Science of the Total Environment 823 (2022) 153667
Contents lists available at ScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
Employing newly developed plastic bubble wrap technique for biofuel
production from diatoms cultivated in discarded plastic waste
⁎
⁎
Mohd Jahir Khan a, Richard Gordon b,c, Sunita Varjani d, , Vandana Vinayak a,
a
Diatom Nano Engineering and Metabolism Laboratory (DNM), School of Applied Sciences, Dr. Harisingh Gour Central University, Sagar, Madhya Pradesh 470003, India
Gulf Specimen Marine Laboratory & Aquarium, 222 Clark Drive Panacea, FL 32346, USA
C.S. Mott Center for Human Growth & Development, Department of Obstetrics & Gynecology, Wayne State University, 275 E. Hancock, Detroit, MI 48201, USA
d
Gujarat Pollution Control Board, Gandhinagar, Gujarat 382010, India
b
c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Bubble farming© using different plastic
bubble wraps® were used to harvest
diatoms.
• No post nutrient uptake and water loss for
40 days to culture diatoms was noted.
• Diatom cell density was maximum in low
density polyethylene (LDPE) bubble wrap.
• Maximum Diafuel™ (37%) and lipid (35
μgmL−1) on 40th day in LDPE bubble
wrap was recorded.
A R T I C L E
I N F O
Article history:
Received 5 January 2022
Received in revised form 28 January 2022
Accepted 31 January 2022
Available online 05 February 2022
Editor: Damià Barceló
Keywords:
Biofuel
Bubble farming©
Diatom
Lipid
Photobioreactor
Plastic air wrap
A B S T R A C T
Algal culturing in photobioreactors for biofuel and other value-added products is a challenge globally specifically due
to expensive closed or open photobioreactors associated with the high cost, problems of water loss and contamination.
Among the wide varieties of microalgae, diatoms have come out as potential source for crude oil in the form of
Diafuel™ (biofuel from diatoms). However, culturing diatoms at large scale hypothesized as diatom solar panels for
biofuel production is still facing a need for facile and economical production of value-added products. The aim of
this work was to culture diatom (microalgae) in a closed system by sealing the reactor rim tightly with very cheap
priced and used plastic bubble wrap material which is generally discarded in a lodging and transportation of goods.
To optimize it, different plastic wraps discarded from a plastic industry were tested first for their permeability to
gases and impermeability to water loss. It was found that among different varieties of plastic bubble wraps, low density
polyethylene (LDPE) bubble wrap material which was used to seal glass containers as photobioreactors allowed harvest of maximum Diafuel™ (37%), lipid (35 μgmL−1), highest cell count (1152 × 102 cells mL−1), maximum CO2 absorbance (0.084) with almost no water loss and nutrient uptake for 40 days of experiments. This was due to its
permeability to gases and impermeability to water. To check usability of such LDPE bubble wrap on other microalgae
it was therefore tested on the red-green microalgae Haematococcus pluvialis, which showed scope to be scaled up for
astaxanthin production using discarded bubble wrap packing material. This study thus would open up a new way
for decreasing plastic disposal and with reuse for sustainable development and application of diatom in biofuel production which could find applications in environmental and industrial sectors.
⁎ Corresponding authors.
E-mail addresses: drsvs18@gmail.com (S. Varjani), kapilvinayak@gmail.com (V. Vinayak).
http://dx.doi.org/10.1016/j.scitotenv.2022.153667
0048-9697/© 2022 Elsevier B.V. All rights reserved.
M.J. Khan et al.
Science of the Total Environment 823 (2022) 153667
1. Introduction
environmental pollution (Varjani et al., 2021). This is essentially not only
good for the cultivation of diatoms but every microalga of potential industrial value cultivated at mass scale for e.g., Haematococcus pluvialis, which is
rich in astaxanthin, a high value product of high demand in the international market. The high cost is probably due to the fact that exorbitant
cost of harvesting/extracting increases the cost of value-added products
like astaxanthin. High cost further is due to the thick cell wall of many
such microalgae which needs pretreatment before extracting value added
products from them (Ahirwar et al., 2021a; Zheng et al., 2022).
In the present study three different qualities of diverse bubble wraps
with different densities of polyethylene were collected and tested for their
CO2 and water permeability. Plastic bubble wraps with the most desirable
permeability to gases and impermeability to water were selected and diatoms were cultivated in conical flasks simulated as photobioreactors
(PBR). The rims of the conical flasks were sealed with such plastic bubble
wrap using a visible colored and robust microalga like H. pluvialis.
With the increase in industrialization and consumer goods, plastic had
been an immense part of industrial and commercial sector (Rajmohan
et al., 2019; Gaur et al., 2022; Varjani et al., 2021). Plastic has somehow entered into every nook and corner of our daily lifestyle (Rajmohan et al.,
2020). Disposing the plastic has been a major cause of environmental pollution leading to its indirect effect on our ecological niche (Khan et al.,
2020; Varjani et al., 2020; Varjani and Upasani, 2021; Tran et al., 2022).
In this manuscript we have considered the reuse of air bubble wraps for
algal culturing not only preventing its dumping into our environment but
economically lowering their culturing cost. For a long time, large scale
algae culture has taken place in closed photobioreactors (Bani et al.,
2021), open raceway ponds (Mohan et al., 2021), horizontal plates
(Cuaresma et al., 2011), tubular equipment (Bani et al., 2021), “Christmas
trees” (Legrand et al., 2021), foil, porous substrate bioreactors (Carbone
et al., 2020) and bubble column photobioreactors (PBR) (Bose et al.,
2021). The exorbitant cost of biofuel from algae like diatoms (about
$3.36/l) (Vinayak et al., 2018) make these methods unsuccessful at a commercial scale, since the market cost is higher (Gordon et al., 2019a;
Jayakumar et al., 2021; Richardson et al., 2012; Singh et al., 2016). This essentially has attracted researchers to look for alternative sources for crude
oil (Ho et al., 2014; Leite et al., 2013). It is projected that by 2030 the global
energy demand will increase 57% (Kowthaman et al., 2022). To meet this
demand for energy, diatoms are good choice as an alternative energy source
as they alone contribute 30% of total crude oil besides synthesizing value
added products like fucoxanthin, fatty acids like eicosanoic acid, and
docosapentaenoic acid (Mourya et al., 2021). These fatty acids vary from
C14:0 to C22:6, and are used extensively in food, personal and health
care markets (Paliwal et al., 2017; Seth et al., 2021). Fucoxanthin is one
of the most common high value metabolites from diatoms; it along with
chlorophyll-a/c, proteins and other metabolites has therapeutic properties
viz; anticancer, antioxidant, antiangiogenic, antidiabetic etc. (Seth et al.,
2021; Leong et al., 2022).
