Journal Pre-proofs

Towards a circular economy: A novel microalgal two-step growth approach to

treat excess nutrients from digestate and to produce biomass for animal feed

Claudio Fuentes-Grünewald, José Ignacio Gayo-Peláez, Vanessa Ndovela,

Eleanor Wood, Rahul Vijay Kapoore, Carole Anne Llewellyn

PII: S0960-8524(20)31623-0
DOI: https://doi.org/10.1016/j.biortech.2020.124349
Reference: BITE 124349

To appear in: Bioresource Technology

Received Date: 8 September 2020

Revised Date: 26 October 2020
Accepted Date: 27 October 2020

Please cite this article as: Fuentes-Grünewald, C., Ignacio Gayo-Peláez, J., Ndovela, V., Wood, E., Vijay
Kapoore, R., Anne Llewellyn, C., Towards a circular economy: A novel microalgal two-step growth approach to
treat excess nutrients from digestate and to produce biomass for animal feed, Bioresource Technology (2020),
doi: https://doi.org/10.1016/j.biortech.2020.124349

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Towards a circular economy: A novel microalgal two-step growth approach to treat

excess nutrients from digestate and to produce biomass for animal feed

Claudio Fuentes-Grünewald*, José Ignacio Gayo-Peláez, Vanessa Ndovela, Eleanor Wood1,

Rahul Vijay Kapoore and Carole Anne Llewellyn

College of Science, Bioscience Department, Swansea University, Singleton Park, SA2 8PP,

Swansea, UK.

1 Present address: SAMS, Scottish Marine Institute, Oban, Argyll, PA37 1QA

*Corresponding author:

e-mail address: c.fuentesgrunewald@swansea.ac.uk

Postal address: College of Science, Bioscience Department, Swansea University, Singleton

Park, SA2 8PP, Swansea, UK.

Abstract

Implementing a circular economy aimed at reusing resources is becoming increasingly

important for industry. Microalgae fit within a circular economy by being able to

bioremediate nutrient waste and as a source of biomass for several commercial applications.

Here, we report a novel validation of a circular economy concept using microalgae at a

relevant industrial scale with a new two-phase process. During the first phase biomass was

grown autotrophically, biomass was then concentrated using membrane technology for the

second phase where mixotrophic conditions were applied to boost growth further. Microalgae

cultures were able to grow (13.8 g/L), uptake and bioremediate nutrients (Nitrogen > 134

mg/L/day) from an anaerobic digestion side-stream (digestate), obtaining high quality

1
microalgae biomass (> 45% protein content) suitable for use as animal feed, closing the

circular economy loop for industrial applications.

Keywords: Circular economy, Microalgae, Digestate, Membrane filtration

Highlights

 A new two-phase microalgal growth process was developed

 Microalgae cultures were able to grow at industrial scale and bioremediate nutrients

 High quality microalgae biomass was obtained suitable for use as animal feed

1. Introduction

A circular economy where resources are kept in use for as long as possible, is a concept that

has been gaining attraction since the early 1970’s. It has been successfully applied on a small

scale since the 1990’s and has recently become practical in several industries. The concept

aims to close loops in industrial activities and minimise waste released into ecosystems. The

approach also aims to reduce greenhouse gas emissions, decrease nutrient discharge that

causes eutrophication to water bodies, and help to create sustainable jobs (Stahel., 2016;

Stiles et al., 2018). Anaerobic Digestion (AD) industrial facilities are the main technology

used in the EU and the UK to treat organic waste. There are more than 17,000 AD plants in

operation in the EU, the main economic driver being the production of electricity from biogas

which has an installing capacity of over 10,500 MW (EBA, 2018). Digestate is a side-stream

of AD plants, with nearly 10 million tons of excess digestate produced. Currently, microalgal

biotechnology is in its youth, but it is increasing its industrial prevalence in a worldwide

context, with applications in industrial fields ranging from pharmaceuticals and

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cosmeceutical to animal feed (e.g. aquaculture) (Khanra et al., 2018; Perez-Garcia & Bashan,

2015; Vaz et al., 2016). The use of microalgae to solve environmental issues, such as

wastewater treatment, eutrophication or treatment of agroindustry by-products (which are one

of the largest sources of wastes produced in the world), has only been demonstrated at a lab

or small scale thus far (Aiba & Ogawa, 1981; Mayhead et al., 2018; Melo et al., 2018;

Savchenko et al., 2017; Xu et al., 2017). Microalgae can grow and produce biomass using

different culture systems, growth strategies and in different locations such as high latitudes

(Fuentes-Grunewald et al., 2015; Savchenko et al., 2017). There are three different growth

modes: autotrophy, heterotrophy and mixotrophy. Autotrophic cultures in closed systems are

the most common and these organisms use light as an energy source, CO2 as a carbon source

and nutrients (mainly nitrogen (N) and phosphorous (P)) to grow (Hwang et al., 2014; Perez-

Garcia & Bashan, 2015). Heterotrophic cultures grow in darkness and with a regular

exogenous carbon source and can achieve high densities of microalgae. Mixotrophic

cultivation is the growth mode where microalgae simultaneously use inorganic CO2 and

organic carbon sources in the presence of light, to increase the biomass over a short period

(Chen et al., 2011).

A combination of two different growth modes (autotrophic-mixotrophic growth) has not been

demonstrated yet in microalgae biotechnology at pilot/industrial scale (Monshupanee et al.,

2016). To apply this new growth approach, the main obstacle is the potential risk of bacterial

contamination during the mixotrophic step due to the high concentration of the organic

carbon source. To minimise the risk and avoid cultures crashing, a non-cell destructive, low

energy consuming method such as membrane technology can be applied (Gerardo et al.,

2014). Membrane technology is a well-established method for water

desalination/purification. In recent years, this method has been applied in microalgae cultures

at different scales, it can be used in the upstream process for water filtration and nutrient

3
preparation, and in the downstream process for biomass dewatering and metabolite

fractionation (Fuentes-Grunewald et al., 2015; Garcia-Lopez et al., 2020; Silkina et al.,

2019).

