Biomass and Bioenergy 145 (2021) 105945

Contents lists available at ScienceDirect

Biomass and Bioenergy

journal homepage: http://www.elsevier.com/locate/biombioe

Biomass production of marine microalga Tetraselmis suecica using biogas

and wastewater as nutrients
Clemens Herold a, b, 1, Tasneema Ishika a, 1, Emeka G. Nwoba a, Stephan Tait c, d, Andrew Ward c,
Navid R. Moheimani a, e, *
a
Algae R&D Centre, Murdoch University, Murdoch, Western Australia, 6150, Australia
b
Department of Applied Biosciences and Process Engineering, Anhalt University of Applied Sciences, Köthen (Anhalt), 06366, Germany
c
Advanced Water Management Centre, The University of Queensland, Brisbane, Queensland, 4072, Australia
d
Centre for Agricultural Engineering, University of Southern Queensland, Toowooomba, Queensland, 4350, Australia
e
Centre for Sustainable Aquatic Ecosystems, Harry Butler Institute, Murdoch University, Murdoch, Western Australia, 6150, Australia

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

Keywords: Anaerobic digestion is a suitable method for treating organic wastes and generating biogas. This biogas contains
Microalgae significant amount of CO2 and some other contaminants. The coupling of wastewater treatment with biogas
T. suecica purification using saline microalgae could effectively upgrade biogas (through photosynthetic CO2 fixation) and
Productivity
concurrently remove nutrients from the effluent, while producing valuable algal biomass. In this context, Tet­
Biogas
Carbon dioxide
raselmis suecica biomass production with the use of an impurity (CO2) in biogas to supply carbon, and nutrients
Anaerobically-digested piggery effluent (nitrogen and phosphorus) from anaerobically-digested piggery effluent (ADPE) was investigated at four oper­
ating pH set points (6.5, 7.5, 8.5 and 9.5). Results showed that pH 7.5 produced the optimum conditions for
T. suecica growth and biogas-based CO2 removal, with the maximum biomass (59.8 mg L− 1 d− 1), lipid (25 mg
L− 1 d− 1) and carbohydrate (6.5 mg L-1 d-1) productivities. Under this condition, CO2, total nitrogen and
phosphorus removal efficiencies were 94.7%, 96% and 72%, respectively. Furthermore, the results showed no
inhibitory effect of dissolved CH4 on the growth of T. suecica at pH 7.5, suggesting the technical feasibility of
harnessing marine T. suecica for simultaneous nutrients removal from wastewaters, biogas upgrading, and
production of energy-rich algal biomass. This process clearly harnesses anaerobically-digested piggery effluent
not only as an asset but also uses an impurity (CO2) in biogas to produce valuable algal biomass.

1. Introduction
Microalgae are prokaryotic or eukaryotic, unicellular or simple
multicellular photosynthetic microorganisms, that exhibit high photo­
synthetic efficiency, rapid growth rate (doubling as short as 3.5 h), and
are capable of year-round production [1]. They can be cultivated on
arable land, in brackish or saline waters to reduce the competition with
food and animal feed crops over farming land and fresh water [2]. They
are able to synthesize and accumulate 20–50% of neutral lipids (of their
dry weight) as lipid bodies (lipid droplets) or lipid inclusions [2] and
their lipid content is higher than that of oil producing conventional
crops [3]. Microalgae have been estimated to have higher biomass
productivity than plant crops in terms of land area required for culti­
vation [1] and are well-recognized as an alternative to existing biofuel
producing crops such as corn and soybean [4]. Apart from lipids,
microalgae can also produce a large range of valuable co-products, such
as fats, polyunsaturated fatty acids, oil, natural dyes, carbohydrates,
pigments, antioxidants, high-value bioactive compounds, and other fine
chemicals and the biomass can even be used as feed/fertilizer [2]. In
spite of its inherent potential, cost-effective and sustainable production
of microalgal biomass at commercial scale has to date been impeded by
various challenges, such as the requirements for large quantities of
water and nutrients.
Microalgae cultivation in open ponds require large amounts of water
(as high as 11–13 million L ha− 1 Year− 1), a major limiting source [5]. In
addition, microalgal growth requires large quantities of nutrients such
as nitrogen, phosphorus and trace elements. These nutrients increase the
operational cost of microalgae cultivation [6]. If microalgae can be

* Corresponding author. Algae R&D Centre, Murdoch University, Murdoch, Western Australia, 6150, Australia.
E-mail address: n.moheimani@murdoch.edu.au (N.R. Moheimani).
1
Equal authorship.

