microalgae culture to treat anaerobic digestion abattoir ... · (2017) and nwoba, ayre, moheimani,...

Microalgae culture to treat anaerobic digestion

abattoir effluent (ADAE)

A thesis submitted as partial fulfilment towards the award of

Master of Environmental Science

Louise Foster

2019

I declare that this thesis is my own account of my own research conducted during my period

of enrolment at Murdoch University for the degree of Master of Environmental Science.

It has not previously been submitted for a degree at this or any other university.

Louise Foster

iii

Abstract

Current abattoir wastewater treatment options involving aerobic and anaerobic digestion

systems are relatively inefficient in reducing the inorganic nutrient load (i.e. ammonium and

phosphate) of the effluent. With anaerobic lagoons favoured in Australia resulting in large

land footprint, loss of environmental and economic value in the form of water and nutrient

recovery. The cultivation of microalgae on undiluted anaerobic digestion abattoir effluent

(ADAE) offers many potential benefits such as the bioremediation of waste nutrients typically

found in the effluent and simultaneous production of valuable algal biomass which

represents the conversion of waste-to-profit. Microalgal culture on undiluted ADAE require

minimal to no freshwater input and does not compete for arable land, it has the potential to

play an important remediation role particularly during the final (tertiary) treatment phase. The

generated biomass can also be a source of revenue for the targeted abattoir.

To the best of my knowledge, to date, there has been no attempt on testing the growth of

algae to treat undiluted ADAE. In this study, the growth, biomass productivity, photo

physiology and nutrient removal rates of mono and mixed culture of Chlorella sp. and

Scenedesmus sp. were evaluated in unfiltered and undiluted ADAE. Chlorella sp. showed

the highest ammonium removal rate of up to 11.93±1.14 mgL-1 d-1 as well as the highest

biomass productivity of 31.52±0.81 mgL-1 d-1 when compared to the other treatments.

Phosphate removal rates were highest in the Scenedesmus cultures while the mixed

cultures varied amongst other parameters measured (COD, Nitrite and Nitrate). Chlorella sp.

with a specific growth rate of 0.260±0.047 d-1 and mixed Chlorella sp. within consortium

(0.294±0.024 d-1) were the dominant species when compared to Scenedesmus sp.

(0.062±0.011 d-1). Overall, Chlorella sp. was identified as the most efficient microalgal

species capable of growing on undiluted ADAE with the potential to generate a waste-to-

profit system.

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Contents

Abstract ..................................................................................................................................... iii

List of figures .............................................................................................................................. v

List of tables ............................................................................................................................... v

Abbreviations ............................................................................................................................ vi

Acknowledgements .................................................................................................................. vii

1. Introduction ............................................................................................................................ 1

1.1 Abattoir wastewater.......................................................................................................... 3

1.2 Current treatment method ................................................................................................ 4

1.3 Gaps in current processes ............................................................................................... 5

1.4 Microalgae ........................................................................................................................ 5

1.4.1 Potential of microalgae wastewater treatment .......................................................... 7

1.4.2 Benefits of microalgae wastewater treatment ........................................................... 9

1.5 Economics ...................................................................................................................... 10

1.6 Aim and objective ........................................................................................................... 11

2. Methods ............................................................................................................................... 12

2.1 Nutrient composition of ADAE and removal rates ......................................................... 12

2.2 Cell count ....................................................................................................................... 13

2.3 Biomass yield ................................................................................................................. 14

2.4 Quantum yield ................................................................................................................ 15

2.5 Temperature ................................................................................................................... 15

2.6 Irradiance ....................................................................................................................... 15

2.7 pH ................................................................................................................................... 15

2.8 Statistical analysis .......................................................................................................... 15

3. Results ................................................................................................................................. 15

3.1 Cell density and biomass ............................................................................................... 15

3.2 Effective quantum yield, pH and temperature ............................................................... 20

3.3 Nutrient concentration .................................................................................................... 22

4. Discussion ............................................................................................................................ 25

5. Conclusion ........................................................................................................................... 26

6. Recommendations ............................................................................................................... 27

7. References ........................................................................................................................... 28

v

List of figures

Figure 1: Current and proposed microalgae integration treatment within abattoirs. ............... 9

Figure 2: Cell comparison of all cultures at 40x magnification............................................... 16

Figure 3: Growth curve showing cell concentration during cultivation period. ...................... 17