Here, we report for the first time, a successful validation of the circular economy concept

using microalgae biotechnology at a relevant scale. The first aim was to uptake excess N & P

from digestate using autotrophic growth of microalgae. The second aim was to use membrane

technology to concentrate the autotrophic biomass and use this in a second mixotrophic

growth step, to improve the quality and quantity of biomass produced. The third aim was to

characterise the microalgae biomass to assess suitability for use as animal feed and to confirm

the technological feasibility of the circular economy concept.

2. Material and methods

2.1 General experimental procedures

Two microalgae strains at different scales (lab: 250 mL to 80 L, pilot: 800 L and industrial:

5000 L) were used to validate the circular economy concept in the microalgae biotechnology

field for the first time. The main goal was to use raw Nutrient Rich Digestate (NRD) from an

AD facility that treats food waste, and that had been processed through membrane

microfiltration. To overcome issues regarding turbidity and high ammonium concentration in

the NRD, Liquid Pure Filtered Digestate (LPFD) was obtained via microfiltration process.

The fate of the sludge digestate obtained from the retentate after microfiltration was spread

onto the land as it was not pre-treatmented. Typically, 10 mᶾ of digestate is spread per acre.

The detailed composition of the sludge digestate obtained after membrane microfiltration is

found in (Fernandes et al., 2020). The microfiltered permeates or LPFD rich in nitrogen (as

ammonium form), phosphate and all trace elements were used for the autotrophic and

mixotrophic growth of the target microalgae.

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The microalgae were acclimatised to LPFD using several dilution trials at indoor lab scale

conditions. Once the optimal LPFD dilution was obtained (2.5% LPFD and 97.5% tap water),

the same dilution was used at the pilot and industrial scales. To apply the new two-step

growth process where the autotrophic growth (used for N & P bioremediation purposes) is

combined with mixotrophic (organic carbon source) growth, a non-disruptive method was

used to preserve the microalgae cells for further growth. Membrane technology and

specifically microfiltration (0.2 μm) was used to concentrate the biomass from the

autotrophic reactor, once concentrated the microalgae biomass was inoculated into the

mixotrophic reactor to produce high microalgae biomass concentration in a short period with

an optimal macromolecular profile for further use as animal feed.

2.2 Strains, medium and pre-cultivation conditions (lab conditions)

The microalgae obtained from Culture Collection of Algae and Protozoa were Chlorella

vulgaris (CCAP 211/11R) and Scenedesmus obliquus (CCAP 276/6A). Both species were

conditioned in indoor cultures at the Centre for Sustainable Aquatic Research (CSAR),

Swansea University (SU), Wales, United Kingdom. Non-axenic cultures of this species were

scaled up (using autoclaved tap water for 20 min at 121°C) from 250 mL to 1 L flasks using

standard F2 commercial medium (Cell-hi F2P™, Varicon Aqua). The flask cultures were

grown with approximately 150 μmol m2 s-1 light irradiance by using cool white fluorescent

tubes perpendicular to the culture, with a light/dark cycle of 18:6 h, light irradiance was

recorded using a light meter (Walz Model US-SQS/L). The average indoor cultures

temperature was 20 ± 1°C. To run all the microalgae culture experiments at pilot-scale (800

L) and at industrial-scale (5 tons) microfiltered LPFD was used as a medium. Raw NRD was

collected directly from the digester tank at Langage AD plant, a 2,500 m2 industrial facility

located in Plymouth (Devon, UK). The Langage AD plant run an engine that produces 600

kw/h, processing 10,000 tons of food waste per year, and produced annually 8,000 m3 of

5
digestate. NRD composition from Langage AD was stable throughout the year,

demonstrating a robust AD process. The NRD was transported to SU (for lab indoor, and

photobioreactor (PBR) pilot experiments) and stored at 4°C, to avoid bacterial development.

Microfiltration (0.2 μm) of NRD was carried out with a ceramic membrane using a trans-

membrane pressure ranging from 1.5 to 2.5 bars (Koch membrane systems Inc.). The

permeate was collected at one end of the membrane. The permeate collected was LPFD that

was used in all the experiments at indoor lab condition, pilot-scale, and industrial scale.

After indoor lab scale growth in the F2 commercial medium the cultures were transferred to

1L flasks, but with LPFD as a nutrient source to acclimatise them, the same indoor abiotic

parameters mentioned before were used. The LPFD concentration in cultures was increased

gradually from 0.5% to 2.5% at each new flask inoculation. The indoor scaling up process

started in flasks from 1L, which were later used to inoculate 20L carboys and finally in

plastic bags of 80L.

2.3 Culture conditions (pilot and industrial scale facilities)

The 80 L bag cultures in indoor conditions were used to inoculate the 800 L pilot-scale

autotrophic PBR (Biofence™ Varicon Aqua) located at SU greenhouse facilities. The initial

Chlorella dry weight concentration at the PBR was 0.17 g/L, using LPFD at 2.5% as a

growth medium. The autotrophic PBR and the mixotrophic plastic columns (3 * 10 L each)

were maintained in a controlled temperature greenhouse at SU facilities. During the 28 days

of autotrophic culture in the PBR, 3 harvests took place (days 9, 14 and 21 during the

exponential growth phase) by removing approximately 30% of the total working volume of

the PBR and adding the calculated volume of pre-filtered (0.2 μm) tap freshwater and LPFD

at 2.5%, so that the total volume was equal to the removed volume. The autotrophic harvests

were carried out to inoculate the 3 mixotrophic columns. For the mixotrophic columns the

6
average temperature recorded during the experiments was 23.9 °C, columns were aerated

continuously by using an air pump (ACO-9730 pump, Hailea, China), with filtered (0.22 μm)

ambient air (0.039% CO2) at a flow rate of 0.2 L/min. The mixotrophic experiments lasted

108 hours and there were three biological replicates which took place over three separate

weeks. Light support was given to the mixotrophic columns continuously (24 hours). To

ensure that no external light sources influenced the algal growth, the columns were wrapped

with three black plastic sheets. The average light intensity of the artificial light source (cool

white fluorescent) applied to the mixotrophic columns was 240 μmol m2 s-1.

