Energy Conversion and Management 256 (2022) 115360

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Energy Conversion and Management

journal homepage: www.elsevier.com/locate/enconman

A novel wastewater-derived cascading algal biorefinery route for complete

valorization of the biomass to biodiesel and value-added bioproducts
Sana Malik a, Ayesha Shahid a, Michael J. Betenbaugh b, Chen-Guang Liu c, Muhammad
Aamer Mehmood a, *
a
Bioenergy Research Centre, Department of Bioinformatics & Biotechnology, Government College University Faisalabad, Faisalabad 38000, Pakistan
b
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA
c
State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and
Biotechnology, Shanghai Jiao Tong University, China

A R T I C L E I N F O A B S T R A C T

Keywords: Integration of multiproduct algal biorefineries with wastewater treatment systems could offer a sustainable
Urban wastewater treatment approach to produce green products in a circular bioeconomy paradigm while keeping the water-energy-
Resource recovery environment nexus sustainable. The present study demonstrated a novel biorefinery route employing a newly
Biological fermentation
isolated self-flocculating microalga Chlamydomonas sp. BERC07. Here, the urban wastewater served as a low-cost
Cascading agal biorefinery
Circular bioeconomy
growth media and improved biomass production (1.24 g/L) by 2-fold with concomitant removal of 100% total
nitrogen, 94% total phosphorus, 56% COD, and 41% BOD. To lower the harvesting cost, an inducible
sedimentation-based harvesting system was developed by employing the lowest dose of a commercial-grade
flocculant, potash alum (0.27 kg/1000L culture) which improved the sedimentation rate of algal flocs by 240-
fold with a biomass recovery efficiency of 96–98%. Later, the wastewater-derived biomass was subjected to
downstream processing in a cascading fashion for its complete valorization. The biomass yielded 1.83 mg/g of
carotenoids and 480 mg/g of lipids. The lipids were transesterified to produce biodiesel which completely met
the preset European and American biodiesel quality standards. After carotenoids and lipids extraction, the re­
sidual biomass was fermented using Aspergillus niger and Aspergillus oryzae for the very first time. Interestingly,
the A. oryzae outcompeted the A. niger and produced 131.6 U/mL of α-amylase along with 375–384 mg/g of
mycoproteins utilizing 75–100 g/L of the residual algal biomass as a sole feedstock. All solvents involved in the
product recovery were recycled to the primary wastewater, leaving no waste at the end of the processing
pipeline. Overall, the data demonstrated a novel and sustainable biorefinery route with the most efficient har­
vesting and the complete valorization of the biomass, in a zero-waste approach. However, further upscaling, life
cycle assessment, and technoeconomic analyses will be required to assess the economic and environmental
sustainability of the proposed biorefinery route.

1. Introduction
Developing novel, renewable, self-sustainable, carbon-neutral, and
zero-waste circular bioeconomy platforms have become crucial to meet
the global demands of bioenergy and biochemicals [1,2]. Integration of
algal biorefineries with the wastewater treatment plants could turn the
problematic wastewater bodies into a resource to produce algal biomass
which is believed as the most promising feedstock to establish the bio­
based economy owing to higher productivity, carbon neutrality, pro­
duction of value-added compounds, and nutrient recycling [3–6].
However, despite these promising features, even after intensive intel­
lectual and financial input into microalgal research, their full potential
could not be harnessed due to various challenges associated with the
upstream, midstream (harvesting), and downstream processing of algal
biomass. Therefore, it is required to establish low-cost and
contamination-free cultivation systems to lower the cost at the upstream
level. While difficult harvesting of microalgae due to their unicellular
nature adds a major share in the cost of processing, hence developing
robust harvesting techniques are needed [7] to improve the cost-
effectiveness at the midstream level. In most cases, algal biomass is

* Corresponding author.
E-mail address: draamer@gcuf.edu.pk (M.A. Mehmood).