https://doi.org/10.1016/j.biombioe.2020.105945
Received 19 June 2020; Received in revised form 12 December 2020; Accepted 13 December 2020
Available online 21 December 2020
0961-9534/© 2020 Elsevier Ltd. All rights reserved.
C. Herold et al.
cultivated using the nutrients present in wastewater or waste sludge, the
production costs can be kept to a minimum. Previous literature has
demonstrated that anaerobically-digested piggery effluent (ADPE) con­
tains high concentrations of growth nutrients, especially nitrogen and
phosphorus, and some microalgal species have been successfully culti­
vated using ADPE [7–9]. Therefore, effluents could be a cost-effective
source of nutrients for microalgae production.
Microalgal photosynthesis is well documented to be one of the most
economical ways to sequester CO2 [10]. Microalgae could effectively
generate 1 kg of dry algal biomass by biofixing about 1.83 kg of carbon
dioxide (CO2) [1]. These days, biogas is commonly produced from
wastewater and used as a fuel to generate electricity and/or heat or, and
also contains approximately 25%–60% CO2 [11]. These gases need to be
first cleaned to achieve a methane content typically >90% [12] to be
used as vehicle fuel or if to be injected into a natural gas grid for supply
of domestic and industrial markets. As microalgae require significant
amounts of CO2 during photosynthesis, biogas could be used as a good
source of CO2 to supply the carbon needed for low-cost microalgal
biomass production [13]. This could help make microalgal biomass
production more sustainable, eco-friendly and cost-effective, with ef­
fluents used as the source of nitrogen and phosphorus, and CO2 from
biogas used as the carbon source.
However, the exploitation of waste agricultural effluents as a
nutrient source could be limited by the salinity of such effluents if
salinity levels become elevated, for example (Fig. 1): (1) if using mod­
erate salinity groundwater as a water source onsite; and (2) if treated
liquid effluent was being repeatedly recycled for in-shed flushing of
animal housing to reduce overall water use onsite, thereby causing a
progressive build-up of salt from evaporation in uncovered effluent
treatment ponds [14,15]. Previous studies on effluent-based microalgal
biomass production utilizing biogas as source of CO2 focused mainly on
freshwater species [16]; very few studies using marine microalgae
(Table 2) [17–19]. In a country like Australia where freshwater is a
limited commodity, cultivating microalgae using freshwater could be
unrealistic [20]. Therefore, marine microalgae is an attractive alterna­
tive, potentially providing similar benefits to freshwater microalgae, e.g.
high biomass productivity, high oil content and ability to tolerate high
nutrient content and CO2 concentrations (Table 2) [21]. Additionally, a
saltwater-based microalgae production system was reported to have a
90% reduction in freshwater requirements [22] and is expected to
Fig. 1. Proposed generic flow sheet showing integration of micro-algal biogas treatment system on-farm.
2
Biomass and Bioenergy 145 (2021) 105945
tolerate the salinity typical of many agricultural production effluents, if
used as cost-effective nutrient supply for the growth of microalgae.
Microalgal uptake of a large proportion of the CO2 from biogas was
found to be positively correlated to CO2 dissolution rate [23]. CO2
dissolution would be dependent on the biogas CO2 partial pressure and
pH of the growth medium. A higher partial pressure and higher pH
would encourage reactive CO2 mass transfer [23]. However, different
microalgal species would also have an optimum pH for biomass growth
and productivity [24]. Therefore, it is expected that an optimum pH
would exist that both encourages CO2 dissolution, and enables micro­
algae to utilize a higher percentage of CO2 supplied in biogas to produce
biomass.
Tetraselmis sp. has been found to endure high concentrations of CO2
and can be cultivated in wastewaters [25,26]. The novelty of this current
research is that it integrates wastewater treatment with biogas purifi­
cation using a saline microalga which has not been reported previously.
The microalgae can be used to treat saline wastewater which cannot be
accomplished with conventional treatment technologies and to remove
impurities from biogas in order to make it useable particularly for in­
dustrial purposes. Hence, this study tests the growth, biochemical con­
tent and maximum quantum yield while growing marine microalga
Tetraselmis suecica using synthetic biogas as a source of CO2, and ADPE
as a source of nutrients, at different operating pH conditions. The effect
of CH4 in biogas is also tested, to assess whether it is inhibitory to
microalgal cultivation of Tetraselmis sp.
2. Materials and methods
2.1. Microalgae and culture media
The marine microalga Tetraselmis suecica (CS187) used for this study
was obtained from the culture collection of CSIRO, Western Australia
(WA). T. suecica was cultivated at natural seawater conditions (35‰
salinity). The seawater was collected from Hillary’s Beach, WA and was
charcoal filtered before use [27]. The ADPE was obtained from an
anaerobic pond of a piggery located about 100 km north of Perth WA,
and was sand filtered prior to use [7]. The ADPE was analysed to
determine total nitrogen, phosphorus and trace metals (Table S1). The
culture medium for T. suecica was prepared using seawater and ADPE,
with seawater used as the base of the culture media and ADPE used as a
C. Herold et al.
nutrient source [7]. Before preparing the media, the total nitrogen
content of F media (as described by Guillard [28]) was determined.
Then, the total nitrogen component of F media was replaced with pig­
gery effluent, by adding the required amount of ADPE to seawater
(Tables S2 and S3). As a result, total nitrogen content in the culture
media (seawater + ADPE) was equivalent to that of F media. The media
was also enriched with vitamin solutions to align with F media
composition as described by Guillard [28].
2.2. Experimental setup
During the experiment, T. suecica was cultivated in 2 L Erlenmeyer
flasks with a working culture volume of 1.5 L. The culture was grown at
temperature 25 ± 1 ◦ C and a 12 h:12 h light/dark period was used. Light
irradiance was 175 ± 25 μmol photons m− 2 s− 1 and was measured using
a Li–185B quantum meter equipped with a PAR quantum sensor,
Li–190SB. This irradiance was based on T. suecica Pmax [29]. Cultures
were mixed using 40 mm magnetic stirrers set at 150 rpm. The cultures
were operated in a batch mode and each treatment had four individual
replicates (n = 4) with an experimental duration of 8 days.
A synthetic test gas was used with a volumetric composition of 40%
CO2 (BOC, Australia). This mimics typical biogas with a CO2 content of
40% available for a microalgal production process [16,30]. For safety,
the remainder gas (60% by volume) was high purity N2 in most of the
experiments, as a substitute for CH4 to eliminate the risk of forming
flammable gas mixtures. In a smaller subset of experiments, a different
synthetic gas mixture was used, comprised of 80% CH4 and 20% CO2
(BOC, Australia) to test potential impacts of dissolved CH4 on microalgal
growth performance, whilst also keeping the experimental conditions
well above the upper flammability limit for CH4 in pure oxygen [31].
Also, the experiments were performed in a fume hood to prevent any
possibility of CH4 accumulation.
The synthetic test gas flow rate was maintained at 100 mL h− 1 using
a gas flow meter (a, Fig. 2) to avoid the coalescence of gas bubbles
typically observed at higher gas flow rates [32]. An on-off solenoid valve
(k) was attached to the synthetic gas cylinder (j) to supply the gas based
on metered pH of the culture media (Fig. 2). A pH probe (d, Fig. 2) was
placed inside the culture flask to determine the pH of the culture media,
which was connected to a miniCHEM-pH Process Monitor (i) used to
control the culture pH to a nominated set-point. If the pH of the culture
media increased to above the pH set point, the controller switched on the
solenoid valve, allowing the test gas to flow to the culture flask. When
Fig. 2. Experimental setup: a – flow meter (mL h− 1), b – one way tube for culture sampling, c – one way tube for gas sampling, d – pH probe, e − gas diffuser, f –
magnetic stirrer, i – pH controller, j – pressurised gas cylinder containing synthetic test gas, k – solenoid valve.
3
Biomass and Bioenergy 145 (2021) 105945
the pH of the culture media decreased to below the pH set point by
dissolving CO2 from the test gas, the solenoid valve turned off the gas
flow. Four treatment pH set points of 6.5, 7.5, 8.5 and 9.5 were tested for
the synthetic gas with N2 and one pH set point of 7.5 was tested for the
synthetic gas with CH4. The pH trigger point was set at 0.3 above the
respective set point. An uncontrolled pH (negative control) was also
tested using only atmospheric air supplied continuously with no addi­
tional CO2. For this, an air pump was used to generate an atmospheric
air flow rate of 100 mL h− 1 through the control culture. As shown in
Fig. 2, there is one gas inlet tube (e), one gas sampling tube (c) and one
culture sampling tube (b). Both sampling tubes were sealed at the outer
end to prevent any gas ingress. The tube for gas sampling only collected
gas from the culture headspace and the culture sampling tube was
submerged into the medium to collect culture (Fig. 2).
2.3. Culture sampling
Microalgal culture samples were collected at 12:30 p.m. for each