Two mixotrophic columns had D-glucose (Sigma Aldrich) as a carbon source and the

concentration, 10 g/L, was based on previous published data (Aiba, 1981; Heredia-Arroyo et

al., 2011). The two D-Glucose mixotrophic columns were called Concentrated Biomass

Glucose (CB-Glucose) and Non-Concentrated Biomass Glucose (NCB-Glucose). For the CB-

Glucose treatment Chlorella biomass harvested from the autotrophic PBR was concentrated

using a microfiltration membrane rig (0.2 μm) reaching an initial average biomass

concentration of approximately 2 g/L. For the NCB-Glucose treatment the average initial

biomass concentration was approximately 0.7 g/L the same concentration reached at harvest

point in the autotrophic PBR. Finally, the third treatment was, Non-Concentrated Biomass

Acetate (NCB-Acetate) where Sodium Acetate (Sigma Aldrich) was the carbon source (10

g/L), based on previous published data (Silva et al., 2016). The initial biomass concentration

was the same as the NCB-Glucose (0.7 g/L). Samples were collected every 12 hours (7 AM –

7 PM), temperature and pH were measured at the same time using a pH/Cond 340i (WTW,

Germany) probe.

For the industrial scale experiment a 7 tons autotrophic PBR (working volume of 5 tons), a

0.3 ton mixotrophic reactor (aerated columns) and all the related downstream equipment (e.g.

membrane rigs, mobile pumps, etc.) were installed and operated during the experiments in a

7
greenhouse at Langage AD facilities. The autotrophic growth mode was the same as

previously described (semi-continuous growth using 2.5% LPFD), but two microalgae were

used, the same Chlorella vulgaris strain used in previous pilot experiments (day 0 to day

146), and Scenedesmus obliquus (day 182 to day 289) (Fig. 1C). Scenedesmus was

acclimatised to LPFD using the same approach as Chlorella (increasing LPFD from 0.5% to

2.5%). For the mixotrophic reactor at industrial scale experiments, a new cheaper option of

carbon source (Dextrose) was used at the same concentration (10 g/L) than previous

mixotrophic D- Glucose pilot-scale experiments. The same approach of autotrophic semi-

continuous growth, harvest and biomass concentration using membrane microfiltration (0.2

μm), prior to inoculating the mixotrophic reactor was used on a regular weekly basis for

Scenedesmus experiments at industrial scale.

2.4 Microalgal analysis (Production parameters)

2.4.1 Dry weight

The microalgal biomass dry weight was measured following the procedure of Sorokin

(Sorokin, 1973) at times 0, 48 and 96 hours for the mixotrophic experiments and every other

day for the autotrophic experiments. 47 mm diameter and 0.22 μm GF/C Whatman filters

were dried in an oven at 80ºC for 4 hours or until a constant weight. The dry filters were

weighed on a Kern ABS80-4N precision balance (Kern & Sohn GmbH, Germany). Dry

filters were used to filter and wash microalgae samples (volume ranged from 5 to 15mL

depending on the culture density) using a vacuum pump XF54 230 50 (Millipore GmbH,

United States). After filtration, the filtered microalgae samples were placed in an oven at

80ºC overnight. After 24 hours, filters were weighed again and the weight difference between

dry filter before and after biomass filtration was calculated according to the following

formula:

8
(X-Y) * 1000/V

Where: X = filter weight (g) after biomass desiccation; Y = pre-weight (g) of clean filter; V =

culture volume (mL)

Optical Density (OD) was used as a proxy of the dry microalgae biomass concentration of the

cultures. OD was recorded at a wavelength of 750 nm with a spectrophotometer

SPECTROstar Nano (BMG Labtech GmbH, Germany).

2.4.2 Growth rate (µ)

The specific growth rate (µ), was estimated during the exponential phase of the cultures. µ

can be calculated as the slope of the linear regression or by the following equation:

µ= LN(N2/N1)/(t2-t1) (1)

where LN is the natural logarithm, N1 and N2 are measured either of dry weight or OD at

750 nm at the initial time (t1) and the final time (t2).

2.4.3 Duplication time (DT)

The duplication time (DT) was defined as the time (days) that the culture needs to double

their cell concentration, and was calculated by the following equation:

DT= LN (2)/µ

where LN is the natural logarithm and µ is the specific growth rate obtained from Eq. (1).

2.4.4 Biomass productivity

9
Biomass productivity was calculated as the difference in terms of dry weight between the

sample day and the previous sample day. Results are expressed in mg/L/day.

2.4.5 Biomass production

Biomass production is the total dry weight obtained at each harvest point and it was

expressed in g/L.

Finally, the microalgae culture samples were observed microscopically (Olympus BX43F,

Japan), providing evidence of cell morphology, behaviour of growing cells (cells in

aggregates or unicellular), and bacterial contamination. Microphotographs were taken at

different times during the growth phases of the experiments. CellSens software was used for

image collection.

2.5 Nutrient analysis (Ammonium and Phosphorus)

Every 12 hours, 50mL of microalgae culture was taken and centrifuged at 4000 rpm for 5

minutes, the supernatant obtained was used to measure the nutrient (N & P) concentrations in

the medium.

For ammonium measurements, the Spectroquant® Kit (Merck KGaA, Germany) was used.

5mL of reactive NH4-1 was added to 100 μL of sample (diluted 1: 4), then a spoon of the

reactive NH4-2 was added and it was stirred vigorously until dissolution, after 15 minutes the

absorbance was measured in the spectrophotometer SPECTROstar Nano (BMG Labtech,

GmbH, Germany) at 690 nm. With a previously developed correlation curve the ammonium

concentration (mg/L) for each sample was obtained using the following equation:

Y = 0.012x + 0.1662 (R2 = 0.993)

For phosphate measurements, the MColortestTM Kit (Merck KGaA, Germany) was used.