https://doi.org/10.1016/j.enconman.2022.115360
Received 5 December 2021; Received in revised form 28 January 2022; Accepted 7 February 2022
Available online 18 February 2022
0196-8904/© 2022 Elsevier Ltd. All rights reserved.
S. Malik et al.
processed for a single product that does not cover the cost and subse­
quently, the product (which is commonly biodiesel) cannot compete
with the market price of petrol-diesel [8]. Hence, instead of a single
product, a complete valorization of the biomass into multiple products
leaving almost no waste at the end of the process may enhance the
profitability of the process to meet the commercial needs.
Besides, a recent upsurge in urbanization and industrialization
especially in developing countries has led to the production of large
volumes of urban wastewater where 44% of the urban wastewater is
discharged into the water bodies untreated. Interestingly, cultivating
microalgae in urban wastewater could be a multifaceted opportunity
offering a low-cost growth media, removal of pollutants, recycling of the
treated water, fixation of atmospheric carbon, and production of high-
value biomass [9]. Different studies have reported the feasibility of
utilizing wastewater for microalgal cultivation as well as high-value
biomass generation [10–15].
Energy and cost-intensive harvesting of microalgae is another chal­
lenge in a biorefinery that increases the cost by 20–30% while among
various methods, self-flocculation is considered to be the most eco-
friendly approach [7,16]. A few algal strains have shown the tendency
of auto-flocculation naturally, while their flocculation efficiency is a
species-specific phenomenon and often does not meet the commercial
robustness. Therefore, inorganic flocculants are often added to the cul­
ture media to induce and/or to increase the flocculation efficiency
[17,18], however, the algal response to the inorganic flocculants is again
a strain-dependent process. Hence, the algal strains either having robust
self-flocculation efficiency or highly responsive to inorganic flocculants
are needed to reduce the cost of harvesting. Besides, induced floccula­
tion has also shown to improve membrane permeance and reduced the
filtration resistance when combined with membrane filtration [19].
Microalgae can turn the inexpensive raw nutrients of wastewater
into valuable biomass components, including carbohydrates, proteins,
lipids, and photosynthetic pigments [20]. However, the bioprocessing of
algal biomass into a single product has made the downstream processing
tedious and expensive. But recently, the focus has been shifted towards
the cascading extraction of multiple products from the same biomass to
achieve economic feasibility and to reduce waste discharge [21–23].
Although this concept has been extensively discussed, it is still in its
infancy and requires substantial research efforts to get it realized. The
present study demonstrated the integration of urban wastewater treat­
ment and recycling with a novel multiproduct biorefinery framework in
a cascading fashion for the complete valorization of biomass into mul­
tiple products including carotenoids, biodiesel, α-amylase, and myco­
proteins based on a zero-waste principle. This is the first study of the
biological transformation of pigment-free and lipid-free residual algal
biomass into industrial enzymes (α-amylase) and mycoproteins which
could open new avenues in the field of circular bioeconomy with the
possibility of achieving maximum economic efficiency of algal
biorefinery.
2. Materials and methods
2.1. Isolation, identification, and basic characterization of the algal strain
The understudy strain was isolated from a water sample collected
from an urban wastewater body located at Kamoke, Punjab, Pakistan
(31.96935◦ N, 74.22865◦ E) which was dominated by the greenish
appearance of the strain. The axenic culture was obtained after several
sub-cultivation stages of serially diluted cultures as described previously
[24]. The purity of the culture was confirmed through microscopy
(Optika Microscope, Italy) and the pure strain was named as BERC07.
The pure culture was enriched using BG11 media at room temperature
28 ± 1 ◦ C with a 12:12 h light: dark cycle of photoperiod. The
morphological features of the strain including size, shape, diameter, and
arrangement of the cells were analysed under the light microscope and
were compared with other species using an online database (http:
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Energy Conversion and Management 256 (2022) 115360
//www.algaebase.org) [25], while detailed microscopy was performed
using the scanning electron microscope (Nova NanoSEM 450). Due to
variation among the morphological features of several algal species
existing in the same or diverse environments, merely microscopic
identification is not adequate to identify an algal species. Therefore,
genomic DNA of the BERC07 strain (which was extracted by using a
commercial kit; Thermo Scientific GeneJET Plant Genomic DNA Puri­
fication Kit) was used as a template to amplify the 18S rRNA gene using
the specific pair of primers (F: 5′ -CCAACCTGGTTGATCCTGCCAGTA-3′
and R: 5′ -CCTTGTTACGACTTCACCTTCCTCT-3′ ) for marker-assisted
molecular identification. The amplified fragment was sequenced, and
the cleaned sequence was subsequently used to find related sequences
using BLAST at NCBI [26].
The basic growth conditions including nutrient media (BG11, BBM,
synthetic wastewater, and urban wastewater), pH (5–12), and light in­
tensity (150–300 μmol m− 2 s− 1) were tested and the set of conditions
which favoured the highest growth were considered as optimum. Later,
the optimum growth conditions were used in subsequent experiments
mainly using urban wastewater as growth media and BG11 as the con­
trol. During basic characterization, the strain BERC07 showed a signif­
icant growth potential in urban wastewater (UWW). Therefore, a time-
course experiment was performed using UWW as growth media to un­
derstand the growth pattern and its impact on the biochemical compo­
sition of BERC07 over a specific course of time. The untreated and non-
sterilized urban wastewater was collected from the local drainage site of
the city. The initial pH of the wastewater was monitored prior to the
cultivation of the strain. The strain was cultured in 1.0 L Erlenmeyer
flasks containing 500 mL UWW in triplicate and the optical density (OD
λ680 nm) was measured on daily basis using the UV-Vis double-beam
spectrophotometer (UH5300, Hitachi). The culture was harvested in
triplicate on the 7th, 14th, 21st, 28th, and 35th day of cultivation, and
the data were used to calculate growth kinetics parameters, and the
biomass from each harvest was subjected to biochemical analyses.
2.2. Self-flocculation-based harvesting
Two different harvesting routes namely SFB (self-flocculation based)
and PFB (Potash Alum-flocculated biomass) were tested to evaluate the
impact of harvesting strategies depending upon their prospective
biotechnological applications. To assess the flocculation efficiency of the
strain in response to different nutrient conditions, the strain was
cultured using BBM, BG11, SWW (synthetic wastewater), and UWW in
1.0 L Erlenmeyer flasks containing 500 mL culture volume at optimized
growth conditions. On the 15th day of cultivation, the standard jar test
was performed according to the American Standard Test Method in
response to each media, and the flocculation efficiency was estimated
[27].
2.3. Developing an induced sedimentation-based harvesting system
Although the molecular mechanism of self-flocculation is not well-
understood however it has been observed that flocculation is often
mediated by the divalent/trivalent cations. Keeping in view the fact,
four different analytical grade salts (Sigma-Aldrich, USA) including
Aluminium sulphate, Iron sulphate, Iron chloride, and a commercial-
grade salt Potash alum (bought from the local market) were selected
to see their impact on the flocculation efficiency of BERC07. Floccula­
tion rate assay was performed with each flocculant solution in triplicate,
separately [17,28]. Briefly, a 100 mL culture suspension was taken into
the 100 mL beaker, and the stock solution of flocculant (10 mg/mL) was
added slowly into the beaker and was mixed for the 30 s using a glass rod
to dissolve it to uniformity. Then the culture was allowed to settle for 4
