. However, the
predicted incubation time, nitrogen, and phosphate concen-
tration at higher desirability weight for biomass is higher in
ANN. The combination of RSM and ANN model to predict
PHA and biomass has reported superiority of ANN model in
comparison to polynomial regression [24, 58, 59].
3.5 Characterization of the biopolymer
The FTIR spectroscopy was performed to investigate the dif-
ferent functional groups of PHA produced by C. necator. The
FTIR spectrum of PHA recorded between 4000 and 400 c­ m−1
(Fig. 4) shows a sharp absorption band at 1730.15 ­cm−1 which
corresponds to carbonyl (C=O) stretching of the ester [60].
The bands of minor relevance at 3446.79 c­ m−1 are related to
a terminal OH group [20]. The band at 1109.07 ­cm-1 is char-
acteristic of C-O-C group stretching. Accompanying bands of
the C-O-C groups emerge in the spectral region from 1100 to
1500 ­cm-1. The absorption band at 619.15 c­ m-1 was assigned
to the C-C stretching vibration in the amorphous phase [61].
1
H NMR analysis (Fig. 5) revealed four resonance
signals indicating the presence of different types of pro-
tons. Triplet and doublet peaks at a chemical shift of
0.842–0.869 ppm and 1.275–1.296 ppm were identified as
terminal methyl (-CH3) group of 3HB unit. Multiplet peak
at 2.447–2.659 ppm represented methylene (-CH2) group
of C2 carbon atom while chemical shift at 5.236–5.301
ppm attributed to methane (-CH) groups of 3HB and 3HV
monomer units. The results obtained were in agreement
with earlier reports [1, 20]. Along with crotonic acid assay,
both 1H NMR and FTIR spectra of the extracted polymer
confirm the PHB form from the production process.
3.6 Practical implication of the work and future
research prospects
The improvements in biopolymer yield while minimizing
biomass accumulation is an important pre-requisite for
sustained PHA production. The optimization of media and
13
Biomass Conversion and Biorefinery
process conditions [62] has been observed to be very effec-
tive to increase the microbial PHA concentration (Table 6),
though proportionate improvement in polymer content in
microbial biomass is not always evident. In some cases,
the PHA content has been observed to decrease due to an
out-of-proportion increase in residual biomass when com-
pared to the polymer [46, 67]. Therefore, multi-objective
optimization with composite desirability is essential to
attain a compromised solution between the two conflict-
ing aspects [53]. ANN models can be adapted to monitor
pilot-scale polymer production in real-time by integrating
them with on-site sensors while using the optimal process
parameters as a reference. Biomass is usually pre-treated to
facilitate polymer recovery through alkali, oxidants, heat,
and mechanical treatment [70] for digestion of non-PHA
cell mass, followed by extraction into an appropriate solvent
and reprecipitation. In the pilot scale, PHA-rich biomass
is harvested through flocculation or simple gravity settling
from which either wet biomass or lyophilized cells are used
for polymer extraction [71]. However, pre-treatment of bio-
mass used on a pilot or commercial scale may not be neces-
sary, and PHA is extracted either with organic solvents (e.g.,
chloroform) or a mixture of organic and aqueous phase [71,
72]. The conditions of extraction protocol can also affect the
polymer recovery where the optimal conditions for the lab
scale might not be suitable for the pilot scale.
Though the use of ANN and multi-objective optimiza-
tion are rarely reported in microbial production process of
PHAs, the results from both the approaches corroborate each
other in optimizing the process conditions as evident from
the total phenolic content and total flavonoid content recov-
ery in phytochemical extraction process [73]. The RSM and
ANN modeling approaches have been successfully adopted
to minimize the hardness while maximizing the toughness of
an egg shell reinforced composite with multi-objective opti-
mization using GA [74]. The ANN model developed from
the CCD matrix integrated with a multi-objective GA has
been used to predict optimal media and process condition to
enhance phycobiliproteins and cell biomass in cyanobacteria
growth [75].
In the present work, RSM and GA-ANN models provide
important insights into the effect of incubation time, urea,
and phosphate concentration while co-optimizing biomass
and PHA accumulation, which can be extended to select
appropriate renewable resources. However, prior economic
analysis of PHA recovery processes at the proposed multi-
objective solution needs to be validated. The improvement
in the upstream process while applying different polymer
recovery routes such as high pressure and heat has been
deemed beneficial in decreasing the production cost [76].
The concept of partial withdrawal of culture medium and
replenishing with a fresh one (referred to as cyclic fed-batch
fermentation), based on the optimized growth condition,
Biomass Conversion and Biorefinery
can increase the PHA productivity [77]. Further, the adop-
tion of a bioprocess model-based optimal feeding strategy
can be invaluable to boost polymer accumulation in hetero-
trophic biomass. The application of optimal feeding strategy
on an industrial-scale becomes essential as it helps opti-
mize the bioprocess conditions which would differ from
that of lab-scale experiments [78]. The optimal process
conditions evaluated for biomass growth and product for-
mation in this laboratory study cannot be extrapolated to
the industrial-scale unless the scale-up parameters relevant
to the mass-transfer process are appropriately considered.
Additional validation studies may be required while con-
sidering the bioreactor operations for polymer production
on an industrial-scale.
4 Conclusions
The selection of appropriate carbon and nitrogen sources and
identifying their optimal concentrations are very important
for an economical microbial biopolymer production process.
In this study, a combination of sucrose and urea offered bet-
ter PHA production by Cupriavidus necator when compared
with other tested carbon and nitrogen sources. CCD-based
analysis suggested a significant influence of incubation time,
urea, and phosphate concentration on the PHA production.
Though both the polynomial and GA-ANN models showed
reasonably good agreement with experimental data, the pre-
dictability of the latter for PHA production was found to be
superior. Multi-objective optimization for increasing PHA
production and decreasing residual biomass predicted an
increase in polymer to biomass ratio in the GA-ANN model.
The exploration of different initial biomass and substrate
concentrations, a detailed cost-benefit analysis, and valida-
tion of the PHA production are highly recommended for the
process to be implemented in an industrial-scale.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 13399-0​ 23-0​ 4043-w.
Acknowledgements PL and BM acknowledge the support from the
Karunya Institute of Technology and Sciences. SKB thanks VIT Vel-
lore (India) for the staff-time support to collaborate with researchers
from the Karunya Institute of Technology and Sciences. Standard ethi-
cal and professional conduct has been followed. There are no conflicts
of interest (financial or otherwise) associated with this publication.
Data availability Experimental data pertinent to this study can be avail-
able from the corresponding author upon reasonable request.
Author contributions PL contributed to conceptualization-supporting,
formal analysis-equal, visualization-lead, writing—original draft-lead,
writing—review and editing-equal. BM contributed to conceptualiza-
tion-equal, project administration-lead, supervision-lead, writing—
original draft-supporting, writing—review and editing-equal. SKB
contributed to editing-equal.
Declarations
Ethical approval Not applicable
Competing interests The authors declare no competing interest.
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