6mL of undiluted sample 700μL of reactive PO4-1 was added, after shaking measures of its

10
colorimetric reaction were recorded using a SPECTROstar Nano spectrophotometer at 410

nm. A correlation curve was used to determine the phosphate concentration (mg/L) for each

sample using the following equation:

Y = 0.0214x + 0.0225 (R2 = 0.999)

2.6 Carbon source concentration in mixotrophic cultures

To verify how the D-glucose added in the medium varied throughout the experiment, a

modified protocol of Dubois et al. (Dubois et al., 1951) was performed for the quantification

of total carbohydrate content. The supernatant obtained after the centrifugation previously

mentioned was used to perform the analysis. This method consists of a sulphuric acid

hydrolysis step to convert polymeric structural and storage carbohydrates into their

constituent monomeric subunits (glucose, mannose, fructose, galactose, xylose). The total

monomer concentration was then measured spectrophotometrically via the reaction between

sulfuric acid and phenol (orange colour, 485 nm).

A sample dilution 1: 100 was used, 2.5mL of 98% H2SO and 0.5 mL of a phenol preparation

(5% w/v phenol) were added to the sample, a waiting time reaction of 20 minutes was set to

read the samples in the spectrophotometer. A correlation curve was used to read the D-

Glucose concentration (mg/L) of the samples by the following equation:

Y = 1.765x + 0.0218 (R2 = 0.990)

2.7 Determination of the biochemical composition

All chemicals and analytical reagents were HPLC grade (Sigma-Aldrich) unless stated

otherwise. A multi-assay procedure (Chen & Vaidyanathan, 2012; Chen & Vaidyanathan,

2013; Kapoore et al., 2019) was modified for the quantification of total protein, carbohydrate,

chlorophylls, and carotenoids as follows. Briefly, freeze-dried microalgae pellets were

11
weighed (1 – 1.5 mg) in 2 mL Safe-Lock microcentrifuge tubes. The dry pellets were

resuspended in 24.3 µL of Phosphate Buffer (pH 7.4) and 1.8 mL of 25% (v/v) methanol in

1N of NaOH along with an equal volume of glass beads (425-600 µm i.d., acid washed).

Cells were disrupted using a cell disruptor (Scientific Industries Inc., NY, USA) for 3 cycles

(ten-minutes bead beating and two-minutes stand). For carbohydrate analysis, two aliquots of

0.2 mL extract were transferred to 2 mL PTFE capped glass vials: for the control by adding

1.2 mL 75% H2SO4; for the experimental samples by adding 0.4 mL 75% H2SO4 and 0.8 mL

anthrone reagent. Samples were incubated at 100°C for 15 minutes followed by measurement

in polystyrene cuvettes (absorbance at 578 nm). The remaining extract after cell disruption

was stored at -80°C in 4 mL PTFE capped glass vials and later saponified by incubating at

100°C for 30 minutes. For the pigment assay, saponified extracts of 0.7 mL were transferred

after vortexing to 2 mL Safe-Lock microcentrifuge tubes containing 1.05 mL 2:1 mixture of

chloroform with methanol. After centrifugation and phase separation, chlorophyll and

carotenoid concentrations were determined colourmetrically. The remaining saponified

extract was stored at -80°C for the subsequent protein assay where saponified extracts of 25

µL were first placed directly into 96-well assay plates with the following additions: controls,

0.2 mL BCA reagent alone (Thermo Scientific); experimental, 0.2 mL BCA/Cu mix (Thermo

Scientific) and incubated at 37°C for 30 minutes, measuring (absorbance at 562 nm).

2.8 FTIR spectroscopy measurements

FTIR attenuated total reflectance (ATR) spectra were collected using a PerkinElmer Model

Spectrum Two instrument, equipped with a diamond crystal ATR reflectance cell with a

DTGS detector scanning over the wavenumber range of 4000–450 cm−1 at a resolution of 4

cm−1 (Fuentes-Grunewald et al., 2015; Mayers et al., 2013). Ethanol (70%) was used to clean

the diamond ATR before the first use and between samples. Approximately 3–5 mg of finely

powdered freeze-dried microalgae biomass was applied to the surface of the crystal and then

12
pressed onto the crystal head. A duplicate (each consisting of an average of 12 scans) of each

sample was conducted, therefore results of 6 ATR spectra were gained and results averaged.

Background correction scans of ambient air were made prior to each sample scan. Scans were

recorded using the spectroscopic software Spectrum (version 10. PerkinElmer, Germany).

2.9 Statistical data analysis

All experiments were carried out with a minimum of three replicates. The results are

expressed as averages ± standard deviation (SD). The effects of the different sources of

carbon were analysed using a one-way analysis of variance (ANOVA), Tukey’s multiple

comparison test with level of significance: (P <0.05). All statistics were performed using

GraphPad Prism version 6.01.

3. Results and Discussion

To demonstrate and validate that it is technically feasible to apply the circular economy

concept to the AD industry, we undertook the main experimental work at an AD industrial

site. Here, we present results obtained from autotrophic and mixotrophic microalgae cultures

by using LPFD at 2.5% dilution as the main source of N & P at pilot (Swansea) and industrial

scale (Plymouth) at two different sites in the UK. The aim was to use microalgae to provide

an environmental service in the first autotrophic phase by uptake of excess N & P from

digestate, and to boost the microalgae biomass concentration in the second mixotrophic phase

to provide high quality microalgae biomass for further quality testing for its potential as

animal feed.