min and an aliquot of the supernatant was pipetted out at a height of
two-third from the beaker. The absorbance of the initial and final culture
aliquots was measured at λ680 nm using a UV-vis double-beam spec­
trophotometer (UH5300, Hitachi), and the sedimentation efficiency was
S. Malik et al.
calculated using Eq. (1).
ODinitial − ODfinal
× 100 (1)
ODinitial
where ODinitial and ODfinal are the optical densities, the aliquots
measured before and after the flocculation. The experiments were car­
ried out during the stationary phase when the biomass concentration
was reached 1.25 g/L, based on dry cell weight (DCW).
To assess the impact of each flocculant on the viability of the cells, a
cell viability assay was performed using 1% Trypan Blue dye. The cells
were then observed under the light microscope (Optika Microscope,
Italy) to differentiate between viable and dead cells [17] in response to
the addition of flocculant. The best flocculant was chosen based on the
flocculation efficiency, settling time, viability of cells, cost-effectiveness,
easy availability, eco-friendliness, and no expected interference during
the downstream processing of biomass. Later, the optimum dosage of the
selected flocculant to concentrate the BERC07 biomass with maximum
efficiency was determined by performing a series of jar tests using a
range of the flocculant (0.03–0.36 g/L) and the relative flocculation
efficiency of the flocculant was calculated. It has been observed that
flocculation efficiency also depends on the cell density, culture volume,
and flocculant dosage. Therefore, a subsequent flocculation efficiency
optimization experiment was employed using 100 mL culture to 20 L
culture using the selected flocculant at the minimum optimum dosage.
To assess the reproducibility of the induced flocculation in BERC07, the
experiments were repeated using three different batch cultures culti­
vated with almost a 30-day interval.
2.4. Experimental design for the cascading multiproduct biorefinery
The data obtained from the aforementioned experiments were used
to design a cascading multiproduct biorefinery bioprocess. The growth
kinetic analyses including the biomass production, biomass productiv­
ity, specific growth rate, doubling time, and biofixation efficiency of
BERC07 were performed based on dry cell weight [29]. The carbohy­
drate and protein content of algal biomass was measured by following
the standard phenol-sulfuric acid [30] and micro-biuret methods [31],
respectively. The lipids were extracted and estimated by a modified
Bligh and Dyer method [32]. Total chlorophyll and carotenoid contents
were estimated to assess the photosynthetic efficiency and biotechno­
logical potential of the strain, as described previously [29].
2.4.1. Stage-I: Use of urban wastewater as growth media
The strain was cultivated using urban wastewater (UWW) as a low-
cost growth media in two locally designed photobioreactors made of
transparent borosilicate glass with a working volume of 10 L each for
proper aeration in the growth room equipped with cool LED (Philips,
Japan) lights where the photoperiod was set at 12:12 h Light: Dark cycle
and the temperature was maintained between 28 and 30 ◦ C. The primary
characteristics of UWW including total nitrogen (TN), and total phos­
phorus (TP) were evaluated before and after the cultivation of strain
following the Kjeldahl method and colorimetric methods, respectively
[33]. The strain was pre-cultured in BG11 media for 7 days and was
inoculated in the UWW-fed photobioreactors with an inoculum size of
0.012 g/L (dry cell weight). The cell density of the culture was period­
ically monitored by measuring the absorbance at λ680nm using the
spectrophotometer (UV-1200, A & E Labs, UK). The growth kinetics and
CO2 utilization efficiency of the strain were analysed after 21 days of
batch cultivation.
2.4.2. Stage-II: Establishing a robust harvesting method and recycling of the
spent media
After 21 days, the total culture (20 L) from both bioreactors (10 L
each) was mixed and then transferred to the two glass beakers (10 L
culture each). Cells in the first beaker were pre-concentrated using the
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Energy Conversion and Management 256 (2022) 115360
selected flocculant, while the cells in the second beaker were pre-
concentrated through self-flocculation and then harvested by using
centrifugation which served as a control. In response to the optimized
dosage of the flocculant (Potash Alum), the cells started flocculating and
made a biomass film/slurry at the bottom of the beaker. The supernatant
and the pre-concentrated biomass were separated through aspiration.
The flocculant-induced concentrated biomass and self-flocculated
biomass (control) were then freeze-dried to estimate the biomass pro­
duction based on dry cell weight (DCW). The algal metabolites were
extracted and estimated from both the Potash Alum-flocculated biomass
(PFB) and self-flocculated biomass (SFB) to analyse the difference
among their contents.
Because Potash Alum was used as a flocculant which might have
released Aluminium ions into the solution. Therefore, the aluminium
present in the various products of the biorefinery including flocculated
biomass, spent media, lipids, and carotenoids were carried out using the
Inductively Coupled Plasma-Mass Spectrometer (ICP-MS) Agilent
7700× (Agilent Technologies, CA, USA). All samples were analysed in
triplicates as described [34]. To see the impact of flocculant on the
FAME profile and biodiesel properties of BERC07, the lipids from PA-
flocculated and self-flocculated biomass (control) were extracted,
trans-esterified, and analysed by following the standard protocol with
little modifications. Based on the relative composition of FAMEs,
carbon-chain length, and position of double bonds, the biodiesel prop­
erties were also evaluated by calculating the key parameters namely
cetane number, cloud point, cold filter plugging point, pour point, high
heating values, saponification value, density, and viscosity [35,36].
The spent media from which algal biomass was recovered through
PA-induced flocculation and self-flocculation was used for the reculti­
vation of the same strain. Accordingly, the strain was inoculated (0.012
g/L) in the spent media (recycled UWW) and fresh UWW (control)
without any addition of nutrients, and pH adjustment to assess the
economic feasibility of the media recycling process. The growth of the
strain was monitored on daily basis. The biomass was again harvested
through PA-induced flocculation and was processed for subsequent
cascading bioprocessing.
2.4.3. Stage-III: Downstream processing of the algal biomass
The efficient downstream processing of algal biomass ensures the
sustainability and profitability of biorefinery. Based on the circular
bioeconomy concept (zero-discharge principle) the biomass of BERC07
was processed in a self-sustainable, multiproduct, and cascading
fashion. The experiment was designed by integrating the bioprocesses to
maximize the resource recovery, recycling of all wastes, and recovering
multiple bioproducts.
2.4.3.1. Extraction of carotenoids. The process chain was initiated with
the extraction of carotenoids from the biomass of BERC07 produced in
the spent media (primarily treated urban wastewater). Carotenoids were
extracted from three grams of lyophilized biomass. After extraction, the
residual biomass was oven-dried at 60 ◦ C for its subsequent use.
2.4.3.2. Extraction of lipids and biodiesel production. The residual
biomass was weighed to record any biomass loss during the extraction of
carotenoids and was subjected to lipid extraction. The lipids were
transesterified to produce biodiesel while the quality and quantity of the
biodiesel were analysed using GC–MS as described previously [36]. To
analyse the commercial feasibility and the impact of flocculant on the
biodiesel composition, the fuel properties were also studied. After
extracting lipid, the residual biomass was again oven-dried (60 ◦ C) and
weighed to observe the losses.
2.4.3.3. Fermentation of the residual biomass. The third stage of the
experiment was aimed to assess the possible utilization of residual
biomass for α-amylase production using Aspergillus niger and Aspergillus
S. Malik et al.
oryzae which are classified as GRAS (Generally Regarded as Safe). Before