analysis. The sampling was undertaken on days 1, 4, 6 and 8. The
collected samples were used to determine biomass yield (both dry
weight and organic weight), cell density, light-adapted maximum
quantum yield (Fqʹ/Fmʹ) and culture media alkalinity (See Analytical
Methods). The lipid, carbohydrate, chlorophyll a content, total nitrogen
and phosphorus were measured on days 1, 4 and 8 (See Analytical
Methods).
Samples for measuring microalgae biomass weight, lipid, carbohy­
drate and chlorophyll a content were filtered using Whatman 2.5 cm GF-
C filters. The filtered microalgae samples were then rinsed with isotonic
ammonium formate solution to remove residual salt [33]. Fresh filtered
samples were used to measure the biomass weight, while samples for
measuring lipid, carbohydrate and chlorophyll a content were stored at
− 80 ◦ C until analysed. Fresh microalgae culture samples were used to
measure cell density and Fqʹ/Fmʹ. Before measuring total nitrogen,
phosphorus content and alkalinity of the culture media, microalgal
biomass was removed from the culture media by centrifugation [34].
2.4. Photosynthetic measurements
The light-adapted maximum quantum yield (Fqʹ/Fmʹ) is the ratio
between variable and maximum quantum yields. Fqʹ shows the
maximum variable fluorescence in light-adapted state, which shows the
difference between Fmʹ and Foʹ (Fqʹ = Fmʹ – Foʹ), where Foʹ is the minimum
C. Herold et al.
fluorescence intensity with all reaction centres of PSII open in the light-
adapted phase and Fmʹ the maximum fluorescence intensity with all
reaction centres of PSII closed in the light-adapted phase [35]. On day 6
of each experiment, the Fqʹ/Fmʹ was measured diurnally, i.e. at 05:30 a.
m. (30 min before the light switched on), 06:30, 09:30, 12:30, 15:30 and
at 18:30 (30 min after the light switched off) [36]. In the subset of ex­
periments with synthetic gas containing CH4, biomass productivity and
effective quantum yield of PSII primary photochemistry (Fqʹ/Fmʹ) were
measured to evaluate the growth potential of the algal cells, with
biomass density determined daily, and Fqʹ/Fmʹ values also measured
daily before illumination (06:00) and at 30 min before the start of the
dark regime (17:30). Light-adapted maximum quantum yield measure­
ments were performed using a hand-held fluorometer AquaPen AP
100-C (Photon Systems Instruments, CZE) paired with the FluorPen 1.1
software. This instrument is equipped with a high-intensity LED-array,
which can emit red light (620 nm). During the measurement, a
high-pulse light (3000 μmol photon m− 2 s− 1) irradiates for less than a
second to stimulate the photosystems of the sample. The emitted fluo­
rescence from the sample is then recorded by the AquaPen.
2.5. CO2 capture efficiency of T. suecica cultures at different pH set points
Gas samples were taken from the culture headspace immediately
after each gas-switching off cycle on days 1, 4, and 8. The gas samples
were withdrawn using an air-tight Luer-Lock gas syringe and were
analysed using a gas chromatograph to determine the concentration of
CO2 in the effluent gas (Section 2.3.11). CO2 concentrations in the
influent and effluent gas were analysed using an Agilent 7820A gas
chromatograph (GC) with flame ionization detector (FID). A total sam­
ple volume of 100 μL was injected directly onto an Altech Econo-CapTM
ECTM-1000 column (30 m length × 0.25 mm internal diameter × 0.25
μm film thickness). The carrier gas was N2, set at a flow rate of 3 mL
min− 1. The oven temperature was programmed as follows: initial tem­
perature 70 ◦ C, increased at 5 ◦ C min− 1 to 100 ◦ C, held for 2.0 min,
increased at 70 ◦ C min− 1 to 250 ◦ C, held for 2.0 min. Injector and de­
tector were set at 250 and 300 ◦ C, respectively. The peak area of the FID
output signal was computed via integration using the EzChrome Elite
Compact Software© (V.3.3.2 SP2) [37]. The CO2 capture efficiency was
calculated as CO2 capture efficiency (%) = [(CO2 in influent – CO2 in
culture headspace gas sample)/CO2 in influent] x 100% [32].
2.6. Analytical methods
The dry weight (DW), organic weight (ash-free dry weight, AFDW)
and biomass productivity were determined using the method described
in Moheimani et al. [38]. Microalgal cell density was determined with
the Improved Neubauer cell counting chamber and light microscope.
Lipid extraction was performed using the Bligh and Dyer method as
modified by Kates and adapted by Mercz [39–41]. The lipid content was
expressed as % of organic weight. Lipid productivity was determined by
multiplying lipid content with biomass productivity (organic weight)
and expressed in units of milligram per liter of culture medium per day.
Total carbohydrate content was determined based on the method
described by Kochert and Ben-Amotz et al. and modified by Mercz
[41–43]. Carbohydrate content was expressed as % of organic weight.
Carbohydrate productivity was determined by multiplying carbohydrate
content with biomass productivity (organic weight), and the value was
expressed in units of milligram per liter of culture medium per day. The
method of Jeffrey and Humphrey was used to determine chlorophyll a
content [44] and was measured using a spectrophotometer at 664 nm
and 647 nm wavelengths. The whole extraction process was carried out
under dim light to prevent degradation of chlorophyll a pigment.
To determine alkalinity, total nitrogen, total phosphorus, iron and
COD content of the filtered culture media, a Multiparameter Photometer
HI 83099 (Hanna Instrument, USA) was used with various Hanna test
kits (Table S4) in accordance with manufacturer’s protocols. Magnesium
4
Biomass and Bioenergy 145 (2021) 105945
and calcium content in ADPE were determined by Spectroquant® Move
100 (Merck KGaA, Germany) using test kits (Table S4) in accordance
with manufacturer’s protocols. The pCO2 of the cultures was determined
from temperature, salinity, phosphate, total alkalinity and pH of the
medium using CO2sys software (v2.1) and the constants of Roy et al. for
seawater [45].
To determine the organic carbon content, at first the culture was
centrifuged for 15 min at 4000 rpm, and pellets dried at 60 ◦ C for 24 h.
Dry biomass was ground finely with a mortar and pestle and stored at
− 20 ◦ C in the freezer until analysis. 1.5–2 mg of ground biomass was
weighed and the organic carbon content was analysed using a Perki­
nElmer 2400 Series II CHNS/O-Analyser [9]. The results were expressed
in units of % of dry biomass.
2.7. Statistical analysis
One-way analysis of variance (ANOVA) and Tukey’s test were con­
ducted using SigmaPlot (version 14.0) to test significant differences
between treatments. For the CH4 tolerance experiment, a t-test was used
to compare the performance of T. suecica at pH 7.5 for the two test gases
(with N2, or with CH4). In each case, a significance threshold of 5% was
used. All measurements were expressed as calculated mean values for
replicates with standard errors (±S.E.). Pearson’s correlations were
performed to determine the direction of relationships between variables.
3. Results
The experiment tested the effect of different pH set points and
associated pCO2 on biomass, lipid and carbohydrate productivity,
chlorophyll a content, and light adapted maximum quantum yield. The
CO2 and nutrients consumption efficiency of T. suecica at different pH set
points were also investigated. Additionally, the effect of methane (CH4)
on the growth of T. suecica was tested.
3.1. Effect of pH and associated pCO2 on growth and light adapted
maximum quantum yield (Fqʹ/Fmʹ)
During each experiment, the exponential log phase biomass pro­
ductivities and cell densities were measured every second day
throughout the experiment (Fig. S1). An overall increase in biomass
productivity was observed with a decrease in the pH set point (Fig. 3).
The highest overall biomass productivity of 59.8 mg L− 1 d− 1 was found
at the pH set point of 7.5 (Fig. 3). Biomass productivity was not signif­
icantly different between the pH 6.5 and pH 7.5 set points (One Way
ANOVA, P > 0.05) (Fig. 3). However, biomass productivity at the pH 7.5
was significantly higher (One Way ANOVA, P < 0.05) than at the pH set
points of 8.5, 9.5 and at the uncontrolled pH (the control test), respec­
tively (Fig. 3). Pearson’s correlations showed that biomass productivity
of microalgae had a strong inverse correlation to pH (correlation coef­
ficient − 0.971, P = 0.029). No correlation was observed between cell
density and pH set points. Overall, the highest cell density was observed
at a pH 8.5 set point (17.4 × 105 cells mL− 1) and was 44% higher than
the cell density recorded at the pH 6.5 set point (One Way ANOVA, P <
0.05) (Fig. S2).
The lipid and carbohydrate productivities were determined on days 1
and 4 and on the final day (day 8) of the experiment (Fig. S3). Overall,
lipid and carbohydrate productivities appeared to mirror the decreasing
trend of biomass productivity with increasing pH set point values.
However, no significant differences were observed in lipid and carbo­
hydrate productivity at the various pH set points (One Way ANOVA, P >
0.05). Lipid content of the uncontrolled pH experiment (55.1% ± 4.2%
of AFDW) was 56% higher than at the 8.5 and 9.5 pH set points (Fig. S3).
Carbohydrate content was significantly higher at the pH set points of 6.5
and 7.5 (13.3 ± 0.5% of AFDW) as compared to the other pH set point
conditions and the uncontrolled pH test (One Way ANOVA, P < 0.05)
(Fig. S3).
C. Herold et al. Biomass and Bioenergy 145 (2021) 105945