3.1 Autotrophic cultures

13
Pilot-scale autotrophic experiments were performed using Chlorella vulgaris, this strain

showed interesting results once the algae were acclimatised to 2.5% LPFD. In Figure 1A

Chlorella showed a short lag phase (acclimation period) when the inoculum was transferred

from the 80L bag indoor culture to the 800L PBR greenhouse conditions. A sharp drop in

biomass concentration during lag phase from day 0 to day 2 was recorded, likely due to the

higher solar radiation under outdoor conditions. From day 2 (once the algae were

acclimatised to new natural light conditions/photoperiod) until day 27 a constant and

continuous growth was recorded, obtaining an average growth rate of 0.32 ± 0.1 d-1 (Fig. 1A).

A similar growth rate (0.33 ± 0.1 d-1) was obtained when the Chlorella cultures were run at

industrial-scale (5 tons) at Langage AD (Fig. 1C, day 0 to day 146). This similar growth

behaviour can be explained because Chlorella was already acclimatised to 2.5% LPFD in the

lab and at pilot-scale, and that is why it was used as a reliable and healthy inoculum for

industrial-scale operation. The autotrophic growth rate obtained during pilot and industrial

experiments using Chlorella, are lower than those reported by (Obata et al., 2008) (0.40 d-1)

or by (Chia et al., 2013) (0.62 to 0.84 d-1), but those authors used standard media (F/2), or

media improved with trace metals, as well as working in indoor controlled conditions at

volumes below 6L. Scenedesmus autotrophic growth at industrial scale are similar (no

significant difference) in terms of growth rate compared to Chlorella in the same industrial

PBR system. Nevertheless, the average biomass production (g/L) was considerably higher (>

103%) in Scenedesmus with an average production of 0.342 ±0.19 g/L compared to Chlorella

with an average of 0.168 ±0.10 g/L (Fig. 1C). It must be noted that industrial cultures of

Scenedesmus also shows robustness, a reliable growth, and no visible contamination (either

predators or competitors) occurred over 108 days of continuous autotrophic growth (Fig. 1C).

3.2 Mixotrophic cultures

14
The new combined autotrophic-mixotrophic growth approach followed in this study allowed

us to fulfil the main requirements of a circular economy approach. Bioremediating side-

streams from an AD plant, up taking N & P from the LPFD during autotrophic growth, and

improving the microalgae growth rate and biomass production in the mixotrophic step, with a

consistent macromolecular composition (mainly protein content), for further use as an

additive in animal feed were all successful. From the results obtained at two different scales

and in two different locations using the model microalgae Chlorella, we can highlight a

significant difference in terms of growth rate (56% increase, p=0.02) in the mixotrophic

reactor (CB-Glucose) compared with the autotrophic reactor. The doubling time in

mixotrophic cultures was reduced by 35% and the biomass (g/L) obtained (48 after

inoculation in the mixotrophic reactor) was one order of magnitude higher or an improvement

of 1097 % (Table 1, Fig. 1B). Clearly the results in the mixotrophic cultures were improved

and better production parameters were obtained compared to the autotrophic cultures,

including results obtained by several authors using autotrophic growth with the same algae

model (Heredia-Arroyo et al., 2011; Liang et al., 2009; Tan et al., 2018).

For the pilot-scale Chlorella mixotrophic experiments, we evaluated different carbon sources

with and without the initial concentration of the biomass, where significant differences

(p=0.004) were found in terms of growth rate and biomass production. Acetate as carbon

source showed slight growth obtaining 1.07 g/L after 48 hours of mixotrophic growth (Fig.

1B). Similar results were observed (Fig. 1E) using absorbance at 750 nm as a proxy of the

dry weight. As shown in Figures 1B and 1E, better growth performance was obtained when

glucose was the carbon source elected. However, we recorded a significant difference

(p=0.004) between the pre-concentrated microalgae biomass (CB-Glucose) and the non-

concentrated biomass (NCB-Glucose). Figure 1B shows an improvement of 104% of the CB-

Glucose when harvested (5.51 g/L) compared with NCB-Glucose (2.70 g/L) at the same time.

15
These mixotrophic NCB-Glucose results are consistent with those obtained by (Heredia-

Arroyo et al., 2011) (1.40 g/L), by (Weibao Kong, 2011) (2.12 g/L) and by (Liang et al.,

2009) (1.69 g/L), using a similar non-concentrated approach, reaching similar dry weight

concentrations, using different glucose concentrations (1 to 4 g/L) and with slightly better

production times in our case (below 72 hours). These low biomass yields for a mixotrophic

growth could be due to the initial low cell density or biomass concentration (0.69±0.02 g/L).

This issue was reported by (J. R. Benavente-Valdés, 2012), who argue that low initial cell

density has several drawbacks, including low productivity, easy bacterial contamination, and

costly product recovery from diluted cultures in closed systems. We have observed the same

phenomena in our non-concentrated cultures regardless of the carbon source used. An

increase in bacterial contamination is detected in cultures where the initial biomass

concentration was low (NCB-Glucose and NCB-Acetate), from 48 hours onward an

explosive boost in bacterial contamination was recorded. By contrast, when the initial

concentration is higher (CB-Glucose; approximately 2g/L as initial concentration) a

negligible bacterial contamination was recorded, and reaching interested final biomass

concentration and growth rates compared to what (Kim et al., 2013) found for instance. In

general, most studies obtain maximum mixotrophic yields in the range of 1.28 to 2.6 g/L

depending on the species chosen or the carbon source (Bhatnagar et al., 2011; Hu et al., 2013;

Liang et al., 2009; Wan et al., 2011; Wang et al., 2010; Zhou et al., 2013). These values are

considerably lower than our Chlorella biomass concentration obtained at harvest point (5.51

g/L). Comparable mixotrophic biomass production results were obtained by (Cheirsilp &

Torpee, 2012) (5.87 g/L) and (Gao et al., 2019) (5,79 g/L), but with Nannochloropsis sp. and

Tetradesmus bernardii respectively.