the fermentation of residual biomass, the biomass was analysed for its
residual carbohydrates, proteins, lipids, and pigments contents. Then,
the oven-dried (60 ◦ C) residual biomass was used as a substrate for the
growth of these commercial strains to analyse their potential to produce
α-amylase enzyme and mycoprotein. For fungi cultivation, the growth
conditions were tested in a preliminary optimization experiment. The
media solely contained the residual biomass as a source of nutrition for
the fungi where 10 g/L, 20 g/L, 30 g/L, 50 g/L, 75 g/L, and 100 g/L of
the residual biomass was autoclaved in distilled water using 100 mL
Erlenmeyer flasks containing 20 mL culture volume. The pH of the
media was adjusted to 5.8–6.0 before inoculation. The inoculum was
prepared by adding 250 µL of 72 h old slant cultures of A. niger and A.
oryzae into the media and the flasks were incubated in the shaking
incubator set at 30 ◦ C temperature and 180 rpm shaking speed [37]. The
potato dextrose broth served as a positive control. While the same
concentrations of residual algal biomass without any fungal inoculum
were used as negative controls. To disrupt the fungal mycelia, small
glass beads were added to each flask. The growth based on dry cell
weight and the α-amylase enzyme activity of both strains were moni­
tored by collecting sample flasks at different time intervals 24 h, 48 h,
72 h, and 96 h [37]. After 96 h of incubation, the culture from each flask
was centrifuged at 6000 rpm for 10 mins and the supernatant enriched
with α-amylase enzyme was transferred to another flask. The pellet
containing the fresh cell mass was weighed, oven-dried (60 ◦ C), and
weighed again to record any change in the biomass after fermentation.
The compositional analyses of the residual biomass were performed
again to estimate the changes in carbohydrates and protein content. The
experiment was performed in triplicates and the data were analysed
using GraphPad Prism 8 (GraphPad Prism, San Diego, USA).
3. Results and discussion
3.1. Identification of the microalgal strain
The preliminary identification and purity of the isolated strain were
confirmed through microscopic analysis, which revealed the presence of
lush green, bi-flagellated, oval-shaped, single cells in the culture. These
characteristics indicated the close resemblance of BERC07 with Chla­
mydomonas sp. But the morphology of microalgae could be altered in
response to cell age and culture conditions for the same strain within
each chemical race [38]. Therefore, a detailed microscopic study was
performed using a light microscope and a scanning electron microscope.
The micrographs showed the closest resemblance with the Chlamydo­
monas species (Fig. 1). Besides, the sequence analysis of BERC07, based
on the 18SrRNA gene, further confirmed that this strain has a 92%
resemblance with Chlamydomonas sp. However, it requires further
identification based on some more molecular markers and fatty acid
profiling in the future.
3.2. Cultivation in urban wastewater improved growth and metabolite
content of the Chlamydomonas sp. BERC07
Interestingly, BERC07 had shown an increasing trend in the biomass
production in UWW till the 21st day of the cultivation with maximum
biomass of 1.24 g/L based on dry cell weight (Fig. 2A), while it produced
0.46 g/L, 0.63 g/L, and 0.87 g/L biomass in BBM, BG11, and SWW,
respectively. This varying growth trend could be due to a relatively
compromised photosynthetic efficiency of strain during the early days of
cultivation owing to the higher nutrient titer, the lag phase. After 7 days,
the cells adapted to the nutrient conditions, and the growth was accel­
erated till the 21st day, during the exponential phase. During the sta­
tionary phase (21st to 28th day), no further increase in the biomass was
recorded, therefore the data for the 35th day of cultivation were
excluded. A similar growth pattern was observed for Chlorella kessleri,
Chlorella vulgaris, Nannochloropsis oculata, Scenedesmus obliquus, and
4
Energy Conversion and Management 256 (2022) 115360
Dunaliella salina when these strains were cultivated in the urban
wastewater [39–41].
After 7 days, the biomass was constituted of 31% carbohydrates,
28% proteins, 22% lipids, and 7.5% pigments, while after 14 days, the
protein and lipid contents were increased to 34% and 29%, respectively,
but the carbohydrate content was decreased to 21%. Later, the lipid
content was rapidly increased to 48% on the 21st day of cultivation.
Overall, a 2.18-fold increase was achieved in lipid content at the cost of a
1.1-fold and 1.5-fold decrease in carbs and proteins content, respec­
tively. It reflected an outstanding potential of this strain to produce
biodiesel. Conversely, the carbohydrate content in the biomass har­
vested on the 28th day was 28% which is quite high with low lipid and
protein content (Fig. 2B). So, harvesting BERC07 on the 21st day of
cultivation would be the best choice because to this day, the strain
concurrently showed the highest biomass (1.24 g/L) and lipid produc­
tion (48 %). This variation in the biomass composition could be attrib­
uted to the fact that urban wastewater had higher concentrations of total
nitrogen and phosphorus, the major nutrients which improved micro­
algal growth and altered the biomass composition. Therefore, at the start
of the cultivation, relatively higher concentrations of nitrogen and
phosphorus in the UWW conferred nutrient stress in the strain which
started accumulating carbohydrates and lipids. After 7 days of cultiva­
tion, the nitrogen and phosphorus concentrations became optimal due to
continued assimilation, the photosynthetic efficiency was high, thus
metabolic flux was shifted towards the protein synthesis. During the
exponential growth phase (14th to the 21st day), the strain encountered
severe nitrogen depletion in the media, in response to which a high
amount of lipids was synthesized and the chlorophyll content was
reduced. During the stationary growth phase, the nitrogen-starved
conditions triggered the strain to store more carbon-rich compounds,
consequently, the lipid content started to decrease [42]. These findings
showed consistency with the previously published reports where nitro­
gen and phosphorus stress triggered the storage of carbohydrates and
lipids in Nannochloropsis sp. [43], C. vulgaris [44], Chlorella zofingiensis
[45], Nannochloropsis oceanica [46], Phaeodactylum tricornutum [47],
C. reinhardtii [48], Isochrysis galbana [49], Spirulina platensis [50], and
S. obliquus [51].
3.3. Inducible flocculation-based harvesting turned out to be the most
robust, cost-effective, and efficient biomass recovery mode
The route-I of harvesting was based on the self-flocculation ability of
the strain BERC07 (Fig. 3A), which has been considered the most eco-
friendly approach to concentrate the algal biomass [52] depending
upon the desired final products (food supplements, animal feed, or
biodiesel). Although, the strain BERC07 exhibited a maximum floccu­
lation efficiency of 95% in wastewater in comparison to the other
growth media, but gravity-based settlement took more time (720 mins)
when compared to the previously reported self-flocculating strains
(Fig. 3B).
Therefore, to reduce the settling time and to induce flocculation at
the desired day of cultivation, an induced flocculation system was devel­
oped for robust harvesting (route-II). The adherence of multivalent
metal ions to negatively charged microalgal cells can be exploited to
induce bridging among the cells whenever needed. As a result, cells
would hold together (the flocs) through charge neutralization and settle
in the media due to gravity sedimentation. For different algal species,
metal ions act differently as flocculants. Here, except for the ammonium
sulphate, all other flocculants were found to be very effective at all
tested concentrations and times. After 2 min of settling time, iron sul­
phate, iron chloride, aluminum sulphate, and commercial-grade potash
alum exhibited the flocculation efficiency of 30%, 78%, 93%, and 94%,
which was increased to 87%, 95%, 96%, and 98% after 4 min, respec­
tively. Some of the flocculants may have a negative influence on cell
viability, therefore, the cell viability assay was carried out by staining
the cells with Trypan blue dye. Interestingly, the cells flocculated
S. Malik et al. Energy Conversion and Management 256 (2022) 115360