Fig. 3. The effect of pH set point on the biomass productivity, pCO2, lipid and
carbohydrate productivity, chlorophyll a content and light adapted maximum
quantum yield (Fq’/Fm’) of T. suecica without CH4 in the test gas. Data pre­
sented are mean values ± S.E. (n = 4). The same letter above each point and Fig. 4. Measured maximum quantum yield (Fqʹ/Fmʹ) across an experimental
column indicate no significant difference (One Way ANOVA, P > 0.05). day for various pH set point conditions and the uncontrolled pH condition
(negative control), in all cases without CH4 in the test gas. The same letter
A gradual increase in chlorophyll a content was observed with below each point indicates no significant difference (One Way ANOVA, P
increasing pH, also supported by a Pearson’s correlation (correlation > 0.05).
coefficient 0.961, P = 0.039). The highest chlorophyll a content was
found at pH 9.5 (1.5% ± 0.06% of AFDW), which was significantly
higher than at the pH 6.5, 7.5 and uncontrolled pH treatments (One Way
ANOVA, P < 0.05) (Fig. 3). The overall light adapted maximum quan­
tum yield (Fqʹ/Fmʹ) was greater than 0.6 at each of the pH set points. The
Fqʹ/Fmʹ value was significantly higher at the pH 7.5 set point than under
the uncontrolled pH conditions (One Way ANOVA, P < 0.05) (Fig. 3).
The light adapted maximum quantum yield (Fqʹ/Fmʹ) of T. suecica was
also measured over the day (during both light and dark photoperiods)
(Fig. 4). With all pH set points, the Fqʹ/Fmʹ increased up to a maximum of
0.74 during light period and decreased to 0.68 during the dark period
(Fig. 4). However, at the uncontrolled pH conditions (negative control),
the Fqʹ/Fmʹ values were found to decrease gradually over the light period
and further decreased to a low of 0.55 during the dark period (Fig. 4).