16
3.3 Biomass concentration approach

We have developed a new approach of biomass pre-concentration using a non-cell destructive

membrane filtration (0.2 µm) technique prior to mixotrophic inoculation. This successful new

method was applied at an industrial scale for the two target species. Interestingly, when we

concentrated the biomass even further, so that the initial biomass concentration was 7.67 g/L

in Scenedesmus mixotrophic experiments, the results were comparable to those for Chlorella

(nearly doubling biomass concentration in a short period), reaching a final biomass

concentration over 12,17 g/L in 48 hours (Table 1, Fig. 1D). We have demonstrated that

membrane technology (microfiltration) is an excellent method when the aim is to concentrate

the biomass obtained from autotrophic cultures, eliminating most of the bacterial

contamination in the concentrated biomass, and leaving the microalgae cells intact for further

mixotrophic growth.

3.4 Bioremediation capacity

Another key aspect we addressed was the environmental service or bioremediation capacity

of the selected target microalgae species. In this case, the aim was to uptake maximum

quantities of excess nutrients from LFPD. The concentration of ammonium (N) presented in

raw digestate from Langage AD plant can be over 4000 mg/L, and this nutrient has reported

toxicity at higher concentrations in photosynthetic unicellular algae (Collos & Harrison,

2014). Therefore, an ammonium dilution was compulsory for growth of the microalgae at the

pilot and industrial scale. Both species independently of scale and growth strategy were able

to uptake over 100 mg/L/day of ammonium in most cases (table 1). In mixotrophic Chlorella

cultures (CB-Glucose), the ammonium was completely removed after 50 hours meanwhile in

the non-concentrated culture the ammonium was not completely removed when the

experiments were finished after 108 hours (Fig. 1F). When we compare our nutrient removal

17
with those observed for the same microalgae genus in other studies, we found better

performance in our cultures when the nitrogen source was ammonium. (Kim et al., 2013) for

instance, recorded an uptake of 19.4 mg-N/L/day for Chlorella sorokiniana, this slow N

uptake could be because the N source was NaNO3, and it is well established that for most of

the photosynthetic organisms, it is easier and less energy demanding to uptake ammonium

rather than nitrate (Glibert et al., 2016). The bioremediation results obtained in the present

work are promising because most of the N-source in the LFPD was taken up by both

microalgae species and in both autotrophic and mixotrophic reactors. Since phosphorus (P) is

considered a limiting element and essential for photosynthetic growth, the results observed

show a higher P uptake in Chlorella than in Scenedesmus in autotrophic conditions (Table 1).

During the mixotrophic experiments the P was not assessed.

Several carbon (C) sources (glucose, acetate, and dextrose) were tested at different scales

(lab, pilot and industrial) with both microalgae species. The best growth performance for

Chlorella was with glucose at a concentration of 10 g/L. Uptake of this sugar was 100% after

72 hours in the CB-Glucose culture, meanwhile NCB-Glucose was not depleted, increasing

the carbohydrate content in the medium after 50 hours (Fig. 1G). One of the drawbacks of

using glucose at an industrial scale is the price, consequently a cheaper source, dextrose, was

used for the mixotrophic growth of Scenedesmus. When we compare the C-source

assimilation of both target microalgae, Scenedesmus have a higher C uptake rate consuming

most of the carbon (dextrose) in 48 hours. By contrast Chlorella took over 72 hours to

achieve the same results using glucose (Table 1). To improve the economic feasibility of

mixotrophic growth at the industrial scale it is useful to test new free side-stream carbon

sources. Therefore, a new free side-stream carbon source, chewing gum powder (a waste

from the chewing gum production process), was tested. This carbon source seems to be

assimilated by the microalgae, but a previous pre-treatment must take place to remove the

18
milky colour (blocking light and prevent algal growth), when the chewing gum powder is

dissolved. Membrane filtration makes it feasible to filter and remove the calcium carbonate

(which give the milky colour) obtaining a clear, colourless carbon source. Ongoing

experiments for mixotrophic growth have been performed and, in the future, this free carbon

source will be utilised in mixotrophic cultures at industrial scale.

3.5 Abiotic parameters in cultures

The main abiotic parameters such as temperature, dissolved oxygen, and pH were recorded

daily during the entire autotrophic experiments for both microalgae species at an industrial

scale. In the autotrophic reactor temperature and pH were set in automatic mode. To maintain

the temperature a heat exchanger was connected to the reactor which allowed a consistent

temperature range across all seasons. The average temperature recorded for the entire period

was 23.56 ±2.5 °C. pH was set in a neutral range, average 7.5 ±0.8, and CO2 was given on

demand to keep the pH at the required level. Finally, the dissolved oxygen concentration was

recorded automatically, were for both species the average O2 concentration during the period

was 16.92 ±3.9 mg/L with the maximum value detected by the O2 probe being 20 mg/L.

which was recorded during the sunniest days.

For the mixotrophic cultures at different scales, the temperature was constant throughout the

experiment (25.5 ºC.) There was a greater fluctuation of temperature in CB-Glucose between

morning and afternoon, still being within the optimum temperature range for the cultivation

of C. vulgaris (Tan et al., 2018). Figure 1H shows that the pH in CB-Glucose treatment

tended to decrease. The increase of pH in microalgae cultures indicates CO2 fixation during

photosynthesis, which releases OH- ions into the medium increasing the pH (Chi et al., 2011).

So, during photosynthesis the pH tends to increase, however, when heterotrophy/mixotrophic

occurs it tends to decrease. In addition, studies by (Weibao Kong, 2011) show how Chlorella

19
vulgaris in mixotrophic culture (glucose) with a different nitrogen source induces sharp drops

in pH levels (̴ 4) causing low level performance of the culture. Consequently, it is necessary

to control the pH of mixotrophic cultures in mixotrophy (Deng et al., 2019).