Like other microalgae, diatom culture in photobioreactors is not easy
nor economic since harvesting of high yield biomass and high value
added products kills the cells (Vinayak et al., 2015). This is mainly due to
thick silica wall of diatoms which makes harvesting/extraction tedious
and exorbitant. Also, making such photobioreactors requires continuous
media supplements to maintain the nutrients and water loss. Furthermore,
diatoms cultured in open systems have high chances of contamination with
spores, bacteria, fungi and other microalgae (Xiang et al., 2017). To conquer these stumbling blocks the possibility of using plastic wraps used in
packaging goods in the form of Bubble Farming© (Gordon et al., 2019a)
stemmed out of earlier research on diatom solar panels for biofuel production to harvest biofuel than to extract (Gautam et al., 2016; Ghobara et al.,
2019; Gupta et al., 2018; Kumar et al., 2016; Kumar et al., 2018; Vinayak
et al., 2014; Mishra et al., 2020). Using bubble wrap in microalgae culture
is advantageous compared to conventional farming as bubble farming
photobioreactors besides recycling the plastic bubble wrap waste maintains
a closed system enabling air exchange with minimum chances of contamination. Hence, bubble farming photobioreactors are economical and prevent water loss and constant feeding of the nutrient media (Gordon et al.,
2019b). Furthermore, bubble farming lowers the cost of culture and additionally, if microalgae of interest like diatoms or any other microalgae are
cultured in wastewaters they not only remediate wastewater pollutants,
but it becomes environment friendly and cheap way for mass cultivation
of microalgae (Khan et al., 2021d, Khan et al., 2021d).
To achieve this, researchers have been continuously doing effort to design different techniques like microbial fuel cell, high rate algal ponds, pyrolysis, etc. using diatoms and other microalgae to harvest value-added
metabolites from them (Mishra et al., 2019; Khan et al., 2021b; Khan
et al., 2021c; Khan et al., 2021e; Lee et al., 2020; Shah et al., 2021;
Vinayak et al., 2021b; Vyas et al., 2022). Plastic bubble wrap, on the
other hand, used in culturing of microalga, can be obtained from goods
transport or the plastic industry. Hence it is economical as well as reduces
2. Materials and methods
2.1. Experimental
In the first step, pond water from local pond in Canada (50.8012° N,
98.9776° W) was filled in wide-mouth glass jars and its mouth was sealed
with AIRplus® air pillow bubble wrap material (Storopack Industry,
Canada; www.storopack.com) using elastic bands. The temperature of
water in the jars was checked to be at 5 °C in the pond. Jars were set
below a west windowsill under a McKenzie Hydrofarm Inc. 24 W Agrobrite
Highoutput 553 cm fluorescent light bulb, on a 12 h on/off cycle starting 6
am (November/December 2020). Ambient temperature of the room was
~21 °C with a relative humidity of ~43%.
2.2. Testing different plastic bubble wraps for CO2 permeability and physical parameters
To evaluate CO2 permeability, three different types of plastic bubble
wraps viz low density polyethylene (LDPE) (Barentsen and Heikens,
1973), high density polyethylene (HDPE) (Khonakdar et al., 2003) and normal polyethylene (NP) (Ingraham et al., 1947) were used. These plastic
bubble wraps having size 20 cm × 20 cm, thickness 20 μm, collected
from disposed plastic packaging material from packaged goods were collected and were used to seal the rim of test tubes in which CO2 permeability
was checked. About 1 mL of calcium hydroxide solution (30 mM) was
added in each culture in test tubes containing about 100 mL of distilled
MilliQ water (Holm and Brien, 1971). Nylon based bubble wrap was
avoided, as these bubble wraps are impervious to atmospheric gases
(Levy, 1998). Each of the above sets was also observed for its quantity of
water retention, UV–Vis absorbance, humidity, pH, conductivity and
Total Dissolved Solids (TDS) after every 5th day for a period of 20 days at
an ambient temperature of 22–25 °C.
2.3. Testing diatom growth in most permeable plastic bubble wrap in a glass flask
photobioreactor
As hypothesized, microalgae would grow in plastic Bubble Farming©
technique without requirement of any additional nutrient feeding
(Gordon et al., 2019a). In this hypothesis we first examined different plastic
bubble wraps showing adequate exchange of gases, especially CO2 and O2.
Thereafter, diatoms added in the flask simulated PBR sealed with the plastic
bubble wraps and were tested for which was most efficient with regard to
permeability to gases and water retention. All experiments had been
planned to be conducted in triplicates with the flasks kept stationary. The
mouth of each conical flask (250 mL) was sealed with different polyethylene bubble wraps using elastic rubber bands, acting as laboratory PBR.
These flasks/PBR were inoculated with a fixed inoculum of the diatom
Nitzschia palea (4.79 × 104 cells mL−1 added to 100 mL of modified f/2
medium) (Vinayak et al., 2021a). A fixed number of cells was added into
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M.J. Khan et al.
Science of the Total Environment 823 (2022) 153667
et al., 2021b). The type of bubble wrap which would be most efficient regarding its permeability and water retention properties would be selected
to grow these green-red microalgae. The selected plastic bubble wrap of
size 6″ × 6″ with each bubble having diameter of 0.25″ was chosen. Each
plastic bubble wrap was filled with microalgae via U-100 injection and
sealed thereafter with clear cuticle hardener (Bwambok et al., 2014). The
maximum amount of H. pluvialis culture was injected per bubble was 160
μL, having about 20 cells/μL. The cell density was analyzed via histograms
of images against a white background. Each image was split into red green
and blue (RGB) (Vrablik et al., 2018) individually using Image J software
since direct counting would lead to loss and leakage of culture (Ferreira
and Rasband, 2021). This would be thereafter studied for a period of 21
days.
100 mL of modified f/2 medium in the test samples (flasks simulated
photobioreactor which were sealed with different plastic bubble wrap material viz NP, LDPE and HDPE) and control (one which was open acted as an
open algal photobioreactor). The diatom cells in these flasks were sealed
with different plastic bubble wraps and were grown at 22 °C temperature
in standard light/dark duration of 8 h/16 h (Vinayak et al., 2014).
2.4. Trans-esterification of lipid into fatty acid methyl ester
Lipid was harvested from the control and three different test samples
(5 mL) in triplicates and transesterified as per the Bligh and Dyer method
(Bligh and Dyer, 1959). Both the test and control samples were centrifuged
and pellets were dried. The biomass was weighed and mixed in chloroform:
methanol: water in a 1:2:0.9 ratio, vortexed and sonicated for 5 min. This
was followed by the addition of chloroform and deionized water to make
a total ratio of chloroform: methanol: water as 2:2:1.8. After that 1 mL of
0.5% NaCl solution was added and the samples were mixed vigorously, centrifuged at 5000 g for 10 min and then kept at room temperature for 1 h to
separate organic and aqueous layers. The organic layer was transferred
carefully into a fresh tube. Solvent was removed from the extract using a
speed-vacuum system and the fatty acid obtained was weighed at a precision of 0.0001 g.
3. Results and discussion
Herein the use of disposed or discarded plastic bubble wrap has shown
significant results. It was seen that the glass jars having pond water algae
with open rim which served as control and test jar rim sealed with
AIRplus® pillow plastics served as test showed some consequential analysis
of algal growth and no water loss. On day 1, volume of pond water in both
the glass jars was same. It was observed that even up to 50th day, the
microalgae remained green in both closed and as well as open glass jar.