3.2. CO2 capture efficiency of T. suecica cultures

The efficiency of marine microalga T. suecica to consume CO2 from
the feed gas was determined by algal growth at various pH set points.
Fig. 5 shows CO2 capture efficiency of the culture at the end of the ex­
periments for the four different pH set points. The average CO2 capture
Fig. 5. The effects of gradual increase in pH on total CO2 capture efficiency of
T. suecica cultures. Mean values are shown with error bars being ± S.E. (n = 4).
The same letter above each point indicates no significant difference (One Way
ANOVA, P > 0.05).

5
C. Herold et al.
efficiencies of T. suecica cultures varied between 83% and 94%, at pH
6.5 and 7.5, respectively. This maximum efficiency at pH 7.5 corre­
sponded to the highest biomass yield. However, no significant differ­
ences were observed in CO2 capture efficiencies between pH 7.5 and 8.5
(One Way ANOVA, P > 0.05), and similarly between pH 6.5 and 9.5
(Fig. 5). The biomass concentration of T. suecica cultures with CO2
addition was significantly higher (One Way ANOVA, P < 0.05) than the
control, in which CO2 was substituted with atmospheric air sparged via
an air pump. The average organic carbon content of dry biomass was
found to be 40.7 ± 1.96% at the different pH set points. The organic
carbon fixed in biomass ranged between 20.7 and 24.2 mg C L− 1 d− 1 at
pH 9.5 and 7.5, respectively and were not significantly different at the
different pH set point values (One Way ANOVA, P > 0.05, Table 1).
3.3. Response of pH, alkalinity and pCO2 at various pH set points
The pH stat system was most active during illuminated periods,
whereas pH remained largely unchanged during dark periods (Fig. S1).
There was no significant difference in measured overall alkalinity at
different pH set points (One Way ANOVA, P > 0.05 and Table 1). The
highest pCO2 (up to 9910 μ atm) was found at pH 6.5. The change in
alkalinity and pCO2 before 6:00 (at 6:00 the light switched on), during
the light period and after 18:00 (at 18:00 the light switched off), during
the dark period, was also measured (Table S5). No statistically signifi­
cant differences were found between the alkalinity values measured
during diurnal cycling for all the various pH set points (One Way
ANOVA, P > 0.05). Similarly, the values for pCO2 at pH set points 6.5
and 7.5 showed no significant differences over the diurnal cycle (One
Way ANOVA, P > 0.05). However, the 12:30 and 15:30 pCO2 values for
the 8.5 and 9.5 pH set points, were up to 99% less than the pCO2 values
recorded at 05:30 (One Way ANOVA, P < 0.05) (Table S5).
3.4. Nutrient uptake rates and COD
Nitrogen and phosphorus consumption rates were evaluated and are
summarized in Table 1. Nitrogen concentrations decreased by 92–99%
at the end of the experiment period for all conditions. The highest ni­
trogen uptake rate was at the pH 8.5 set point and was 50% higher than
at the pH 6.5 and 7.5 set points (One Way ANOVA, P < 0.05 and
Table 1). The phosphorus uptake rate appeared to vary between the
different pH set points, but not significantly (One Way ANOVA, P > 0.05
and Table 1).
The COD removal rate was also measured during cultivation, to
determine the concentration of oxidizable substrates present; results are
summarized in Table 1. The COD removal rate appeared to increase with
increasing pH, with the pH 8.5 set point showing the highest COD
removal rate at 152.4 mg L− 1 d− 1. However, COD removal rates were
not significantly different between the pH 7.5 and pH 8.5 set points (One
Table 1
Average (mean ± S.E., n = 4) overall alkalinity, organic carbon content, total COD and nitrogen and phosphorus removal rates at different pH limits and without CH4 in
the test gas. The same letter in each column indicate no significant difference (One Way ANOVA, P > 0.05).
CO2 Alkalinity [mg Carbon fixed in biomass [mg C
supply L− 1CaCO3] L− 1d− 1]
6.5 + 200.0 ± 47.5a 22.9 ± 1.58a
7.5 + 178.1 ± 31.2a 24.2 ± 0.76a
8.5 + 171.9 ± 33.2a 21.2 ± 1.49a
9.5 + 146.9 ± 33.4a 20.7 ± 1.15a
Negative – 153.1 ± 64.4a 13.4 ± 1.86b
control*
Overall alkalinity was calculated from values measured on days 1, 4, 6, 8. Carbon fixed in biomass was obtained as product of the overall organic carbon content and
biomass productivity. Removal rates were calculated as the ratio of the difference between the initial and final values, and the cultivation duration. TN means total
nitrogen; TP means total phosphorus. *Negative control was aerated with a constant supply of air and did not have a controlled pH.
6
Biomass and Bioenergy 145 (2021) 105945
Way ANOVA, P > 0.05) (Table 1).
3.5. The effect of CH4 on T. suecica growth performance
Fqʹ/Fmʹ value was observed to rapidly decrease initially after the
supply of test gas with CH4 on day 0 (data not shown), which may have
represented an acute response to new culture conditions. However,
across the whole experiment, CH4 in the test gas did not show a signif­
icant effect on the growth rate of T. suecica at the pH 7.5 set point (t-test
P > 0.05) (Fig. 6). Likewise, there were no significant differences (t-test,
P > 0.05) between Fqʹ/Fmʹ values measured between the tests with and
without CH4 in the test gas (Fig. 6). With test gas containing CH4, values
of PSII operating efficiency at 06:00 were similar to that at 17:30 on days
1, 2 and 3.
4. Discussion
4.1. Growth and productivity
The biomass productivity of T. suecica was found to be slightly higher
at a lower pH set point which could be influenced by high cell weight at
that pH (Fig. S2). Lipid and carbohydrate productivity of T. suecica was
found to follow the same trend as biomass productivity but did not differ
significantly between pH set points (Section 3.3). The lipid productivity
of T. suecica achieved in this study aligned well with the values 20.9 mg
L− 1 d− 1 [19] and 26.2 mg L− 1 d− 1 [18] reported for freshwater Chlorella
sp. and Scenedesmus sp., respectively. Lipid content was found to be
slightly higher at the pH 6.5 and 7.5 set points, when compared to the
carbohydrate content which was significantly higher at the pH 6.5–8.5
set points than at the pH 9.5 set point, and this result agreed with
findings of Sakarika and Kornaros; and Khalil et al. [46,47]. A lipid
content of 27% for Scenedesmus sp. was previously found while grown
with a gas mixture containing 40% CO2 and 60% CH4 [18] and a lipid
content of 28% for Chlorella sp. was reported in a biogas comprising 50%
CO2 and 50% CH4 [19]. In our study, the lipid content (28–38% ash-free
dry weight) of T. Suecica under the test pH conditions are higher than
literature values. However, the highest lipid content was achieved at
uncontrolled pH (negative control test, Section 3.3). According to the
findings of Pal et al. and Gu et al. under unfavourable conditions, many
algal species change their lipid biosynthetic pathways toward the for­
mation and accumulation of free fatty acids as the stored form of lipids
[48,49]. This may explain the increased lipid content recorded at the
uncontrolled pH treatment (negative control). An incremental increase
in chlorophyll a content was observed with an increase in pH. Ismaiel
et al. reported that at higher pH ranges, the amount of dissolved CO2 in
the media decreases significantly, resulting in the microalgae cells
experiencing some form of oxidative stress created by a carbon defi­
ciency, which in turn influences the ability of microalgae to produce
COD [mg L− 1] TN removal [mg TP removal [mg
L− 1d− 1] L− 1d− 1]
Day 1 Day 8
2173.3 ± 2396.7 ± 1.39 ± 0.58c 0.08 ± 0.03a
212.3ab 106.8ab
2020.0 ± 0.0a 1823.3 ± 1.51 ± 0.79bc 0.07 ± 0.01a
171.7b
3260.0 ± 0.0b 2193.3 ± 3.00 ± 0.53d 0.03 ± 0.00a
450.6ab
2450.0 ± 0.0ab 3180.0 ± 40.4a 2.19 ± 0.81a 0.03 ± 0.00a
2206.7 ± 3260.0 ± 2.41 ± 0.85ab 0.11 ± 0.02a
150.6ab 150.4a
C. Herold et al. Biomass and Bioenergy 145 (2021) 105945