Figure 4: Biomass yield of all algae cultures on ADAE. ......................................................... 18

Figure 5: Specific growth rate of each culture within growth phase. Each of the same letter

represents no significant difference. Each different letter showing significant difference. ..... 19

Figure 6: Biomass productivity of each culture up to plateau period. Each of the same letter

represents no significant difference. Each different letter showing significant difference. ..... 20

Figure 7: Effective quantum yield of each culture measured every second day. .................. 21

Figure 8: pH at day 0 and day 15 of cultivation. ..................................................................... 21

Figure 9: Culture room temperature recorded daily. .............................................................. 22

Figure 10: Nutrient concentration during algae growth period of seven days in ADAE. ....... 23

Figure 11: (a) Ammonium removal rate and (b) Phosphate removal rate. Each of the same

letter represents no significant difference. Each different letter showing significant difference.

.................................................................................................................................................. 24

List of tables

Table 1: Typical regulatory concentration and load based limits for nutrients. ........................ 3

Table 2: Nutrient Composition of Raw ADAE before inoculation. .......................................... 13

Table 3: Nitrite and nitrate nutrient concentration and increase rates from day 0 after

inoculation to day 7. ................................................................................................................. 24

https://d.docs.live.net/85b173614435f462/Uni/ENV690%20Enviro%20Masters%20Project/thesis/Thesis%20Louise%202019%20FINAL%20Library%20submission.docx#_Toc20320469

vi

Abbreviations

AD Anerobic digestion

ADAE Anaerobic digestion abattoir effluent

AFDW Ash free dry weight

ANOVA Analysis of variance

COD Chemical oxygen demand

CER Certified emission reduction

CDM Clean development mechanism

CH4 Methane

CO2 Carbon dioxide

DI Deionized

DW Dry weight

Fqʹ/Fmʹ Effective quantum yield

GHG Greenhouse gasses

GWP Global warming potential

LED Light emitting diodes

MPPs Meat processing plants

NaNO2 Sodium nitrite

NH3 Ammonia

NH3-N Ammonia nitrogen

NH4+ Ammonium

NO2- Nitrite

NO2--N Nitrite nitrogen

NO3--N Nitrate nitrogen

NO3- Nitrate

OM Organic matter

P Phosphorus

PO43- Phosphate

P2O5 Phosphorus pentoxide

PSII Photosystem II

RMP Red meat processing

SWW Slaughterhouse wastewater

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Acknowledgements

Firstly, I would like to thank my supervisor Dr Navid Moheimani and Dr Ashiwin Vadiveloo for

all their support, advice and wisdom. Dr Angelo Matos for his assistance, as well as all

researchers at the algae and research centre. Secondly, I’d like to thank the abattoir for

supplying the ADAE sample. Finally, I’d like to thank my partner Gregory Walker who

provided substantial support throughout.

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1. Introduction

The meat processing industry is one of the leading industries within Australia (Bustillo-

Lecompte & Mehrvar, 2015; FAO, 2018). It produces significant volumes of effluent termed

as slaughterhouse wastewater (SWW) which is primary composed of animal fluids (i.e.

blood, manure, and urine), the water used for cleaning the slaughterhouse facilities and

meat processing plants (MPPs). Up to 85% of freshwater used in MPPs is directly

discharged as wastewater (AMPC, 2017), establishing it as one of the major consumers of

freshwater (24%) among various food and beverage processing facilities worldwide (Bustillo-

Lecompte & Mehrvar, 2015). With up to 29% of freshwater consumed by the agricultural

sector worldwide (Bustillo-Lecompte & Mehrvar, 2015; Mekonnen & Hoekstra, 2012) beef

and cattle production significantly contribute the largest total global water footprint of farm

animal production per animal at 33% (Mekonnen & Hoekstra, 2012). Australia is one of the

world’s driest continent with average rainfall of below 600 mm for over 80% of the continent

and below 300 mm for more than 50% (Pink, 2012). Water scarcity is a growing concern not

only in Australia but worldwide (Cuellar-Bermudez et al., 2017).