However, it was seen that there was no water loss in the jar sealed with
plastic bubble wrap while about 50% water loss was seen in an open jar
(Fig. S1). On the offset, there was full water retention in the sealed jar,
with almost equal algal growth in both control and test samples analyzed
via histograms of RGB images studied using Image J software which
showed no change in the peak's positions (Fig. S1). This demonstratedd
that the sealing of glass jar rim with plastic bubble wrap didn't starve the
algae for exchange of gases (CO2 and O2) which is required for photosynthesis and respiration and simultaneously maintained the water level in
growth media. This is significant since water loss and contamination are
the major drawbacks of microalgae cultivation in open raceways
(Branyikova and Lucakova, 2021; de Carvalho Silvello et al., 2021). Furthermore, bubble wraps can be used in making cost effective
photobioreactor for algal farming and obtained value added products
from these microalgae could be commercialized. Plastic bubble wrap
which is generally used to pack the goods is thrown away once the goods
items are unpacked, projected at US10.7 billion in 2023 (https://www.
statista.com/statistics/1013196/global-market-value-of-bubble-wrap/,
covering over 2 million hectares, per Gordon et al., 2019b). These disposed
bubbles wraps from lodging goods or from plastic industry caused environmental pollution. These bubble wraps can be thus utilized for economical
algal farming. Therefore, sealing of an open raceway by large sheets of bubble wrap plastic or customizing its production in large size bubble wrap for
mass scale diatom cultivation could probably avoid most these stumbling
blocks. The quest was thus to quantitatively check the CO2, O2 availability
and water retention of different bubble wrap plastic materials viz NP, LDPE,
HDPE and compare them with the control. This was done by recording
physical parameters such as humidity, pH, conductivity and TDS.
It was found that the pH in all the test samples sealed with NP, LDPE,
HDPE bubble wraps was alkaline (pH 12) on 20th day. The pH change in
control and test samples is associated with growth of microalgae as well exchange of gases through the different bubble wraps (Siddiki et al., 2022).
The pH of control was recorded slightly lower than test samples (pH 11.3
± 0.25) on the 20th day (Fig 1A). However, there were only minor changes
in the conductivity and TDS value in all bubble wrapped test samples.
Conductivity of the control sample on the other hand decreased from
5.64 ± 0.02 μS on day 1 to 2.42 ± 0.32 μS on 20th day, whereas conductivity of test samples increased from ~5.64 ± 0.10 μS on the 1st day to 5.74
± 0.25 μS for NP, 6.95 ± 0.24 μS for LDPE and 7.23 ± 0.27 μS for HDPE on
20th day (Fig 1B). These results were in concordance with the previous reported data (Grishagin, 2015; Mostafa et al., 2012). Control samples without any bubble wrap showed maximum TDS value (3.88 ppt) on 1st day
and decreased (1.61 ppt) thereafter till 20th day (Fig 1C). This was possibly
2.5. Lipid estimation by sulpho-phospho vanillin (SPV) method
The amount of lipid in diatom samples was estimated from the FAME by
the colorimetric sulfo-phospho-vanillin (SPV) method (Khan et al., 2019).
Linseed oil (Supelco, PA, USA; 1000 mg) was used as a standard. In each
sample, 100 μL of concentrated sulfuric acid was added and incubated at
90 °C for 10 min followed by cooling at 4 °C for 5 min. After cooling, 2.4
mL of phosphovanillin (PV) reagent was added (prepared by addition of
120 mg vanillin (Sigma-Aldrich, USA) into 100 mL of hot 85% phosphoric
acid). The mixture was then kept for 30 min at room temperature so as to
develop pink color. The absorbance was thereafter recorded at 530 nm
using a UV–Vis spectrophotometer (LabIndia, UV-3000). The amounts of
oil in diatom samples were determined with the help of a standard plot prepared using different concentrations of linseed oil. At the start, the neutral
lipids in live diatoms were stained using Nile red stain and imaged via confocal microscopy to confirm their presence.
2.6. Estimation of chlorophyll content
Microalgal culture having diatoms (5 mL) were taken on 1st, 10th, 20th,
30th and 40th day and the absorbances were recorded at 680 nm using a
UV–Vis spectrophotometer (Lab India UV-3000, India) (Khan et al.,
2020). This analyzes the physiological state as well as growth of diatom
cells. The amount of chlorophyll in control and experimental samples
were determined using the UV–Vis spectrophotometer for different time intervals as per Eq. (1) (Angioni et al., 2018):
ð20:2 A645 Þ þ ð8:02 A663 Þ
Chlorophyll μg mL−1 ¼
2
(1)
Control and test diatom culture (5 mL) of 1st, 10th, 20th, 30th and 40th
day were taken and centrifuged at 3000 ×g for 10 min. Supernatant was
discarded and 2 mL absolute ethanol was added in the pellet and vortexed
followed by boiling in a water bath for 5 min. Cell debris was removed by
centrifugation and the absorbance of clear ethanol layer having pigments
was recorded at 645 nm and 663 nm using the UV–vis spectrophotometer
(Angioni et al., 2018).
2.7. Employing bubble wrap as plastic waste for growing H. pluvialis
In order to test if bubble wrap could actually be used for in-vitro culturing of microalgae, rapidly growing microalgae H. pluvialis (a green-red
microalgae) in its red encyst stage was taken for cross checking (Ahirwar
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M.J. Khan et al.
Science of the Total Environment 823 (2022) 153667
Fig. 1. CO2 absorption and desorption in different experimental conditions at temperature 25 ± 0.237 °C for 20 days.
because of high availability of CO2 and other gases due to no hindrance and
mingling of CO2 gases. This was a unlike behavior seen in NP, LDPE and
HDPE test samples where water was turbid on last 20th day as there were
gradual increase in TDS due to less permeability of gases (Fig 1C). However,
TDS was near 0.07 ± 0.04 ppt on 1st day for NP, LDPE and HDPE whereas
on 20th day it increased and reached near ~2.5 ± 0.25 ppt in NP and LDPE
while it was least for HDPE bubble wrap as it was 2.12 ± 0.24 ppt on the
20th day.
The changes in conductivity and TDS with the carbon dioxide absorption and desorption were concordant in pattern as explained by Al-Hindi
and Azizi in a bubble column reactor tank (Al-Hindi and Azizi, 2020). However, there were minor changes in pH which is concordant with the earlier
reported studies in which pH changed with CO2 absorption (Al-Hindi and
Azizi, 2020). Increase in CO2 absorption results in rise in salinities with
the effect of different water types studied for CO2 absorption and desorption studies in bubble column reactors (Al-Hindi and Azizi, 2020)
(Navaza et al., 2009). Lastly, relative humidity of the culture room where
the experiment was conducted was stable for all days reaching between
15 and 17% which is a significant factor for the maintaining concentration
of gases present in the atmosphere throughout the experiment.