Fig. 1. Morphological and molecular characterization of the BERC07: The micrographs of BERC07 strain taken at 10X (A), 40X (B), 100X (C), and 5000X (D)
indicating the resemblance of this strain with Chlamydomonas sp., (E) Phylogenetic tree based on 16S rRNA gene sequence.

5
S. Malik et al.
Fig. 3. Self-flocculation potential of Chlamydomonas sp. BERC07: Com­
parison of self-flocculation efficiency and settling time of BERC07 with other
self-flocculating strains (Chlorella vulgaris JSC-7[53], Scenedesmus obliquus AS-6-
1 [54], Ankistrodesmus falcatus, Tetraselmis suecica [55], Ettlia texensis [56],
Chlorococcum sp. [57], Scenedesmus rubescens [58], Desmodesmus sp. [59], and
Monoraphidium sp. [60]) reported in the literature. The self-flocculation effi­
ciency of the strain was the highest when compared to all published strains,
however, the settling time was 720 min which required further optimization.
through aluminum sulphate and potash alum were found to be perfectly
viable (Fig. 4A and C). However, a few cells flocculated through iron
chloride, and iron sulphate showed the dye uptake indicating the death
6
Energy Conversion and Management 256 (2022) 115360
Fig. 2. Time-course variation of the biomass produc­
tion and biochemical composition: The variation in the
biomass production (A) and composition of urban
wastewater-produced biomass of BERC07 (B) over the
specific course of time. The biomass was harvested on 7th,
14th, 21st, and 28th day. Total carbs, proteins, lipids, and
pigments were measured using standard methods as
described in Materials and Methods. The strain showed a
clear variation in the metabolite variation, but the highest
lipid content was achieved on the 21st day.
of those cells (Fig. 4G). While the iron-based flocculants also changed
the color of the microalgal culture to rusty-orange (Fig. 4H and J), which
indicated that these may cause the rusting and choking of the bio­
reactors if employed at a large scale. Moreover, iron-based flocculants
are reported to interfere with the quality of microalgae-derived pig­
ments and biodiesel [61,62].
Due to its higher relative flocculation efficiency, cost-effectiveness,
and easier availability, the commercial-grade potash alum was
selected for further experiments. Upon testing different concentrations
of potash alum, it was found that 270 mg/L was the optimal minimum
dose to induce flocculation in BERC07 (Fig. 5A) which was quite lower
when compared to other studies based on potash alum-mediated floc­
culation. Previously, two different doses of 300 mg/L and 700 mg/L of
potash alum were used to flocculate I. galbana and Chlorella emersonii
biomass which took 960 and 80 mins with an overall flocculation effi­
ciency of >95%, respectively [63,64]. Whereas Chlorella sorokiniana had
shown a flocculation efficiency of 98% in 18–28 mins in response to the
800 mg/L of potash alum [65]. Similarly, 2500 mg/L of aluminum
sulphate and potash alum were used to harvest the C. vulgaris in 10 mins
with >90% flocculation efficiency [66,67]. Besides, to date, most of the
flocculant-based harvesting methods were developed using small cul­
ture volumes in the jar (100 mL). In the present study, the scale-up
feasibility of the algal harvesting process was appraised by floccu­
lating from 100 mL to 20 L culture with a similar dose of potash alum
(270 mg/L). Interestingly, the increasing culture volume did not influ­
ence the dose requirement and flocculation efficiency of the flocculant
to harvest BERC07, however, settling time was increased from 2 to 3 min
to 6–8 min (Fig. 5B). Overall, this inducible flocculation system
improved the flocculation rate by 240-folds, which is the highest rate
reported so far which turned out to be the most efficient, robust, and
cost-effective harvesting efficiency when compared to previously pub­
lished self-flocculating algal strains.
Fig. 4. Induced flocculation and impact of the
flocculants on cell viability: The impact of metal-
based flocculants on the cell viability and floccula­
tion efficiency of BERC07 using a 100 mL jar was
performed. The micrographs reflect the impact of
Aluminum sulphate (A), Potash alum (C), Iron chlo­
ride (G), and Iron sulphate (I). It was shown that iron-
based flocculants had a negative impact on the cell
viability when compared to the control (E), while
Aluminum-based flocculants had no negative impact
on cell viability. However, all flocculants induced a
substantial flocculation effect on BERC07 cells. The
highest flocculation efficiency was achieved using the
least concentration of the commercial-grade Potash
Alum, reported so far.
S. Malik et al. Energy Conversion and Management 256 (2022) 115360