3.6 Biomass production and quality

From the mixotrophic experiments at industrial scale we were able to produce over 3.75 Kg

and 1.6 Kg of dry weight biomass for Scenedesmus and Chlorella respectively, twice a week

during several consecutive weeks. Chlorella mixotrophic biomass was then fully

characterised and compared to the biomass obtained from autotrophic reactors using 2.5%

LPFD and a standard commercial F2 medium. Figure 2A shows a clear increase in the protein

content in 2.5% autotrophic, 56% higher content compared to the F2 and 88% more protein

concentration than mixotrophic cultures. The results are confirmed by FTIR analysis (Fig.

2C). This increase is also detected for Chlorophyll a where the maximum obtained in

autotrophic cultures was with 2.5% LPFD. These results are expected because in autotrophic

mode chlorophyll is needed for photosynthesis and there is more protein formation using

ammonium as a N-source. The protein results in mixotrophic conditions are in the range

expected (approximately 45%), and these concentrations are interesting when the final aim is

to use the biomass (with high protein concentration) as feed for animals. It is also interesting

to note that the mixotrophic biomass shows an increase in carotenoids respect to chlorophyll

(Fig. 2B). Carotenoids are molecules with health boosting properties (Gong & Bassi, 2016).

These results reinforce the potential use of quality microalgae biomass produced under

mixotrophic conditions for animal feed purposes.

Our work highlights the technical feasibility of the circular economy concept applied to

industrial activities by using microalgae biotechnology. As (Stahel., 2016) suggests, there is a

need for “research and innovation at all levels” in the circular economy approach. Therefore,

20
researchers must apply cutting-edge science to tackle environmental issues that are impacting

the ecosystem. We suggest a microalgae circular economy model (graphical abstract) to

improve one of the biggest environmental issues, eutrophication, which is caused by the

release of the excess nutrients (N & P) from AD plants onto the soil. AD plants are the main

technology used in the EU and the UK to treat organic waste. Currently, there are more than

17,000 AD plants in operation in the EU, the main economic driver being the production of

electricity from biogas which has an installing capacity of over 10,500 MW (EBA, 2018).

Digestate is a side-stream of AD plants, with nearly 10 million tons of excess digestate

produced in a yearly basis. As highlighted by Stiles et al. (Stiles et al., 2018), the EC Nitrates

Directive (91/676/EEC) and Nitrate Vulnerable Zone (NVZ) legislation, restrict the amount

of N that AD plant can dispose of onto soil. Nevertheless, minimal research has been done to

solve this increasing pollution problem. Microalgae technology has been targeted as a

possible solution to protect the environment against eutrophication (Aiba, 1981; Mayhead et

al., 2018; Melo et al., 2018; Savchenko et al., 2017; Silkina et al., 2019; Xu et al., 2017). The

main advantages of this technology are the low-energy requirements, greenhouse gas

emission removal (CO2 uptake) and cost-effectiveness (Chisti, 2007; Sayre, 2010). Besides

the undoubtedly good performance of microalgae to treat different side-streams, the

macromolecular composition of these cells is of high interest (proteins, lipids, and

carbohydrates). The microalgae biomass have known food and feed applications, plus others

high value metabolites that can be extracted from microalgae such as pigments, omega-3 oils,

amino acids, and exopolysaccharides, that can be used in a range of industrial applications

(Fuentes-Grunewald et al., 2015; Khanra et al., 2018; Rumin et al., 2020; Silva et al., 2016;

Weibao Kong, 2011; Zaslavskaia, 2001). In our work, we have been able to bioremediate an

AD side-stream and produce microalgal biomass consistently and reliably, with a high

content of proteins suitable for use in animal feed. Recent reports have also highlighted the

21
potential of cultivating microalgae on digestate as a feedstock and later proposed the

suitability of such biomass for animal feed purposes, owing to its high nutritional contents

such as proteins, lipids, fatty acids and carotenoids (Stiles et al., 2018). However, as far as the

authors know, there is no clear legal framework in the EU nor in the UK on the use of

microalgae biomass produced from industrial side-streams that could be used for animal feed.

Considering the suggestion of (Stahel., 2016), we have advanced on the circular economy

approach, and have demonstrated that it is technically feasible to apply microalgae

biotechnology under this concept. Now it is time for collaboration with policy makers to

progress on this subject and establish a legal framework that will help to deliver this

technology to AD stakeholders across the EU and the UK. From the results of this work,

interesting projections on the impact of N & P bioremediation and biomass production can be

made. Forecast calculations that include a microalgae plant of 0.12-hectare (half of the

Langage AD plant footprint) coupled to an AD plant, a 36m3 autotrophic PBR, a 2 x 4m3

mixotrophic reactor, and the use of Scenedesmus as an algae model, show that it is feasible to

treat over 50 tons of raw digestate and produce over 6 tons of dry weight microalgae biomass

per year, suitable for the production of feed for animal. Extrapolating these results to the

numbers of AD plants currently in operation in the EU, potentially over 850,000 tons of raw

digestate could be treated yearly. This new circular economy industrial approach could help

to alleviate the eutrophication pressure of NVZ, produce large quantities of microalgae

biomass for animal feed production, and create thousands of new sustainable jobs.

4. Conclusions

In this work we have successfully validated the circular economy concept applied to

microalgae biotechnology in a relevant industrial scale. We have demonstrated

bioremediation of excess nutrients in digestate (ammonium form), producing microalgae

biomass in a reliable and consistent way to ensure a quality suitable for animal feed.

22
Applying new two-step approach where we have combined autotrophic growth in a first stage

to uptake nutrient excess, then, by using membrane technology, we were able to concentrate

the microalgae biomass for second growth stage (mixotrophic) with the goal of high

concentration microalgae biomass production with an improved macromolecules profile

(high protein).

"E-supplementary data for this work can be found in e-version of this paper online"

Acknowledgments

This work was funded by the Interreg North West European Regional development fund,

project ALG-AD. https://www.nweurope.eu/projects/project-search/alg-ad-creating-value-

from-waste-nutrients-by-integrating-algal-and-anaerobic-digestion-technology/

Author contributions

C. Fuentes-Grünewald: Conceptualization, methodology, validation, formal analysis,

investigation, visualization, supervision, writing original draft, writing-review, and editing.