The UV–Vis spectroscopic observations of control and test for CO2 absorption and desorption showed that the control changed its turbidity
after addition of calcium hydroxide solution into it. Figure 2 illustrates
that on 5th day, control showed maximum absorbance of 1.77 Au at
208 nm whereas NP, LDPE, and HDPE showed maximum absorbance's of
1.75 Au, 1.73 Au and 1.72 Au at 212 Au, respectively. As the time of exposure increased, absorbance of CO2 increased from 10th to 20th day. The
highest absorbance (1.91) was reported in control sample on 20th day
whereas there were significant changes in the absorbance in NP, LDPE
and HDPE observed as 1.72 Au, 1.68 Au and 1.67 Au, respectively. The
high absorbance in control was due to the fact that maximum CO2 was absorbed thus forming CaCO3 precipitate (Gettens et al., 1974). This is clearly
elaborated in Fig. 2D due to maximum area under curve occupied by control compared to test samples. It was further seen that the increase in CO2
absorption results not only decrease in TDS but also decrease in absorption
coefficient as is seen from the UV–Vis spectral studies for control sample
which is concordant with earlier reported studies (Al-Hindi and Azizi,
2020). Even though LDPE bubble wrap test samples showed comparatively
better absorption of CO2, all the bubble wraps test samples were further
tested for growth of diatoms, water loss, chlorophyll content, cell density,
lipids and biofuel from diatoms. It was further seen that cell growth in
the LDPE test sample was linearly proportional to the CO2 gases absorbed
in LDPE samples as seen in Fig. 2.
Henceforth, fixed number of diatom cells contained in 250 mL conical
flasks acting as PBR was sealed with NP, LDPE and HDPE bubble wraps
along with control in triplicates. These experimental sets were run for 40
days and showed varied diatom cell counts as shown in Fig 3A. It was observed that the control showed a fall in diatom growth on the 40th day
(197 × 102 cells mL−1). On the 40th day, the growth of diatoms was
lower than on the 30th day in the case of NP (429 × 102 cells mL−1) and
HDPE (856 × 102 cells mL−1) bubble wrap. However, only LDPE showed
a stable quasi-exponential behavior in growth of diatom cells (1152 ×
102 cells mL−1) until 30th to 40th day as compared to NP and HDPE. The
growth condition was further verified by recording the absorbance at
680 nm using UV–Vis spectrophotometer (Fig 3B). On 10th day, LDPE sample displayed a higher absorbance (0.4) with cell count of 499 × 102 cells
mL−1 as compared to other bubble wrap because of active photosynthesis.
Active photosynthesis in LDPE bubble wrap is associated with efficient absorption of CO2 gases exchanged via its permeable plastic membrane. However, at 40th day, lowest absorbance (0.052 Au) was found in the NP
samples with minimum cell count of 429 × 102 cells mL−1. Similarly,
cell count was low in HDPE (0.018 Au) with 856 × 102 cells mL−1on
40th day as seen in Fig 3B. On the other hand, LDPE on the 40th day
displayed maximum absorbance (0.084 Au) with highest cell count of
about 1152 × 102 cells mL−1. The results were in concordance with the
theory that cell growth is directly proportional to the absorbance of light
getting absorbed due to scattered particles in the cuvette (Ritchie and
Sma-Air, 2020).
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Science of the Total Environment 823 (2022) 153667
Fig. 2. Absorbance of water mixed with calcium hydroxide in test tubes marked as Control, NP, LDPE and HDPE bubble wrap/s on 5th; 10th; 15th and 20th day of exposure to
atmospheric CO2.
Furthermore, it was found that the amount of lipid on 10th day was
26.17 μgmL−1, 22.40 μgmL−1, 25.09 μgmL−1 and 25.97 μgmL−1 for
control, NP, LDPE and HDPE, respectively (Fig 4A). Lipid content further increased substantially in LDPE 31.21 μgmL −1 on 20th day,
33.88 μgmL−1 on 30th day and 34.89 μgmL−1 on 40th day. The control
sample showed decrease in lipid content about 2.33 μgmL−1 on 40th
day which is possibly due to decrease in cell count and contaminations
in open culture flask. The fall in lipid was also observed in NP (10.24
μgmL −1 ) and HDPE (15.83 μgmL −1 ) on 40th day. However, lipid
yield continued to rise in LDPE. Comparing these results, it was analyzed that even though on 10th day LDPE showed lowest lipid content
(22.40 μgmL−1), it increased gradually which was well in accordance
with cell growth analyzed spectrophotometrically and via and cell
count. This was probably due to scarcity in CO2 and decline in photosynthesis during initial days after inoculation. Hence, CO2 plays
an important role in photosynthesis to make carbohydrates required
for its fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase
(RUBISCO) enzyme in C4 pathway photosystem (Pandey et al., 2021;
Riebesell, 2000). Its deficiency may ultimately result in failure to synthesize carbohydrates required for further energy generating pathways.
Furthermore, when these lipids were transesterified they showed the
formation of Diafuel™ (biofuel from diatoms) i.e. fatty acid methyl esters (FAME) (Fig 4B). However, maximum Diafuel™ percentage was
37.27% per dry weight (DW) for the LDPE sample, less in HDPE about
28.1% per DW and minimum for NP 2.35% per DW and least in control
sample (1.6%) per DW on 40th day.
Diatoms were further stained with Nile red dye for the detection of
neutral lipids on day 1 and 40th day in LDPE. Neutral lipid in diatom
samples was visualized by confocal microscopy (Fig 4). The optical images clearly showed that lipid in diatoms increased on 30th day in LDPE
It was seen that the chlorophyll concentration on the 10th day was
highest for control (0.835 μg mL −1 ) as compared to NP (0.456 μg
mL−1), LDPE (0.752 μg mL−1) and HDPE (0.555 μg mL−1) test samples
inoculated with fixed number of diatom cells (Fig. 3C). The chlorophyll
concentration of the control increased until the20th day and decreased
thereafter. However, on 20th day, chlorophyll concentration of control
decreased to 0.735 μg mL−1. Decrease in chlorophyll concentration in
the control sample after 20th day is because of water loss and microbial
contaminations concordant with open raceway culturing results
(Borowitzka and Moheimani, 2013; Shekh et al., 2021). On the offset,
the chlorophyll concentration in LDPE and HDPE on 40th day reached
to 3.34 μg mL−1 and 2.5 μg mL−1, respectively. The amount of chlorophyll is also directly proportional to cell growth and the results obtained were in accordance with the cell count in each set (Lim et al.,
2021). This was also in concordance with earlier research demonstrating a direct correlation between the chlorophyll content and cell
count (Groß et al., 2021; Myers and Kratz, 1955). The chlorophyll content constitutes the major carotenoids which are a group of high value
metabolites being synthesized in a diatoms (Seth et al., 2021). The
water content was measured for each sample on 1st, 10th, 20th, 30th
and 40th day and it was found that there was no remarkable water
loss in bubble wraps closed test samples except control which displayed
significant water loss (~37%) (Fig. 3D) and microalgal contamination
as evident from the microscopy. This is the reason closed system PBRs
are superior to open raceway ponds but due to their high operational
cost they have limited use (Gordon et al., 2019a); therefore, a variety
of techniques have been inspired for their economical harvesting
(Khan et al., 2021c; Vinayak et al., 2021b) while simultaneously
cleaning the environmental plastic pollution (Dang et al., 2022; Khan
et al., 2020).