Fig. 5. Potash alum-based flocculation efficiency: The highest flocculation efficiency (96–98%) was achieved at 270 mg/L of Potash alum (A: left Y-axis) leaving
almost no biomass in the supernatant (A: right Y-axis), and upscaling of the culture volume from 100 mL to 20 L culture showed that 270 mg/L Potash alum remained
effective, but sedimentation time was little delayed from 3 min to 6–8 min (B).

3.4. The multiproduct cascading biorefinery design in a circular

bioeconomy paradigm

3.4.1. Stage-I: Contamination-free, low-cost cultivation, and wastewater

treatment/recycling
The wastewater-based cultivation improved the growth and biomass
productivity of the strain where it grew exponentially and resulted in a
biomass production of 1.25 g/L (dry cell weight) which was 2-fold
higher when compared to the control media (BG11). The higher
biomass production usually implies a higher CO2 utilization rate.
Accordingly, a carbon utilization efficiency of 2.35 g/L was observed
during cultivation in urban wastewater. The biomass production of
BERC07 was higher when compared to different microalgal strains
including C. vulgaris, Neochloris sp., and Scenedesmus sp. which produced
0.46 g/L, 0.52 g/L, and 0.84 g/L of biomass when cultivated using Fig. 6. Pollutant removal efficiency of BERC07 during cultivation in
different wastewaters, respectively [68–70]. Interestingly, the BERC07 UWW: The pollutants including Total Nitrogen, Total phosphorous, BOD, and
removed 100% of TN, 94% of TP, 56% COD, and 41% BOD, on the final COD were measured in the water samples collected on the 7th, 14th, and 21st
day (day-21) of cultivation (Fig. 6). A comparison of biomass composi­ day while the highest removal efficiency was observed on the 21st day.
tion and nutrient removal efficiency of BERC07 with other reported
microalgae is given in Table 1. Cultivating Scenedesmus obliquus and Various alkaliphilic microalgal/cyanobacterial strains have been shown
C. vulgaris also resulted in 99% removal of TN and TP in municipal to increase the pH of the media through a carbon concentration mech­
wastewater [71]. A similar trend was also observed for the Chlorella sp. anism and thus offer a contamination-free cultivation [36,78].
[72]. The nutrient removal rates of BERC07 were found to be higher
when compared to the various strains of Chlorella sp. cultured in urban 3.4.2. Stage-II: Robust harvesting approach
wastewater [73–75]. During its cultivation in the wastewater, this strain
had shown a tendency to increase the pH of the wastewater from 7.9 3.4.2.1. Impact of inducible flocculation on the biomass processing. The
(normal pH of UWW) to 11.7 on the final day (day 21) of cultivation. harvesting method should not affect the biochemical composition and
This interesting feature of BERC07 could be the main reason that we processing of the microalgal biomass. The contents of primary metab­
never found any bacterial/fungal contamination during batch cultiva­ olites recovered from SFB (biomass collected through self-flocculation)
tions. It could also be the reason that there was no significant reduction and PFB (biomass collected through potash-alum induced flocculation)
in COD/BOD levels, because of poor microbial activity at high pH [76]. were as follows; carbohydrate content was 20.34 ± 0.32% and 19.51 ±
The rise in pH can be attributed to the photosynthetic assimilation of 0.27%, the protein content was 22.44 ± 0.23% and 21.34 ± 0.35%, the
CO2 and can reach beyond 11 if no external CO2 is provided [77]. pigment content was 5.64 ± 0.41% and 5.61 ± 0.37%, while the lipid

7
S. Malik et al. Energy Conversion and Management 256 (2022) 115360

Table 1
Comparison of biomass composition and nutrient removal efficiencies of BERC07 with other microalgae grown on various wastewaters.
Microalgae species Wastewater type Biomass Composition (%) Nutrient Removal (%) References

Carbs Proteins Lipids TN TP COD

Chlamydomonas sp. BERC07 Urban 19 21 48 100 94 56 This study

Chlorella sorokiniana POME – – 11.21 62.02 30.77 47.09 [79]
C. sorokiniana – – 15.07 91.54 83.25 63.85 [80]
C. pyrenoidosa Olive oil mill 65 11 23 – 97 96.2 [81]
Scenedesmus sp. Meat market – 41.2 23.2 90 85 – [82]
C. sorokiniana Swine – 56 – 97 92.8 90.1 [83]
Spirulina sp. Aquaculture 69.77 – 12.77 81.10 99.97 89.34 [84]

content was 47.45 ± 0.28% and 48.68 ± 0.34%, respectively. These data
indicated that the harvesting method did not interfere with the extrac­
tion process of primary metabolites from the algal biomass. The same
findings have been documented in the literature, where the use of alum
and other techniques to harvest the algal biomass, did not induce any
modifications in the metabolite profile of Ankistrodesmus falcatus [85].
The extraction and yield of fucoxanthin pigment were not affected when
P. tricornutum biomass was harvested using different flocculants
including the alum [86].
The ICP-MS analysis was performed to determine the total amount of
aluminium in the harvested biomass, carotenoids, lipids before deciding
their biotechnological applications. The data from the ICP-MS analysis
reflected that the filtrate contained an aluminium content of 0.74 mg/L,
which is acceptable if this water is going to be used for irrigation pur­
poses. A negligible amount of residual aluminium was detected in the
lipids and carotenoids extracted from the biomass of BERC07, which
indicated their suitability for vast biotechnological applications. These
findings were consistent with another study, in which no detectable
amount of aluminium was found in the lipids after harvesting the
biomass of Nannochloropsis salina using aluminium potassium nitrate
[87].
derived filtrate media (SFM), and PA-induced flocculation derived
filtrate media (PFM), respectively (Fig. 7). These results indicated that
after harvesting the biomass, residual filtrates could be recycled in algal
cultivation which will further improve the cost-effectiveness of the
process. These findings are in line with previous reports where no sig­
nificant difference was recorded in growth rates of different microalgae
using the filtrate and control media [17,63,87].
3.4.2.3. Impact of inducible flocculation on the FAME profile. The ideal
chemical flocculant should not interfere with the yield and composition
of lipids. Here, the impact of potash alum was studied on the fatty acid
composition of lipids extracted from the PA-flocculated biomass, and the
comparison was made with self-flocculated biomass. The FAME profile
of lipids extracted from both types of biomasses did not show any sig­
nificant deviation in the composition in contrast to a study that reported
a slight reduction in the lipid content after using metal sulphates to
harvest the C. vulgaris [67]. Qualitative and quantitative analyses
revealed that the monounsaturated fatty acids (52%) were present in

3.4.2.2. Recycling of harvesting filtrates. To improve the cost-

effectiveness of this biorefinery route, the filtrates recovered from self-
flocculation and PA-induced flocculation were tested for their abilities
to support the growth of the BERC07 strain in the next round of culti­
vation. It was shown that BERC07 produced 0.99 g/L, 0.85 g/L, and
0.73 g/L in the fresh UWW (which served as a control), self-flocculation

Fig. 7. Biomass production in recycled media. The BERC07 strain was

cultured in recycled media after harvesting the biomass through self-
flocculation (SFM; green curve with squares), and through induced floccula­
tion using Potash alum (PFM; purple curve with triangles). It was shown that
recycled media could be used to culture the microalgae without having a
substantial negative impact on the growth when compared to the UWW (con­
trol, orange curve with filled circles). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of
this article.)
Fig. 8. FAME analyses of the BERC07-derived lipids: Fatty acid composition
of lipids derived from the biomass harvested through self-flocculation (the inner
circle of A), and the biomass harvested through Potash alum induced floccu­
lation (the outer circle of B) showed that harvesting had no impact on the
composition (9A) of saturated fatty acids (SFA), polyunsaturated fatty acids
(PUFA), branched-chain fatty acids (BCFA), and monounsaturated fatty acids
(MUFA), and quantity of dominant fatty acids (9B). Each value represents a
mean of three biological replicates.