Table 2
Comparisons between the present study and other relevant studies.
Microalgal species Light source, Biomass Nutrient Biogas CO2 CH4 CO2 CH4 content TN, TP Ref
and type of species irradiance and pH productivity source source content in content in removal in upgraded removal
(mg L− 1 d− 1) biogas biogas efficiency biogas (%v/ efficiency
(%) (%) (%) v) (%)

Chlorella sp., LEDs, 350 μmol 103 Biogas ADPL 26 71 86 92 84, 80 [10]
Freshwater m− 2 s− 1, 14 h:10 h effluent
photoperiod, pH
6.8
Chlorella vulgaris, LEDs, 100 μmol 165 Biogas Synthetic 45 55 80 92 61, 69 [11]
Freshwater m− 2 s− 1, 16:8 h effluent
photoperiod, pH
7.4
Scenedesmus LEDs, 100 μmol 177 Biogas Synthetic 45 55 80 93 63, 74 [11]
obliquus, m− 2 s− 1, 16:8 h effluent
Freshwater photoperiod, pH
7.2
Neochchloris LEDs, 100 μmol 149 Biogas Synthetic 45 55 75 91 55, 66 [11]
oleoabundans, m− 2 s− 1, 16:8 h effluent
Freshwater photoperiod, pH
7.3
Nannochloropsis Fluorescent lamps, 30 f/2 Synthetic 28 72 81 – – [13]
gaditana, 100 μmol m− 2 s− 1,
Marine pH 7.5–8.0
Chlorella sp., Fluorescent lamps, 78 Chu 13 Synthetic 50 50 69 – – [19]
Marine 3000 lux, 16:8 h
photoperiod, ph 6.8
Chlorella vulgaris, Fluorescent lamps, 0.014 (9.4 g ADV Synthetic 29.5 70 81 – 37, 75 [30]
Freshwater 104 μmol m− 2 s− 1, m− 2 d− 1)
16:8 h photoperiod
Chlorella sp., Florescent lamps, 322 f/2 Synthetic 20 70 86 91 – [32]
Marine 300 μmol m− 2 s− 1
Chlorella sp., LEDs, 300, 600, 900 3.6 Biogas slurry Synthetic 38 61 85 – 87, 92 [54]
Freshwater μmol m− 2 s− 1, 16:8,
14:10, 12:12 h
photoperiod, pH
6.4
Tetrasemis suecica, LEDs, 175 ± 25 59.8 (at pH ADPE Synthetic Up to 40 Up to 80 94.7 ± 0.18 – Up to 99, up This
Marine μmol m− 2 s− 1, 7.5) containing to 73 study
12:12 h modified F
photoperiod, pH media
6.5, 7.5, 8.5 and 9.5

ADPL: Anaerobic digestion plant, ADV Anaerobically digested vinasse, ADPE: Anaerobic digested piggery effluent.

Fig. 6. Biomass productivity (bars) and Fqʹ/Fmʹ (filled circle) of T. suecica
culture aerated with 80% CH4/20% CO2 and 60% N2/40% CO2 for a cultivation
period of 5 days. Cultures were set at pH = 7.5 using a pH stat system. Bars and
circles with same letter are not significantly different (data are average ± SE, n
= 4).
more chlorophyll a [50].
4.2. An assessment of stress
An Fqʹ/Fmʹ value over 0.6 is generally used to represent ‘healthy’
cultures while values under 0.6 typically suggests some form of stress
experienced by the algal cells [51]. In this study, cells grown at each pH
set point treatment condition showed Fqʹ/Fmʹ values of over 0.6 which
indicates that T. suecica was not subjected to any significant stress
(Section 3.3). The Fqʹ/Fmʹ values were also measured at different times
over the day and all four pH set point treatments showed that the
microalgae T. suecica was not stressed; not even at the pH 9.5 set point.
However, Fqʹ/Fmʹ values of uncontrolled pH (negative control) obtained
from diurnal cycle testing showed a declining trend over the light
period, and at the end of the light period it was as low as 0.55, indicating
stress experienced by the algal cells under these conditions. Hart et al.
showed that under stressed conditions, the growth of some marine
microalgae decreased due to a reduced photosynthetic rate [52]. In the
current study, this was reflected at the uncontrolled pH condition
(negative control, Section 3.3) by an overall lower biomass productivity.
Biogas-based microalgae cultivation systems also produce oxygen as
a photosynthetic and metabolic co-product that may intoxicate the
culture and inhibit biomass growth and CO2 uptake from biogas. In the
present study, the chance of oxygen intoxication was minimised by
increasing the amount of inorganic carbon, e.g. CO2, HCO−3 and CO2− 3
(up to 200 ± 47.5 mg L− 1) (Table 1). According to van Den Hende et al.,
the increase of CO2:O2 ratio by adding more inorganic carbon (e.g. by
adding more raw biogas) to the culture can be an effective way to
overcome the photosynthetic inhibition by oxygen evolved during
photosynthesis [53]. Additionally, high COD of the culture media also
would have contributed to removal of dissolved oxygen by biological
oxidation; thereby, further minimising the negative effects of oxygen
evolution on the T. suecica culture.