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Science of the Total Environment 823 (2022) 153667
Fig. 3. Diatom samples in different bubble wrap plastics sets at different time periods showing their A: Cell count mL−1; B: Absorbance of diatom cells at 680 nm, C: Extracted
Chlorophyll content and D: Water retention.
sealed flask PBRs without requiring any extra post-inoculation nutrient
feed. Nile red staining has been used in analyzing neutral lipids in diatoms and is one of the most reliable method for in situ testing for amount
of neutral lipids in cells (Jayakumar et al., 2021; Wu et al., 2014). The
confocal microscopy of the amount of neutral lipids stained in diatoms
on day 1 and 40th day as seen in Fig 4C and D clearly showed increase in
amount of neutral lipids in diatoms cultured in LDPE flask PBR in accordance with earlier published results with neutral red dye (Khan et al.,
2021a). It indicates that LDPE is most efficient plastic bubble wrap material to seal PBR while simultaneously maintaining a closed microcosm, thus avoiding contamination and water loss. Its scale up in
bubble farming would definitely bring down the cost of harvesting
value added products, which is probably due to the high capital cost
and investment in running the PBR whether it's closed or open type.
Table 1 shows a comparison of closed, open and LDPE bubble farming
PBR for various parameters which are involved while cultivating different types of algae.
Not limiting to our study on diatoms it was necessary to check this
novel practice on in situ bubble wrap as such, henceforth an already optimized LDPE bubble wrap material measuring 6″ × 6″ with each bubble having diameter of 0.25″ was chosen for inoculation of a visibly
distinguishable red-green microalgae, H. pluvialis for 21 days inoculated during its condition of stress from the mother liquor. H. pluvialis
is rich in astaxanthin which gives it its red color and is highly distinguishable in bubble wrap (Ahirwar et al., 2021b). However, using plastic bubble wrap having a bubble diameter of 0.25″ made cell counting
cumbersome due to frequent puncturing of the bubble wrap. Hence,
cell density was measured by histogram of each photographed image
spilt into red, green and blue and processed via Image J software
(Grishagin, 2015). Fig 5 shows the blue channel of the image shifted
from in its mean value of 143 ± 7.77 at day 1 towards 155 ±
11.17 at 21st day. This method gave concordant analysis as given for
yeast biomass concentration where cell counting was not possible
(Acevedo et al., 2009). This gives a scope for light weighted economical
algal foods and cultivations in space missions too (Vinayak, 2022).
However, this was just a small-scale check and the same could be
done using bigger air pillows of LDPE plastic wrap material wherein
the main aim was to keep the system closed, have enough permeability
for CO2 and other gases while simultaneously preventing water loss,
avoiding contamination and frequent inputs of nutrient media. This
gives us reason to start a pilot scale project with Air pillow mass cultivation of any microalgae of interest and analyzing all the desired parameters for industrial scale economic start up.
4. Conclusions
The results demonstrated that the desirable gaseous exchange takes
place through discarded low density polyethylene (LDPE) plastic bubble wrap to support diatom growth without loss of water and any
extra nutrient uptake. The LDPE had highest diatom cell count on
40th day (1152 × 102 cells mL−1). The chlorophyll content was maximum for LDPE compared to that in the control; NP and HDPE. NP and
HDPE also exhibited no significant water loss. The lipid and Diafuel™
had the highest values in LDPE at the 40th day, 34.89 μg mL −1 , and
37.27% per 1152 × 102 cells mL −1 , respectively whereas a fall in
lipid content in the control and rest of the test samples was noted.
This not only demonstrates a facile way of making a diatom solar
panel for Diafuel™ production but also an economical way to bridge
the gap between the cost of operating expensive photobioreactors
which in turn increase the cost of high value metabolites obtained
from them. On the offset, algal Bubble Farming© would be envisaged
on fields under natural, uncontrolled weather conditions by utilizing
discarded plastic bubble wraps from plastic industry or lodging goods
and materials. The weight of the water might protect the bubble wrap
6
M.J. Khan et al.
Science of the Total Environment 823 (2022) 153667
Fig. 4. Diatoms cultured in different bubble wrap plastics at different time periods producing A: Lipids; B: Diafuel™ percentage and confocal microscopy of Nile red stained
diatoms in LDPE bubble wrap showing lipid accumulation at C: day 1 and D: 40th day.
Declaration of competing interest
from wind, and their shape may clean their top surface during rain.
Field test are in order. This research work serves an economical method
for harvesting algae by recycling plastic waste for their biomass and
value-added products.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2022.153667.
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
CRediT authorship contribution statement
VV is thankful to DST-Nanomission (Govt. of India) project number
(SR/NM/NT-1090/2014(G) and PPMB 7133 CEFIPRA Indo French project for financial support. MJK thanks DST-Nanomission for Postdoc
Fellowship. Authors are also thankful to DST-Nanomission Lab
Assistant, Deepesh Sinha and SIC, Dr. Harisingh Gour Central
University, Sagar, India for providing necessary research facilities.
MJK: Literature review, Writing - original draft, Data curation; RG: Conceptualization, Design of experiment; SV: Writing - original draft, Review &
editing Resources; VV: Conceptualization, Supervision; Writing - original
draft, Review & editing, Funding acquisition.
Table 1
Parameters and their role in different photobioreactors vs bubble farming.
S. no.
Parameter
Closed system photobioreactors
Open system photobioreactors
Bubble farming (LDPE)
(this study)
1
2
3
4
5
6
7
8
9
10
11
12
Capital cost
Rate of Contaminations
Gases exchange rate
Scale up to higher reactors
Land area required
Feeding
Algae type
Operation and handling
Water loss due to evaporation
Cultivation period
Productivities
Power/energy to run the reactor
High
Low
Fair/high
Easy
Small
Required regularly
Any
Excellent
Low/prevented
Long
High
Required
Low
High
Poor
Possible
Large
Required regularly
Limited
Excellent
High
short
Low
Not required
Zero
Zero
Fair/high
Easy
Small
Not required regularly
Any
Excellent
Zero
Long
High
Not required
7
M.J. Khan et al.
Science of the Total Environment 823 (2022) 153667
Fig. 5. Histogram segregation of red, green, blue (R, G, B) images of Haematococcus pluvialis inoculated in LDPE bubble plastic wrap of size 6″ × 6″ into A1 (R), A2 (G), A3
(B) on day 1 and B1 (R), B2 (G) and B3 (B) on 21st day by ImageJ software.
Bose, A., O'Shea, R., Lin, R.C., Murphy, J.D., 2021. Design, commissioning, and performance
assessment of a lab-scale bubble column reactor for photosynthetic biogas upgrading
with Spirulina platensis. Ind. Eng. Chem. Res. 60 (15), 5688–5704.