8
S. Malik et al. Energy Conversion and Management 256 (2022) 115360

higher amounts in the BERC07-derived biodiesel in comparison to the standards of commercial biodiesel. All these parameters were found to
polyunsaturated fatty acids (24%) and saturated fatty acids (20%) agree with the commercial biodiesel and better than the other strains
(Fig. 8A). The strain under study showed the presence of a higher pro­ namely C. vulgaris [44], Chlorella minutissima [95], Scenedesmus sp. [96],
portion of short-chain fatty acids (C14-C18), making the FAME profile Chlorella sp., and Chlorolobion sp. [97].
favorable for biodiesel. Additionally, the FAME profile of BERC07
marked the presence of three dominating fatty acids namely, palmitic 3.4.3. Stage-III: Cascading biomass processing and complete valorization of
acid (34.7 mg/g), followed by oleic acid (2.95 mg/g), and myristic acid the residual biomass in a zero-waste fashion
(1.64 mg/g) (Fig. 8B). These results are in line with other studies To improve the process economics in an algal biorefinery, the valo­
reporting the dominance of palmitic acid in the FAME profile of different rization of algal biomass for multiple products following a cascading
microalgae [29,88,89]. approach has become crucial. Here, a novel biorefinery scheme based on
a zero-waste approach was followed, by utilizing the biomass harvested
3.4.2.4. Impact of inducible flocculation-based harvesting on the biodiesel through both routes (self-flocculation and PA-induced flocculation) in a
properties. The dominance of palmitic acid in the FAME composition of cascading fashion to ensure the highest possible resource recovery.
any biodiesel specifies the high-quality fuel with ideal parameters
including the higher oxidative stability and CN, and lower emission of 3.4.3.1. Product-I: High-value carotenoids. Microalgae are known to
nitric oxide. Whereas the oleic acid in the FAME profile contributes produce carotenoids, which are a group of high-value natural pigments
towards balanced fuel parameters such as HHV, density, viscosity, and having immense medical, cosmeceutical, and nutraceutical applications.
ignition quality [90]. It was found that the harvesting had no negative Lutein and β-carotene are the predominant carotenoids in Chlamydo­
impact on the biodiesel properties of the BERC07 strain. The biodiesel monas sp., which have copious health applications because of their
produced from the lipids obtained from the BERC07 biomass met all the unique antioxidant properties [98]. In this study, a 2-fold increase in the
quality standards set by European (EN14214) and American carotenoid content of BERC07 was observed when cultivated using
(D6751ASTM) commercial biodiesel (Table 2). The combustion perfor­ urban wastewater as compared to the BG11 media. Here, carotenoids
mance of an engine is determined by cetane number (CN) where higher were extracted as the first product of the cascading bioprocess owing to
CN corresponds to delay in the ignition process [91]. The minimum their high-value nature. The maximum yield obtained was recorded as
required CN value specified by ASTM is 47, and the CN and SV of the 1.83 mg/g and 1.78 mg/g from the SFB and PFB, respectively. The level
biodiesel were 47.29 and 194.18, respectively, which reflects good of production of carotenoids varies from strain to strain and depends
ignition quality. The oxidative stability of the fuel is assessed by iodine upon the physicochemical factors. The carotenoid profiling showed a
value (IV) which reflects the number of double bonds present in it. The great difference in the carotenoid yields of five different microalgae
IV of the biodiesel was 83.27, which is due to the high unsaturation in including Porphyridium cruentum, Tetraselmis suecica, Isochrysis galbana,
Chlamydomonas sp., but still, it was within the specified limits (≤120) Nannochloropsis gaditana, and P. tricornutum when cultivated under
and found to be less than the other Chlamydomonas sp. [92]. The degree similar conditions [99]. Studies have shown that different stress condi­
of unsaturation concentrations of linolenic acid (1.23%) and linoleic tions such as high salinity, high temperature, high light, or varying
acid (not detected) indicated good oxidative stability when compared to nutrient concentrations can be applied to increase the carotenoid as well
the biodiesels reported in Chlorella sp. [92], D. tertiolecta, C. vulgaris, and as lipid content in microalgae [100] which will help to enhance the
Phormidium sp. [93]. The high heating value (HHV) of the BERC07- economic feasibility of the proposed biorefinery scheme.
dervied biodiesel was found to be 38.98 MJ/kg, which is comparable
to the HHVs of other reported strains [94]. The presence of the higher 3.4.3.2. Product-II: High-quality biodiesel. The compositional analyses of
amount of saturated fatty acids (SFA) in the fuel can cause the clogging BERC07 showed that lipids were the major constituent that accounted
of the fuel lines at lower temperatures, that is why the cold flow prop­ for 480 mg/g of the biomass (48%) on a dry cell mass basis. During the
erties including cold point (CP), pour point (PP), and Cold Filter Plug­ 2nd stage of the proposed biorefinery scheme, lipids were extracted
ging Point (CFPP) of any fuel must be determined to evaluate its from the oven-dried pigment-free residual biomass harvested via both
feasibility in countries with cold climates. The CFPP value of BERC07- routes, separately. Interestingly, there was no difference in the lipid
biodiesel was found to be lesser than 0◦ which is considered as the content of pigment-extracted biomass when compared to the lipid con­
most suitable value for the flow of biodiesel. Fuel viscosity and density tent acquired from the whole-cell biomass following the reference pro­
are two other important parameters determining the combustion ability tocol. The extracted lipids were then transesterified into biodiesel.
of any fuel. Less viscous fuels can cause leakage in the internal pump Besides, the biodiesel properties were shown to meet the international
during the process of spray atomization. The viscosity of the biodiesel biodiesel standards prescribed by the European and American labs.
derived from BERC07 was recorded as 3.69 mm/s, which meets the
3.4.3.3. Products III & IV: Valorization of the waste biomass into valuable
industrial products. To ensure maximum resource recovery and to
Table 2
Fuel properties of BERC07-derived biodiesel with commercial diesel.
minimize waste, the pigment-free and lipid-free residual biomass was
subjected to fermentation. Among both fungal strains, A. oryzae per­
Fuel properties BERC07 BERC07 Algal ASTM European
formed better with an α-amylase production of 131.6 U/mL utilizing the
(SFB) (PFB) biodiesel D6751 14,214
75 g/L of biomass concentration after 96 h. The strain showed the
Cetane number 48.4 47.29 71.2 >47 >51
highest enzyme productivity of 3.92 U/mL/h when 75 g/L of the waste
Iodine value (gI2) 84.3 83.27 – – ≤120
Saponification 192.1 194.18 – – –
biomass was provided as a sole source of nutrition (Fig. 9A). Interest­
value (mg ingly, it was 3-fold higher when compared to the control PDB media.
KOH/g) However, the A. niger produced 33 U/mL of the α-amylase after 72 h
High heating 39 38.98 24–40 – – where 30 g/L of residual algal biomass was provided as the sole source of
value (MJ/kg)
nutrition. However, after 72 h the enzyme productivity started to
Cloud point (◦ C) − 3.92 − 3.651 − 15 to 2 3 –
Cold filter − 11.91 − 12.04 − 8 to – – decline till the end of the cultivation. The maximum enzyme produc­
plugging point − 10 tivity of 0.53 U/mL/h was achieved using 30 g/L of biomass after 96 h of
(◦ C) fermentation which was comparable to the productivity achieved using
Pour point (◦ C) − 10.52 − 10.78 − 14 – 3–15 PDB media (0.54 U/mL/h), which served as a control (Fig. 9B). These
Density (g/cm3) 0.84 0.83 0.80 – 0.86–0.9
Viscosity (mm/s) 3.67 3.69 3.83 1.9–6 3.5–5
results indicated that the biomass loadings higher than 30 g/L and 75 g/