7
C. Herold et al.
4.3. Uptake of carbon dioxide and removal of nutrients
CO2 uptake efficiency from biogas would generally positively
correlate with microalgae growth [54]. The result of the present study
clearly indicates that the microalga T. suecica can grow efficiently on
40% CO2 (Table 2). During photosynthesis, the uptake of dissolved CO2
by microalgae increases the culture pH, because CO2 is removed from
the medium [55]. Hence, to maintain the pH set point in the experi­
ments, the flow of test gas was automatically turned on to dissolve
additional CO2 from the test gas into the culture medium. The process
rationale for a higher pH set point is to maintain a lower CO2 concen­
tration in the culture medium, thereby encouraging the dissolution of
CO2 [16].
pH plays a critical role during microalgal growth. In certain growth
systems, pH regulates the ionization and uptake of biochemical metab­
olites and chemical compounds [33]. Most Microalgal species have their
own optimal pH range and for maximized biomass production, optimal
pH is necessary [56]. The species Tetraselmis suecica is a well charac­
terized species, this characterization determined that biomass and lipid
productivities followed the following scenario pH 7.5 > pH 7 > pH 8 >
pH 6.5 [57]. It has also been reported that when the pH was reduced to
pH 6 for T. suecica, the algae cells started sticking to the glass walls of
photobioreactors and also began to clump; therefore, it would be diffi­
cult to maintain semi-continuous culture systems under these condi­
tions. On the other hand, increased pH was found to reduce this
clumping significantly [57]. Also low pH has been correlated with high
pCO2, and detrimental to algal growth. Therefore, as the main aim of
this study was to find a middle ground for concurrent high CO2 utili­
zation and biomass production, therefore pH values below 6 were not
tested.
Algal cells might generally prefer CO2 as inorganic carbon source,
with uptake of bicarbonate being energetically less favourable [56] and
uptake of carbonate being unlikely [35,55]. Increasing culture medium
pH from 6.5 to 8.5 will reduce dissolved CO2 and increase the proportion
of bicarbonate. Above pH 8.5, very minimal dissolved CO2 would be
available for algal uptake and the proportion of bicarbonate would
progressively be reduced relative to carbonate as pH is further increased.
In this study, as expected, pCO2 was found to be higher at the low pH set
point treatments. However, even though more CO2 is available at the
lower pH set point, a low pH can negatively affect algal growth [56].
This was indeed observed in our study. Where the pCO2 in the culture
medium was almost 100 times higher when T. suecica was grown at a pH
6.5 set point when compared to the pH 7.5 set point, yet at the pH 6.5 set
point algal growth was significantly lower (Section 3.3).
During the growth of T. suecica at different pH set points, nitrogen
uptake rate was highest at the pH 8.5 set point (Section 3.4), possibly
due to the higher cell number at this pH set point as compared to other
pH set points (Fig. S2). Lau et al. showed that cell densities were posi­
tively correlated with the nutrient uptake rate [58]. It is worthy to note
that ammonia volatilization could also be higher under alkaline condi­
tions as at the pH 8.5 and 9.5 set points [7–9]. Importantly, the ADPE
added to supply nutrients did not show any apparent inhibitory effects
on the growth of T. suecica, in line with the findings of Ward et al. [26];
however, it is important to note that this is among the pioneer studies
looking into the cultivation of marine T. suecica using ADPE. The ability
of microalgae (e.g. T. suecica) to consume nutrients from digestate whilst
sequestering CO2 from biogas means that systems can be designed for
efficient microalgal production. Specifically, microalgal plants can be
co-located with biogas facilities to utilize the CO2 in biogas as a source of
carbon for effective biomass production, resulting in the reduction of
raw material costs associated with carbon capture, storage and trans­
portation unlike other carbon capture measures such as absorption by
organic solvents [16,59].
8
Biomass and Bioenergy 145 (2021) 105945
4.4. CH4 tolerance of T. suecica
Biomass productivity achieved in the culture in the present study
sparged with 80% CH4/20% CO2 at the pH 7.5 set point was 1.5 times
higher than the value reported for Arthrospira platensis cultures (41 mg
L− 1 d− 1) sparged with biogas composed of 70.5–76.0% CH4 and
13.2–19.5% CO2 [60]. Similarly, Kao et al. [32] cultured Chlorella sp.
MB-9 in a digestate sparged with 80% CH4 and 20% CO2, and reported a
high biomass productivity of 0.24 g L− 1 d− 1. These results show that the
performance of microalgae in CH4 rich biogas is species-specific. In our
work, T. suecica photosynthetic activity was uninhibited by the CH4
exposure with the Fqʹ/Fmʹ as high as 0.69 (Section 3.5). The data showed
that T. suecica cultured under direct intermittent sparging of biogas with
high CH4 content can efficiently grow, consuming CO2 from the biogas.
This is in agreement with a general suggestion that many microalgal
strains are tolerant of high levels of CH4 found in biogas [16].
4.5. Implications
There would in general be a preferred operating pH to encourage
dissolution of CO2 from biogas to supply CO2 for growth and to promote
microalgal growth. The results from the present study suggest that for
the marine microalga T. suecica a pH set-point of 7.5 is preferred to
maintain high biomass productivity (Section 3.3) and showing no in­
hibition by dissolved CH4 (Section 3.5). The CO2 removal efficiency was
found to be 94% at the pH 7.5 set point (Section 3.2).
Apart from high CO2 and CH4 in biogas, H2S is a trace gas also found
in biogas that is known to inhibit microalgal growth at high concen­
trations [16]. However, with the oxygen evolution and the concentra­
tion of H2S limited to an extent by dissolution from the gas phase, sulfide
is expected to be chemically oxidised to sulfate, so that biogas-based
microalgae cultivation system have generally shown resilience to H2S
concentrations in biogas up to 200 ppm or even higher [61]. Further,
because the applications focus of the present study will likely include
upfront biological oxidation or similar H2S removal treatment of biogas
prior to the algal treatment step [62], the anticipated effects of H2S
would be minimal, and were therefore not explored in the present study.
As freshwater is a major limiting resource in microalgae cultivation,
exploitation of marine microalgae T. suecica will likely significantly
reduce the requirement and use of freshwater. In addition, this study
used ADPE as a nutrient source, which not only reduces the cost related
to fertilizer inputs but also, converts a waste into an asset. According to
the proposed design, biomass production via microalgal growth is free of
fertilizers and CO2 costs, which typically make up approximately 76.2%
of the total raw material cost for producing microalgal biomass [63];
therefore, if applied at large scale it could greatly reduce the biomass
production cost. In addition, a biogas-based microalgae cultivation
system also offers upgraded biogas purity and provides treatment of
wastewaters.
Based on the results, the proposed process can provide up to 61 mg
L− 1 d− 1 of low-cost biomass that may be suitable as a commercial
aquaculture feed or as feed for livestock. Whilst the market price would
dictate cost feasibility, T. suecica biomass in general could have a sig­
nificant commercial value, especially if derived with minimal raw ma­
terial costs using biogas and wastewater as nutrient. The developed
process can be implemented to any climatic region that can support algal
cultivation. Obviously the environmental and climatic conditions will
affect the growth and productivities [64]. The process described in this
study can be applied to other salt-tolerant microalgal species; however, a
wider spectrum of microalgae would need to be tested considering their
physiological responses are species-specific [18,19].
5. Conclusion
To date, almost all studies on the treatment of anaerobic digestates
using microalgae, focused on freshwater microalgal species such as
C. Herold et al.
Chlorella and Scenedesmus. To the best of our knowledge, this is the first
study examining the potential use of marine microalgae to treat such
effluents. The potential of using biogas as a source of carbon for marine
algal cultivation can not only strip CO2 from biogas but also lead to
significantly higher biomass productivity. This study demonstrates that
the marine microalga T. suecica can be grown successfully utilizing CO2
from biogas and nutrients (nitrogen and phosphorus) from anaerobic
digested piggery effluent (ADPE). An efficient CO2 uptake of 94% was
recorded when this alga was grown at 7.5 pH treatment set point. The
process operation at pH 7.5 also supported high microalgal biomass
productivities (59.8 mg L-1 d-1) through photosynthetic CO2 assimila­
tion with higher lipid and carbohydrate productivities. Most impor­
tantly, no inhibitory effects from CH4 were observed on the growth of
T. suecica. This process harnesses a waste (ADPE) not only as an asset but
also uses an impurity (CO2) in biogas to produce potentially valuable
algal biomass which can generate significant revenue for farmers. We
have no doubts that further studies, especially at pilot scale, could
further validate the suitability of biogas-based microalgae cultivation
systems.
Acknowledgements
This work was funded by the CRC for High Integrity Australian Pork
(Pork CRC) via the Australasian Pork Research Institute Limited (APRIL)
Project 4C-119. We are thankful to Dr. Ralf Cord-Ruwisch, Senior
Lecturer and Dr. Md Iqbal Hossain, Research Associate, School of En­
gineering and Information Technology, Murdoch University, Western
Australia, for their assistance with the gas chromatography analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biombioe.2020.105945.
Authors contributions
Clemens Herold: Investigation, Writing-Original draft. Tasneema
Ishika: Methodology, Formal Analysis, Investigation, Writing-Original
draft, Writing-Review and Editing, Supervision. Emeka G. Nwoba: Meth­
odology, Investigation, Writing-Original draft, Writing-Review and Editing.
Stephan Tait: Conceptualization, Funding acquisition, Writing-Review
and Editing. Andrew Ward: Writing-Review and Editing. Navid R Mohei­
mani: Conceptualization, Funding acquisition, Methodology, Writing-
Review and Editing, Supervision.
References
[1] L. Brennan, P. Owende, Biofuels from microalgae—a review of technologies for
production, processing, and extractions of biofuels and co-products, Renew.
Sustain. Energy Rev. 14 (2) (2010) 557–577.
[2] T.M. Mata, A.A. Martins, N.S. Caetano, Microalgae for biodiesel production and
other applications: a review, Renew. Sustain. Energy Rev. 14 (1) (2010) 217–232.
[3] E.W. Becker, Microalgae: Biotechnology and Microbiology, Cambridge University
Press, 1994.
[4] J. Sheehan, T. Dunahay, J. Benemann, P. Roessler, A Look Back at the U.S.
Department of Energy’s Aquatic Species Program—Biodiesel from Algae, National
Renewable Energy Lab, USA, 1998, p. 325.
[5] S. Chinnasamy, A. Bhatnagar, R.W. Hunt, K.C. Das, Microalgae cultivation in a
wastewater dominated by carpet mill effluents for biofuel applications, Bioresour.
Technol. 101 (9) (2010) 3097–3105.
[6] J.K. Pittman, A.P. Dean, O. Osundeko, The potential of sustainable algal biofuel
production using wastewater resources, Bioresour. Technol. 102 (1) (2011) 17–25.