Branyikova, I., Lucakova, S., 2021. Technical and physiological aspects of microalgae cultivation
and productivity—spirulina as a promising and feasible choice. Org. Agric. 11 (2), 269–276.
Bwambok, D.K., Christodouleas, D.C., Morin, S.A., Lange, H., Phillips, S.T., Whitesides, G.M.,
2014. Adaptive use of bubble wrap for storing liquid samples and performing analytical
assays. Anal. Chem. 86 (15), 7478–7485.
Carbone, D.A., Olivieri, G., Pollio, A., Melkonian, M., 2020. Biomass and phycobiliprotein production of Galdieria sulphuraria, immobilized on a twin-layer porous substrate
photobioreactor. Appl. Microbiol. Biotechnol. 104 (7), 3109–3119.
Cuaresma, M., Janssen, M., Vílchez, C., Wijffels, R.H., 2011. Horizontal or vertical
photobioreactors? How to improve microalgae photosynthetic efficiency. Bioresour.
Technol. 102 (8), 5129–5137.
Dang, B.-T., Bui, X.-T., Tran, D.P., Ngo, H.H., Nghiem, L.D., Nguyen, P.-T., Nguyen, H.H., Lin,
C., Lin, K.Y.A., Varjani, S., 2022. Current application of algae derivatives for bioplastic
production: a review. Bioresour. Technol. 126698.
de Carvalho Silvello, M.A., Gonçalves, I.S., Azambuja, S.P.H., Costa, S.S., Silva, P.G.P., Santos,
L.O., Goldbeck, R., 2021. Microalgae-based carbohydrates: a green innovative source of
bioenergy. Bioresour. Technol. 126304.
Ferreira, T., Rasband, W., 2021. ImageJ.
Gaur, V.K., Gupta, S., Sharma, P., Gupta, P., Varjani, S., Srivastava, J.K., Chang, J.-S., Bui, X.T., 2022. Metabolic cascade for remediation of plastic waste: a case study on microplastic
degradation. Curr.Pollut.Rep. 1–21.
Gautam, S., Kashyap, M., Gupta, S., Kumar, V., Schoefs, B., Gordon, R., Jeffryes, C., Joshi, K.B.,
Vinayak, V., 2016. Metabolic engineering of TiO2 nanoparticles in Nitzschia palea to
form diatom nanotubes: an ingredient for solar cells to produce electricity and biofuel.
RSC Adv. 6 (99), 97276–97284.
Gettens, R.J., FitzHugh, E.W., Feller, R.L., 1974. Calcium carbonate whites. Stud. Conserv. 19
(3), 157–184.
Ghobara, M.M., Mazumder, N., Vinayak, V., Reissig, L., Gebeshuber, I.C., Tiffany, M.A.,
Gordon, R., 2019. On light and diatoms: a photonics and photobiology review. In:
Seckbach, J., Gordon, R. (Eds.), Diatoms: Fundamentals And Applications. Wiley-Scrivener. Beverly, Massachusetts, USA, pp. 129–189.
Authors also thank Prof Clifford R Merz and Prof Benoit Schoefs for
critic comments to improve the manuscript.
References
Acevedo, C.A., Skurtys, O., Young, M.E., Enrione, J., Pedreschi, F., Osorio, F., 2009. A nondestructive digital imaging method to predict immobilized yeast-biomass. LWTFood
Sci.Technol. 42 (8), 1444–1449.
Ahirwar, A., Meignen, G., Khan, M., Khan, N., Rai, A., Schoefs, B., Marchand, J., Varjani, S.,
Vinayak, V., 2021a. Nanotechnological approaches to disrupt the rigid cell walled
microalgae grown in wastewater for value-added biocompounds: commercial applications, challenges, and breakthrough. Biomass Convers.Biorefin. 1–26.
Ahirwar, A., Meignen, G., Khan, M.J., Sirotiya, V., Scarsini, M., Roux, S., Marchand, J.,
Schoefs, B., Vinayak, V., 2021b. Light modulates transcriptomic dynamics upregulating astaxanthin accumulation in haematococcus: a review. Bioresour. Technol.
125707.
Al-Hindi, M., Azizi, F., 2020. The effect of water type on the absorption and desorption of carbon dioxide in bubble columns. Chem. Eng. Commun. 207 (3), 339–349.
Angioni, S., Millia, L., Mustarelli, P., Doria, E., Temporiti, M.E., Mannucci, B., Corana, F.,
Quartarone, E., 2018. Photosynthetic microbial fuel cell with polybenzimidazole membrane: synergy between bacteria and algae for wastewater removal and biorefinery.
Heliyon 4 (3), e00560.
Bani, A., Fernandez, F.G.A., D'Imporzano, G., Parati, K., Adani, F., 2021. Influence of
photobioreactor set-up on the survival of microalgae inoculum. Bioresour. Technol.
320, 9.
Barentsen, W.M., Heikens, D., 1973. Mechanical properties of polystyrene low density polyethylene blends. Polymer 14 (11), 579–583.
Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can.
J. Biochem. Physiol. 37 (8), 911–917.
Borowitzka, M.A., Moheimani, N.R., 2013. Open pond culture systems. In: Borowitzka, M.A.,
Moheimani, N.R. (Eds.), Algae for Biofuels And Energy. Springer, Netherlands.
Dordrecht, pp. 133–152.
8
M.J. Khan et al.
Science of the Total Environment 823 (2022) 153667
Mourya, M., Khan, M.J., Ahirwar, A., Schoefs, B., Marchand, J., Rai, A., Varjani, S., Rajendran,
K., Banu, J.R., Vinayak, V., 2021. Latest trends and developments in microalgae as potential source for biofuels: the case of diatoms. Fuel 122738.
Myers, J., Kratz, W.A., 1955. Relations between pigment content and photosynthetic characteristics in a blue-green alga. J. Gen. Physiol. 39 (1), 11–22.
Navaza, J.M., Gómez-Díaz, D., La Rubia, M.D., 2009. Removal process of CO2 using MDEA
aqueous solutions in a bubble column reactor. Chem. Eng. J. 146 (2), 184–188.
Paliwal, C., Mitra, M., Bhayani, K., Bharadwaj, S.V.V., Ghosh, T., Dubey, S., Mishra, S., 2017.
Abiotic stresses as tools for metabolites in microalgae. Bioresour. Technol. 244,
1216–1226.
Pandey, A., Tyagi, R.D., Varjani, S., 2021. Biomass, Biofuels, Biochemicals: Circular
Bioeconomy—Current Developments And Future Outlook. Elsevier.
Rajmohan, K.V.S., Ramya, C., Viswanathan, M.R., Varjani, S., 2019. Plastic pollutants: effective waste management for pollution control and abatement. Curr.Opin.Environ.Sci.
Health 12, 72–84.
Rajmohan, K.S., Yadav, H., Vaishnavi, S., Gopinath, M., Varjani, S., 2020. Perspectives on biooil recovery from plastic waste. In: Varjani, S., Pandey, A., Gnansounou, E., Khanal, S.K.,
Raveendran, S. (Eds.), Resource Recovery From Wastes. Elsevier, Amsterdam,
Netherlands, pp. 459–480.