J.I. Gayo-Peláez & V. Ndovela: Methodology, validation, investigation, data curation,

writing review, and editing. E. Wood & R. V. Kapoore: Validation, formal analysis,

investigation, and visualisation, writing-review, and editing. C. Llewellyn: Funding

acquisition, conceptualization, project administration, supervision, writing-review, and

editing.

23
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26
 Chlorella Auto & Mixotrophic gr
Chlorella Auto & Mixotrophic growth 8
Figures
8 Captions

Dry weight [g/L]

6
Dry weight [g/L]

6
Figure 1. Overview of the main biotic and abiotic parameters in autotrophic and mixotrophic
4
cultures at pilot and industrial scale using Chlorella vulgaris and Scenedesmus obliquus. A.
Chlorella
4 autotrophic growth at pilot scale (800 L). B. Chlorella autotrophic growth compared
with mixotrophic growth (day 28 onward). C. Autotrophic growth 2at industrial scale (5m3),
day 02 to 146 for Chlorella, day 182 to 290 for Scenedesmus. D. Scenedesmus mixotrophic
growth. E. Growth (abs 750nm) comparison of three C sources in Chlorella mixotrophic
0
experiments. F. Ammonium uptake in mixotrophic Chlorella experiments. 0 G. Glucose
10 uptake
20 30
0
in mixotrophic Chlorella cultures. H. pH trend in mixotrophic Chlorella cultures. * <0.05; Days **
<0.005;0 *** <0.001.
10 20 30 40
CB Glucose (10 g/L) NCB Glucose (10 g/L)
Days
CB Glucose (10 g/L) NCB Glucose (10 g/L) NCB Acetate (10 g/L) Autotrophic growth (g/L)

Autotrophic growth (g/L)

Figure 2. Chlorella biomass characterisation from mixotrophic and autotrophic experiments

performed at industrial scale. F2 and 2.5% dig are experiments that were run in autotrophic
conditions A. Main macromolecule composition B. Main pigment concentrations C. Fourier
Transformed Infra-Red analysis (used as a proxy of macromolecules composition analysis
showing) the different areas under each of the main peaks obtained from 12 scans for each
sample (n=3).

Table caption

Table 1. Main biotic and abiotic parameters in autotrophic and mixotrophic cultures of
Chlorella vulgaris and Scenedesmus obliquus. Chlorella data obtained from pilot-scale
experiments, 800L for autotrophic cultures and 30L for mixotrophic cultures. Scenedesmus
data obtained from industrial scale experiments, 5m3 for autotrophic cultures and 0.3m3 for
mixotrophic cultures. Pilot-scale experiments were performed in greenhouse conditions at
Swansea University, Swansea, Wales, UK. Industrial scale experiments were performed in
greenhouse condition at Langage AD farm, Plymouth, England, UK. All the cultures were run
in closed systems, photobioreactor system for autotrophic cultures and bubble columns for
mixotrophic cultures.

27
 Figures
Chlorella Auto & Mixotrophic growth

1.0 8 8
B
A
0.8

Dry weight [g/L]

Dry Weight [g/L]

Dry weight [g/L]

6 6
0.6
4

**
4

***
0.4

0.2 2 2

*
0.0 0
0 10 20 30 0
0 0 10 5020 30100 40 150
Days Days
Hours
15CB Glucose (10 g/L) NCB Glucose (10 g/L) NCB Acetate (10 g/L)

C D
CB Glucose (10 g/L)
Autotrophic growth (g/L)
NCB Glucose (10 g/L) NCB Acetate (10 g/L)

Dry weight [g/L] 10

0
0 20 40 60 80
Hours
30 80
E F

60
Abs 750 nm

NH4 [mg/L]

20

40

10
20

0 0
0 50 100 150 0 50 100 150
Hours Hours
CB Glucose (10 g/L) NCB Glucose (10 g/L) NCB Acetate (10 g/L) CB Glucose (10 g/L) NCB Glucose (10 g/L) NCB Acetate (10 g/L)
10 10
G H
8
9
6
[g/L]

pH

8
4

7
2

0 6
0 50 100 150 0 50 100 150
Hours Hours
CB Glucose (10 g/L) NCB Glucose (10 g/L) CB Glucose (10 g/L) NCB Glucose (10 g/L) NCB Acetate (10 g/L)
Figure 1

28
 A B

Figure 2

Tables
Table 1
Autotrophic Mixotrophic
Parameters Chlorella Scenedesmus Chlorella Scenedesmus units
Growth rate 0.32 0.33 0.50 0.42 d-1
Duplication time 2.16 2.13 1.39 1.67 days
Nitrogen uptake 104.6 134.9 115.5 98.8 mg/L/day
Phosphorus uptake 1.89 0.95 - - mg/L/day
Glucose uptake - - 2692 - mg/L/day
Dextrose uptake - - - 4980 mg/L/day
Biomass productivity 84.27 43.69 1621 1591 mg/L/day
Biomass production (average) 0.46 0.35 5.32 12.50 g/L
Maximum biomass production 1.01 0.73 5.51 13.83 g/L
Nitrogen concentration 370.5 97.45 140.8 82.72 mg/L
Maximum Ammonium 910 165.57 254.9 153.82 mg/L
Phosphorus concentration 9.53 5.24 37.4 - mg/L
N:P (average) 39 18.6 6.8 -

29
C. Fuentes-Grünewald: Conceptualization, methodology, validation, formal analysis,

investigation, visualization, supervision, writing original draft, writing-review, and editing.

J.I. Gayo-Peláez & V. Ndovela: Methodology, validation, investigation, data curation,

writing review, and editing. E. Wood & R. V. Kapoore: Validation, formal analysis,

investigation, and visualisation, writing-review, and editing. C. Llewellyn: Funding

acquisition, conceptualization, project administration, supervision, writing-review, and

editing.

30
Graphical abstract

31