[7] E.G. Nwoba, J.M. Ayre, N.R. Moheimani, B.E. Ubi, J.C. Ogbonna, Growth
comparison of microalgae in tubular photobioreactor and open pond for treating
anaerobic digestion piggery effluent, Algal Research 17 (2016) 268–276.
[8] E.G. Nwoba, N.R. Moheimani, B.E. Ubi, J.C. Ogbonna, A. Vadiveloo, J.R. Pluske, J.
M. Huisman, Macroalgae Culture to Treat Anaerobic Digestion Piggery Effluent
(ADPE), Bioresource Technology, 2016.
[9] E.G. Nwoba, B.S. Mickan, N.R. Moheimani, Chlorella sp. growth under batch and
fed-batch conditions with effluent recycling when treating the effluent of food
waste anaerobic digestate, J. Appl. Phycol. 31 (6) (2019) 3545–3556.
9
Biomass and Bioenergy 145 (2021) 105945
[10] C. Yan, Z. Zheng, Performance of photoperiod and light intensity on biogas
upgrade and biogas effluent nutrient reduction by the microalgae Chlorella sp,
Bioresour. Technol. 139 (2013) 292–299.
[11] S. Sun, Z. Ge, Y. Zhao, C. Hu, H. Zhang, L. Ping, Performance of CO2 concentrations
on nutrient removal and biogas upgrading by integrating microalgal strains
cultivation with activated sludge, Energy 97 (2016) 229–237.
[12] J. Moran, S. Koonphapdeelert, A. Bunkham, W. Rojanaratanangkule, P.
Aggarangsi, Development of a national standard for a biogas grid in Thailand,
Journal of Enviromental Science 7.
[13] L. Meier, R. Pérez, L. Azócar, M. Rivas, D. Jeison, Photosynthetic CO2 uptake by
microalgae: an attractive tool for biogas upgrading, Biomass Bioenergy 73 (2015)
102–109.
[14] P. Bohutskyi, S. Chow, B. Ketter, C.F. Shek, D. Yacar, Y. Tang, M. Zivojnovich, M.
J. Betenbaugh, E.J. Bouwer, Phytoremediation of agriculture runoff by filamentous
algae poly-culture for biomethane production, and nutrient recovery for secondary
cultivation of lipid generating microalgae, Bioresour. Technol. 222 (2016)
294–308.
[15] P. Bohutskyi, S. Chow, B. Ketter, M.J. Betenbaugh, E.J. Bouwer, Prospects for
methane production and nutrient recycling from lipid extracted residues and whole
Nannochloropsis salina using anaerobic digestion, Appl. Energy 154 (2015)
718–731.
[16] D. Nagarajan, D.-J. Lee, J.-S. Chang, Integration of Anaerobic Digestion and
Microalgal Cultivation for Digestate Bioremediation and Biogas Upgrading,
Bioresource technology, 2019, p. 121804.
[17] M. Erkelens, A.J. Ward, A.S. Ball, D.M. Lewis, Microalgae digestate effluent as a
growth medium for Tetraselmis sp. in the production of biofuels, Bioresour.
Technol. 167 (2014) 81–86.
[18] S. Srinuanpan, B. Cheirsilp, W. Kitcha, P. Prasertsan, Strategies to improve
methane content in biogas by cultivation of oleaginous microalgae and the
evaluation of fuel properties of the microalgal lipids, Renew. Energy 113 (2017)
1229–1241.
[19] W. Tongprawhan, S. Srinuanpan, B. Cheirsilp, Biocapture of CO2 from biogas by
oleaginous microalgae for improving methane content and simultaneously
producing lipid, Bioresour. Technol. 170 (2014) 90–99.
[20] T. Ishika, N.R. Moheimani, P.A. Bahri, Sustainable saline microalgae co-cultivation
for biofuel production: a critical review, Renew. Sustain. Energy Rev. 78 (2017)
356–368.
[21] L. Jiang, S. Luo, X. Fan, Z. Yang, R. Guo, Biomass and lipid production of marine
microalgae using municipal wastewater and high concentration of CO2, Appl.
Energy 88 (10) (2011) 3336–3341.
[22] J. Yang, M. Xu, X. Zhang, Q. Hu, M. Sommerfeld, Y. Chen, Life-cycle analysis on
biodiesel production from microalgae: water footprint and nutrients balance,
Bioresour. Technol. 102 (1) (2011) 159–165.
[23] T.M. Sobczuk, F.G. Camacho, F.C. Rubio, F.G.A. Fernández, E.M. Grima, Carbon
dioxide uptake efficiency by outdoor microalgal cultures in tubular airlift
photobioreactors, Biotechnol. Bioeng. 67 (4) (2000) 465–475.
[24] K. Ying, W.B. Zimmerman, D.J. Gilmour, Effects of CO and pH on growth of the
microalga Dunaliella salina, J. Microb. Biochem. Technol. 6 (2014) 167–173.
[25] M. Olaizola, E.O. Duerr, D.W. Freeman, Effect of CO2 enhancement in an outdoor
algal production system usingTetraselmis, J. Appl. Phycol. 3 (4) (1991) 363–366.
[26] A. Ward, D. Lewis, F. Green, Anaerobic digestion of algae biomass: a review, Algal
Research 5 (2014) 204–214.
[27] T. Ishika, N.R. Moheimani, P.A. Bahri, D.W. Laird, S. Blair, D. Parlevliet, Halo-
adapted microalgae for fucoxanthin production: effect of incremental increase in
salinity, Algal Research 28 (2017) 66–73.
[28] R.R.L. Guillard, Culture of Phytoplankton for Feeding Marine Invertebrates,
Culture of Marine Invertebrate Animals, Springer, Boston, MA1975, pp. 29-60.
[29] F. Abiusi, G. Sampietro, G. Marturano, N. Biondi, L. Rodolfi, M. D’Ottavio, M.
R. Tredici, Growth, photosynthetic efficiency, and biochemical composition of
Tetraselmis suecica F&M-M33 grown with LEDs of different colors, Biotechnol.
Bioeng. 111 (5) (2013) 956–964.
[30] M.L. Serejo, E. Posadas, M.A. Boncz, S. Blanco, P. García-Encina, R. Muñoz,
Influence of biogas flow rate on biomass composition during the optimization of
biogas upgrading in microalgal-bacterial processes, Environ. Sci. Technol. 49 (5)
(2015) 3228–3236.
[31] C.-C. Chen, A study on estimating flammability limits in oxygen, Ind. Eng. Chem.
Res. 50 (17) (2011) 10283–10291.
[32] C.-Y. Kao, S.-Y. Chiu, T.-T. Huang, L. Dai, L.-K. Hsu, C.-S. Lin, Ability of a mutant
strain of the microalga Chlorella sp. to capture carbon dioxide for biogas upgrading,
Appl. Energy 93 (2012) 176–183.
[33] S. Fon Sing, Strain Selection and Outdoor Cultivation of Halophilic Microalgae
with Potential for Large-Scale Biodiesel Production, PhD Thesis, Murdoch
University, Western Australia, 2010, p. 221.
[34] J.M. Ayre, N.R. Moheimani, M.A. Borowitzka, Growth of microalgae on undiluted
anaerobic digestate of piggery effluent with high ammonium concentrations, Algal
Research 24 (2017) 218–226.
[35] J. Cosgrove, M.A. Borowitzka, Chlorophyll Fluorescence Terminology: an
Introduction, Chlorophyll a Fluorescence in Aquatic Sciences: Methods and
Applications, Springer, Dordrecht2010, pp. 1-17.
[36] E.G. Nwoba, D.A. Parlevliet, D.W. Laird, A. Vadiveloo, K. Alameh, N.
R. Moheimani, Can solar control infrared blocking films be used to replace
evaporative cooling for growth of Nannochloropsis sp. in plate photobioreactors?
Algal Research 39 (2019) 101441.
[37] M.I. Hossain, A. Paparini, R. Cord-Ruwisch, Direct oxygen uptake from air by novel
glycogen accumulating organism dominated biofilm minimizes excess sludge
production, Sci. Total Environ. 640–641 (2018) 80–88.
C. Herold et al.
[38] N.R. Moheimani, M.A. Borowitzka, A. Isdepsky, S.F. Sing, Standard Methods for
Measuring Growth of Algae and Their Composition, Algae for biofuels and energy,
Springer2013, pp. 265-284.
[39] E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and purification,
Can. J. Biochem. Physiol. 37 (1) (1959) 911–917.
[40] M. Kates, B. Volcani, Lipid components of diatoms, Biochim. Biophys. Acta Lipids
Lipid. Metabol. 116 (2) (1966) 264–278.
[41] T.I. Mercz, A Study of High Lipid Yielding Microalgae with Potential for Large-
Scale Production of Lipids and Polyunsaturated Fatty Acids, PhD Thesis, Murdoch
University, Westerm Australia, 1994, p. 278.
[42] G. Kochert, Carbohydrate determination by the phenol-sulfuric acid method,
Handbook of phycological methods, Phycological and biochemical methods 95
(1978).
[43] A. Ben-Amotz, T.G. Tornabene, W.H. Thomas, Chemical profile of selected species
of microalgae with emphasis on Lipids 1, J. Phycol. 21(1) 72-81.
[44] S.W. Jeffrey, G.F. Humphrey, New spectrophotometric equations for determining
chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton,
Biochem. Physiol. Pflanz. (BPP) 167 (2) (1975) 191–194.
[45] R.N. Roy, L.N. Roy, K.M. Vogel, C. Porter-Moore, T. Pearson, C.E. Good, F.
J. Millero, D.M. Campbell, The dissociation constants of carbonic acid in seawater
at salinities 5 to 45 and temperatures 0 to 45 C, Mar. Chem. 44 (2–4) (1993)
249–267.
[46] M. Sakarika, M. Kornaros, Effect of pH on growth and lipid accumulation kinetics
of the microalga Chlorella vulgaris grown heterotrophically under sulfur
limitation, Bioresour. Technol. 219 (2016) 694–701.
[47] Z.I. Khalil, M.M. Asker, S. El-Sayed, I.A. Kobbia, Effect of pH on growth and
biochemical responses of Dunaliella bardawil and Chlorella ellipsoidea, World J.
Microbiol. Biotechnol. 26 (7) (2010) 1225–1231.
[48] D. Pal, I. Khozin-Goldberg, Z. Cohen, S. Boussiba, The effect of light, salinity, and
nitrogen availability on lipid production by Nannochloropsis sp, Appl. Microbiol.
Biotechnol. 90 (4) (2011) 1429–1441.
[49] N. Gu, Q. Lin, G. Li, Y. Tan, L. Huang, J. Lin, Effect of salinity on growth,
biochemical composition, and lipid productivity of Nannochloropsis oculata CS 179,
Eng. Life Sci. 12 (6) (2012) 631–637.
[50] M.M.S. Ismaiel, Y.M. El-Ayouty, M. Piercey-Normore, Role of pH on antioxidants
production by Spirulina (Arthrospira) platensis, Braz. J. Microbiol. 47 (2) (2016)
298–304.
[51] V.K. Bhola, F.M. Swalaha, M. Nasr, S. Kumari, F. Bux, Physiological responses of
carbon-sequestering microalgae to elevated carbon regimes, Eur. J. Phycol. 51 (4)
(2016) 401–412.
10
Biomass and Bioenergy 145 (2021) 105945
[52] B.T. Hart, P. Bailey, R. Edwards, K. Hortle, K. James, A. McMahon, C. Meredith,
K. Swadling, A review of the salt sensitivity of the Australian freshwater biota,
Hydrobiologia 210 (1–2) (1991) 105–144.
[53] S. Van Den Hende, H. Vervaeren, H. Saveyn, G. Maes, N. Boon, Microalgal bacterial
floc properties are improved by a balanced inorganic/organic carbon ratio,
Biotechnol. Bioeng. 108 (3) (2011) 549–558.
[54] C. Yan, L. Zhu, Y. Wang, Photosynthetic CO2 uptake by microalgae for biogas
upgrading and simultaneously biogas slurry decontamination by using of
microalgae photobioreactor under various light wavelengths, light intensities, and
photoperiods, Appl. Energy 178 (2016) 9–18.
[55] S.C. Doney, V.J. Fabry, R.A. Feely, J.A. Kleypas, Ocean acidification: the other CO2
problem, Annual Review of Marine Science 1 (2009) 169–192.
[56] B. Zhao, Y. Su, Process effect of microalgal-carbon dioxide fixation and biomass
production: a review, Renew. Sustain. Energy Rev. 31 (2014) 121–132.
[57] N.R. Moheimani, Inorganic carbon and pH effect on growth and lipid productivity
of Tetraselmis suecica and Chlorella sp (Chlorophyta) grown outdoors in bag
photobioreactors, J. Appl. Phycol. 25 (2) (2013) 387–398.
[58] P.S. Lau, N.F.Y. Tam, Y.S. Wong, Effect of algal density on nutrient removal from
primary settled wastewater, Environ. Pollut. 89 (1) (1995) 59–66.
[59] E.G. Nwoba, D.A. Parlevliet, D.W. Laird, K. Alameh, N.R. Moheimani, Light
management technologies for increasing algal photobioreactor efficiency, Algal
Research 39 (2019) 101433.
[60] A. Converti, R. Oliveira, B. Torres, A. Lodi, M. Zilli, Biogas production and
valorization by means of a two-step biological process, Bioresour. Technol. 100
(23) (2009) 5771–5776.
[61] L. Meier, D. Stará, J. Bartacek, D. Jeison, Removal of H2S by a Continuous
Microalgae-Based Photosynthetic Biogas Upgrading Process, Process Safety and
Environmental Protection, 2018.
[62] A. Skerman, S. Heubeck, D. Batstone, S. Tait, On-farm trials of practical options for
hydrogen sulphide removal from piggery biogas, Process Saf. Environ. Protect. 117
(2018) 675–683.
[63] E.M. Grima, E.-H. Belarbi, F.A. Fernández, A.R. Medina, Y. Chisti, Recovery of
microalgal biomass and metabolites: process options and economics, Biotechnol.
Adv. 20 (7–8) (2003) 491–515.
[64] M.A. Borowitzka, Algal physiology and large-scale outdoor cultures of microalgae,
in: M. Borowitzka, J. Beardall, J. Raven (Eds.), The Physiology of Microalgae,
Springer, Cham, Switzerland, 2016, pp. 601–652.