Richardson, J.W., Johnson, M.D., Outlaw, J.L., 2012. Economic comparison of open pond
raceways to photo bio-reactors for profitable production of algae for transportation
fuels in the southwest. Algal Res. 1 (1), 93–100.
Riebesell, U., 2000. Photosynthesis: carbon fix for a diatom. Nature 407 (6807), 959–960.
Ritchie, R.J., Sma-Air, S., 2020. Using integrating sphere spectrophotometry in unicellular
algal research. J. Appl. Phycol. 32 (5), 2947–2958.
Shah, A.V., Srivastava, V.K., Mohanty, S.S., Varjani, S., 2021. Municipal solid waste as a sustainable resource for energy production: state-of-the-art review. J.Environ.Chem.Eng. 9,
105717. https://doi.org/10.1016/j.jece.2021.105717.
Seth, K., Kumar, A., Rastogi, R.P., Meena, M., Vinayak, V., 2021. Bioprospecting of fucoxanthin from diatoms—challenges and perspectives. Algal Res. 60, 102475.
Shekh, A., Sharma, A., Schenk, P.M., Kumar, G., Mudliar, S., 2021. Microalgae cultivation:
photobioreactors, CO2 utilization, and value-added products of industrial importance.
J. Chem. Technol. Biotechnol. (In press).
Siddiki, S.Y.A., Mofijur, M., Kumar, P.S., Ahmed, S.F., Inayat, A., Kusumo, F., Badruddin, I.A.,
Khan, T.Y., Nghiem, L., Ong, H.C., 2022. Microalgae biomass as a sustainable source for
biofuel, biochemical and biobased value-added products: an integrated biorefinery concept. Fuel 307, 121782.
Singh, V., Tiwari, A., Das, M., 2016. Phyco-remediation of industrial waste-water and flue
gases with algal-diesel engenderment from micro-algae: a review. Fuel 173, 90–97.
Tran, H.T., Lin, C., Bui, X.T., Nguyen, M.K., Cao, N.D.T., Mukhtar, H., Hoang, H.G., Varjani, S.,
Ngo, H.H., Nghiem, L.D., 2022. Phthalates in the environment: characteristics, fate and
transport, and advanced wastewater treatment technologies. Bioresour. Technol.
126249, 344. https://doi.org/10.1016/j.biortech.2021.126249.
Varjani, S., Mishra, B., Yadavalli, R., Bui, X.-T., Taherzadeh, M.J., Agrawal, D.C., You, S.,
Chang, J.-S., 2021. Petroleum waste biorefinery: a way towards circular economy.
Waste Biorefinery. Elsevier, pp. 375–389.
Varjani, S., Upasani, V.N., 2021. Bioaugmentation of Pseudomonas aeruginosa NCIM 5514 - a
novel oily waste degrader for treatment of petroleum hydrocarbons. Bioresour. Technol.
319, 124240. https://doi.org/10.1016/j.biortech.2020.124240.
Varjani, S., Upasani, V.N., Pandey, A., 2020. Bioremediation of oily sludge polluted soil
employing a novel strain of Pseudomonas aeruginosa and phytotoxicity of petroleum hydrocarbons for seed germination. Sci. Total Environ. 737, 139766. https://doi.org/10.
1016/j.scitotenv.2020.139766.
Vinayak, V., 2022. Algae as sustainable food in space missions. Biomass, Biofuels, Biochemicals. Elsevier, pp. 517–540.
Vinayak, V., Gordon, R., Gautam, S., Rai, A., 2014. Discovery of a diatom that oozes oil. Adv.
Sci. Lett. 20 (7–9), 1256–1267.
Vinayak, V., Gordon, R., Joshi, K., Schoefs, B., 2018. Diafuel. Trademark Application no
3778882; Trade Marks Journal No: 1846(Class 4.).
Vinayak, V., Khan, M.J., Jha, A.N., 2021a. Photosystem I P700 chlorophyll a apoprotein A1 as
PCR marker to identify diatoms and their associated lineage. J. Eukaryot. Microbiol.
e12866.
Vinayak, V., Khan, M.J., Varjani, S., Saratale, G.D., Saratale, R.G., Bhatia, S.K., 2021b. Microbial fuel cells for remediation of environmental pollutants and value addition: special
focus on coupling diatom microbial fuel cells with photocatalytic and photoelectric fuel
cells. J. Biotechnol. 338, 5–19.
Vinayak, V., Manoylov, K.M., Gateau, H., Blanckaert, V., Herault, J., Pencreac'h, G.,
Marchand, J., Gordon, R., Schoefs, B., 2015. Diatom milking: a review and new approaches. Mar.Drugs 13 (5), 2629–2665.
Vyas, S., Prajapati, P., Shah, A.V., Srivastava, V.K., Varjani, S., 2022. Opportunities and knowledge gaps in biochemical interventions for mining of resources from solid waste: a special
focus on anaerobic digestion. Fuel 311, 122625. https://doi.org/10.1016/j.fuel.2021.
122625.
Vrablik, A., Hidalgo-Herrador, J.M., Cerny, R., 2018. RGB histograms as a reliable tool for the
evaluation of fuel oils stability. Fuel 216, 16–22.
Wu, S.C., Zhang, B.Y., Huang, A.Y., Huan, L., He, L.W., Lin, A.P., Niu, J.F., Wang, G.C., 2014.
Detection of intracellular neutral lipid content in the marine microalgae Prorocentrum
micans and Phaeodactylum tricornutum using Nile red and BODIPY 505/515. J. Appl.
Phycol. 26 (4), 1659–1668.
Xiang, X.W., Ozkan, A., Chiriboga, O., Chotyakul, N., Kelly, C., 2017. Techno-economic analysis of glucosamine and lipid production from marine diatom Cyclotella sp. Bioresour.
Technol. 244, 1480–1488.
Zheng, H., Wang, Y., Li, S., Nagarajan, S., Varjani, S., Lee, D.J., Chang, J.S., 2022. Recent advances in lutein production from microalgae. Renew. Sustain. Energy Rev. 153, 111795.
https://doi.org/10.1016/j.rser.2021.111795.
Gordon, R., Merz, C.R., Gurke, S., Schoefs, B., 2019a. Bubble farming: scalable microcosms for
diatom biofuel and the next Green Revolution. In: Seckbach, J., Gordon, R. (Eds.), Diatoms: Fundamentals And Applications. Vol. 1. Wiley-Scrivener, Beverly, Massachusetts,
USA, pp. 583–654.
Gordon, R., Merz, C.R., Gurke, S., Schoefs, B., 2019b. Bubble farming: scalable microcosms for
diatom biofuel and the next Green Revolution. Diatoms Fundam. Appl. 583–654.
Grishagin, I.V., 2015. Automatic cell counting with ImageJ. Anal. Biochem. 473, 63–65.
Groß, E., Boersma, M., Meunier, C.L., 2021. Environmental impacts on single-cell variation
within a ubiquitous diatom: the role of growth rate. Plos One 16 (5), 17.