9
S. Malik et al.
Fig. 9. Complete valorization of the residual biomass into industrial bioproducts: Production of α-amylase enzyme and mycoproteins after the fermentation
process by A. oryzae (A, C) and A. niger (B, D) when cultured using the pigment-free and lipid-free residual algal biomass.
L have caused the substrate inhibition in the case of A. niger and
A. oryzae respectively.
The fungal biomass at the base of each flask was shown to be the rich
source of mycoproteins because it mainly contained proteins and was
collected after 96 h of incubation. The carbohydrate content of the
fermented biomass was decreased with increasing time of incubation
which indicated that both fungal strains utilized the carbohydrates for
their growth and enzyme production. Conversely, protein content
showed a steady increase over the time of the fermentation, indicating
the biomass transformation of carbohydrates into fungal proteins, while
no change in the protein/carbs content was observed in the control
flasks. The protein content of the supernatant of each culture flask was
also assayed which exhibited the α-amylase production. The A. oryzae
produced 375 mg/g and 384 mg/g proteins by using 75 g/L and 100 g/L
of the residual biomass, respectively (Fig. 9C). However, the A. niger
produced 278 mg/g and 290 mg/g of proteins by using 30 g/L and 50 g/
L of residual algal biomass, respectively (Fig. 9D). There was a 1.5-fold
increase in the protein content when compared to the protein content of
the biomass before fermentation. These data could not be compared
with the literature because this was the first study of its kind where
pigment-free and lipid-free residual biomass was valorized to α-amylase
and mycoproteins via biological fermentation.
Various studies have demonstrated the use of different industrial
wastes as feedstock for fermentation with A. niger to produce the value-
added industrial enzymes including proteases and amylases which have
many applications in textile, food, and paper industries. For instance
The brewery and meat wastewaters were used as substrate to produce
70.29 and 60.12 EU/mL of α-amylase, respectively, using A. niger after
supplementing both wastewaters with 40 g/L of starch [101]. Recently,
cassava has also been found to be a potential substrate for the A. niger,
resulting in the production of 14.01 U/mL of α-amylase [102]. Inter­
estingly, the biotransformation of residual biomass into α-amylase and
mycoproteins has shown relatively higher enzyme units (especially with
A. oryzae) when compared to these previous studies. Here, the highest
possible biomass valorization efficiency was achieved due to robust
biomass utilization capabilities of the Aspergillus spp. To our knowledge,
this is the first study based on the fermentation of residual algal biomass
by utilizing commercial fungal strains.
10
Energy Conversion and Management 256 (2022) 115360
4. Conclusion and prospects
The self-sustainable biorefinery framework employing a newly iso­
lated self-flocculating Chlamydomonas sp. BERC07 has facilitated the
best possible resource recovery along with wastewater treatment,
recycling of product extraction solvents, and complete valorization of
the biomass into multiple products in a cascading fashion following a
zero-waste approach (shown in schematic Fig. 10). The strain has shown
promising potential for urban wastewater treatment (removal of 100%
total nitrogen, 94% total phosphorus, 56% COD, and 41% BOD) along
with a higher and contamination-free growth owing to its alkaliphilic
nature (pH 10–11). The self-flocculation ability of this strain aided in
developing an inducible flocculation system (a harvesting switch) using
the lowest dosage reported so far (0.27 kg/1000 L of culture) of a
commercial-grade flocculant which improved the flocculation rate by
240-fold. In the end, wastewater-grown biomass was valorized into
multiple products including the carotenoids, lipids, α-amylase, and
mycoprotein in a cascading fashion. Here, fungal fermentation of the
residual biomass to produce industrial enzymes (131 U/mL) and
mycoproteins (375–384 mg/g) was another novel approach for valo­
rizing the residual biomass. All solvents involved in the biomass pro­
cessing were recycled to the wastewater being used for the cultivation of
BERC07. Overall, the whole biorefinery route resulted in a minimum
waste during the cultivation, harvesting, and processing the biomass.
Fixation of atmospheric carbon, treatment of urban wastewater,
maximum resource recovery, and minimum waste production were the
added benefits of this novel eco-efficient biorefinery route. This work
opens new avenues in the concept of multiproduct-based algal bio­
refineries by proposing the most beneficial and productive biorefinery
route so far in a circular bioeconomy paradigm. However, detailed
techno-economic and life-cycle assessment-based studies would be
required in the future to have a clear assessment on the technical and
commercial fronts.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
CRediT authorship contribution statement
Sana Malik: Investigation, Writing – original draft, Methodology.
Ayesha Shahid: Methodology, Software, Validation. Michael J.
S. Malik et al. Energy Conversion and Management 256 (2022) 115360

Fig. 10. Schematic diagram of the cascading algal biorefinery in a circular bioeconomy paradigm: 1. Low-cost upstream processing; A schematic diagram
indicates the low-cost cultivation of the BERC07 in urban wastewater along with pollutant removal and atmospheric carbon fixation (shown in 1st rectangular box).
2. Robust midstream processing; Later an inducible flocculation system was developed using a low concentration (0.27 g/L) of commercial-grade Potash alum (shown
in 2nd rectangular box). 3. Efficient downstream processing; a novel route of complete biomass valorization into valuable industrial products including carotenoids,
biodiesel, α-amylase, and mycoproteins (shown in 3rd rectangular box). The circles labelled as A, B, & C indicate the possibilities of recycling of filtrates and solvents.

Betenbaugh: Writing – review & editing, Resources. Chen-Guang Liu:
Writing – review & editing, Software, Validation. Muhammad Aamer
Mehmood: Conceptualization, Supervision, Writing – review & editing,
Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
Authors are obliged to Higher Education Commission, Pakistan for
providing funds to Ms. Sana Malik under Indigenous Ph.D. Fellowship
program. Partial support in MJB laboratories was rained from NSF grant
90078340.
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