Gupta, S., Kashyap, M., Kumar, V., Jain, P., Vinayak, V., Joshi, K.B., 2018. Peptide mediated
facile fabrication of silver nanoparticles over living diatom surface and its application.
J. Mol. Liq. 249, 600–608.
Ho, D.P., Ngo, H.H., Guo, W., 2014. A mini review on renewable sources for biofuel.
Bioresour. Technol. 169, 742–749.
Holm, L.W., Brien, L.J., 1971. Carbon dioxide test at the Mead-Strawn field. J. Pet. Technol.
23 (04), 431–442.
Ingraham, F.D., Alexander, E., Matson, D.D., 1947. Polyethylene, a new synthetic plastic for
use in surgery: experimental applications in neurosurgery. J.Am.Med.Assoc. 135 (2),
82–87.
Jayakumar, S., Bhuyar, P., Pugazhendhi, A., Rahim, M.H.A., Maniam, G.P., Govindan, N.,
2021. Effects of light intensity and nutrients on the lipid content of marine microalga (diatom) Amphiprora sp. for promising biodiesel production. Sci. Total Environ. 768,
145471.
Khan, M.J., Bawra, N., Verma, A., Kumar, V., Pugazhendhi, A., Joshi, K.B., Vinayak, V.,
2021a. Cultivation of diatom pinnularia saprophila for lipid production: a comparison
of methods for harvesting the lipid from the cells. Bioresour. Technol. 319, 124129.
Khan, M.J., Das, S., Vinayak, V., Pant, D., Ganghrekar, M., 2021b. Live diatoms as potential
biocatalyst in a microbial fuel cell for harvesting continuous diafuel, carotenoids and bioelectricity. Chemosphere 132841.
Khan, M.J., Mangesh, H., Ahirwar, A., Schoefs, B., Pugazhendhi, A., Varjani, S., Rajendran, K.,
Bhatia, S.K., Saratale, G.D., Saratale, R.G., 2021c. Insights into diatom microalgal farming
for treatment of wastewater and pretreatment of algal cells by ultrasonication for value
creation. Environ. Res. 111550.
Khan, M.J., Rai, A., Ahirwar, A., Sirotiya, V., Mourya, M., Mishra, S., Schoefs, B., Marchand,
J., Bhatia, S.K., Varjani, S., Vinayak, V., 2021d. Diatom microalgae as smart
nanocontainers for biosensing wastewater pollutants: recent trends and innovations. Bioengineered 12 (2), 9531–9549.
Khan, M.J., Singh, N., Mishra, S., Ahirwar, A., Bast, F., Schoefs, B., Marchand, J., Rajendran,
K., Banu, J.R., Saratale, G.D., 2021e. Impact of light on microalgal photosynthetic microbial fuel cells and removal of pollutants by nanoadsorbent biopolymers: updates, challenges and innovations. Chemosphere 132589.
Khan, M.J., Singh, R., Joshi, K.B., Vinaya, V., 2019. TiO2 doped polydimethylsiloxane (PDMS)
and Luffa cylindrica based photocatalytic nanosponge to absorb and desorb oil in diatom
solar panels. RSC Adv. 9 (39), 22410–22416.
Khan, M.J., Singh, R., Shewani, K., Shukla, P., Bhaskar, P., Joshi, K.B., Vinayak, V., 2020.
Exopolysaccharides directed embellishment of diatoms triggered on plastics and other
marine litter. Sci. Rep. 10 (1), 1–11.
Khonakdar, H.A., Morshedian, J., Wagenknecht, U., Jafari, S.H., 2003. An investigation of
chemical crosslinking effect on properties of high-density polyethylene. Polymer 44
(15), 4301–4309.
Kowthaman, C., Kumar, P.S., Selvan, V.A.M., Ganesh, D., 2022. A comprehensive insight from
microalgae production process to characterization of biofuel for the sustainable energy.
Fuel 310, 122320.
Kumar, V., Gupta, S., Rathod, A., Vinayak, V., Joshi, K.B., 2016. Biomimetic fabrication of biotinylated peptide nanostructures upon diatom scaffold; a plausible model for sustainable
energy. RSC Adv. 6 (77), 73692–73698.
Kumar, V., Singh, R., Thakur, S., Joshi, K.B., Vinayak, V., 2018. Doping of magnetite nanoparticles facilitates clean harvesting of diatom oil as biofuel for sustainable energy. Mater.
Res.Express 5 (4), 8.
Lee, S.Y., Khoiroh, I., Vo, D.-V.N., Kumar, P.S., Show, P.L., 2020. Techniques of lipid extraction from microalgae for biofuel production: a review. Environ. Chem. Lett. 1–21.
Legrand, J., Artu, A., Pruvost, J., 2021. A review on photobioreactor design and modelling for
microalgae production. React.Chem.Eng. 6 (7), 1134–1151.
Leite, G.B., Abdelaziz, A.E.M., Hallenbeck, P.C., 2013. Algal biofuels: challenges and opportunities. Bioresour. Technol. 145, 134–141.
Leong, Y.K., Chen, C.Y., Varjani, S., Chang, J.S., 2022. Producing fucoxanthin from algae – recent advances in cultivation strategies and downstream processing. Bioreosour.Technol.
344, 126170. https://doi.org/10.1016/j.biortech.2021.126170.
Levy, J.H., 1998. Nylon 6 barrier coextrusions - a cost-effective packaging route. Polymers,
Laminations & Coatings Conference, Books 1 and 2. Tappi Press, Atlanta, pp. 163–194.
Lim, H.R., Khoo, K.S., Chew, K.W., Chang, C.-K., Munawaroh, H.S.H., Kumar, P.S., Huy, N.D.,
Show, P.L., 2021. Perspective of Spirulina culture with wastewater into a sustainable circular bioeconomy. Environ. Pollut. 117492.
Mishra, B., Varjani, S., Agarwal, D.C., Mandal, S.K., Ngo, H.H., Taherzadeh, M.J., Chang, J.S.,
You, S., Guo, W., 2020. Engineering biocatalytic material for the remediation of pollutants: a comprehensive review. Environ. Technol. Innov. 20, 101063. https://doi.org/
10.1016/j.eti.2020.101063.
Mishra, B., Varjani, S., Iragavarapu, G.P., Ngo, H.H., Guo, W., Vishal, B., 2019. Microbial fingerprinting of potential biodegrading organisms. Curr.Pollut.Rep. 1–17. https://doi.org/
10.1007/s40726-019-00116-5.
Mohan, N., Rao, P.H., Boopathy, A.B., Rengasamy, R., Chinnasamy, S., 2021. A sustainable
process train for a marine microalga-mediated biomass production and CO2 capture: a
pilot-scale cultivation of Nannochloropsis salina in open raceway ponds and harvesting
through electropreciflocculation. Renew. Energy 173, 263–272.
Mostafa, S.S., Shalaby, E.A., Mahmoud, G.I., 2012. Cultivating microalgae in domestic wastewater for biodiesel production. Not.Sci.Biol. 4 (1), 56–65.
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