Slaughterhouses are part of a large industry, which is common to numerous countries

worldwide where meat consumption is an important part of the local population diet

(Bustillo-Lecompte & Mehrvar, 2015). The global processing of cattle has intensified

consistently over the past 50 years, in which production has more than doubled at the

expense of waste mitigation techniques which have lagged behind (Harris & McCabe, 2015;

Mekonnen & Hoekstra, 2012). The Australian red meat processing (RMP) industry is

currently working on a range of innovative measures in an effort to reduce carbon pollution

and improve energy efficiency through actively seeking renewable resources of energy and

water recovery. This has been largely in response to a variety of factors including an

increase in community focus, prolonged drought, water restrictions, rising fuel, water and

energy costs, and greenhouse gas (GHG) emissions (Harris & McCabe, 2015). The

continuing decrease in the availability of freshwater resources has rearranged the objectives

in the wastewater treatment field from disposal to reuse and recycle. As a result, a high level

of treatment efficiency has to be achieved within such facilities (Bustillo-Lecompte &

Mehrvar, 2015).

A significant portion of SWW streams are produced through cleaning of facilities and

slaughtering of animals resulting in high concentration of nutrients, suspended solids, oil and

grease. Many methods have been and are currently in use to treat wastewater to reduce its

adverse effect on the environment. In particular, anaerobic treatment methods have been

mostly preferred and employed due to the composition of these wastewaters which is rich in

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organic matter (OM), with the added benefit of energy generation (i.e. methane) and low

surplus sludge production (Chan, Chong, Law, & Hassell, 2009). However, the failure of

treated effluent arising from anaerobic systems to meet environmental discharge standards

has restricted the use of these systems as a complete treatment method. In practical

applications, anaerobic treatment suffers from the low growth rate of the microorganisms, a

low settling rate, process instabilities and the need for subsequent post treatment (Chan et

al., 2009). Therefore, it is important to develop and evaluate other innovative methods that

can be integrated to current systems to improve wastewater mitigation in MPPs (Taskan,

2016). With high concentration of nutrients (i.e. nitrogen and phosphorus), SWW may

provide a suitable environment for the cultivation of microalgae. As SWW are different to

other industrial wastewaters which are rich in pathogens and heavy metals (Maizatul, Radin

Mohamed, Al-Gheethi, & Hashim, 2017). Wastewater treatment by microalgae for the

removal of organic and/or inorganic pollutants has been reported since 1953 (Oswald,

Gotaas, Ludwig, & Lynch, 1953).

Wastewater represents a continuous and abundant source of water and nutrients for algal

biomass production. Whilst algae cultivation on wastewater represents a simple and cost

effective option to improve the treatment of wastewater by removing organic pollutants and

reducing nutrient concentration, hence generating clean water as the by product (Fornarelli,

Bahri, & Moheimani, 2017). Algae biomass produced through the cultivation in SWW can be

used for producing biogas through anerobic digestion (AD), thus offsetting some of the

abattoir energy demand and revenue stream, or can be used on site as fertilizer or animal

feed which represent the conversion of waste to profit (Fornarelli et al., 2017). In general,

certain microalgae species such as Chlorella and Scenedesmus have been well identified

and established as a suitable treatment option for various wastewater and the simultaneous

production of biomass (Su, Mennerich, & Urban, 2012). Ayre, Moheimani, and Borowitzka

(2017) and Nwoba, Ayre, Moheimani, Ubi, and Ogbonna (2016) reported the ability of local

microalgae strains such as Chlorella sp. and Scenedesmus sp. that could grow efficiently on

undiluted AD piggery effluents at high ammonium concentrations of up to 1600 mg L-1. This

outcome is highly promising as the dilution of wastewater for microalgae growth is not

considered to be viable with limitation of freshwater supply in some places and potential

problems with disposal of this overall larger volume of water (Ayre et al., 2017).

A niche opportunity has been identified in the integration of algae cultivation processes with

wastewater treatment, where algal biomass is grown as a by-product of wastewater

treatment (Craggs, McAuley, & Smith, 1997; Fornarelli et al., 2017). Both fundamental and

3

field-scale research is needed to optimise algal production and harvest from wastewater

treatment while maintaining high effluent water quality (Park, Craggs, & Shilton, 2011).

The over-arching aim of this study is using microalgal natural ability to bioremediate and

produce valuable biomass through cultivation in wastewater providing waste-to-profit

initiatives. This will provide the potential for the treated ADAE after microalgal cultivation to

be reused and recycled within abattoir’s as a source of non-potable water, reducing the cost

of freshwater consumption and disposal, providing it meets environmental standards

(Fornarelli et al., 2017; Maizatul et al., 2017). Typical environmental discharge nutrient

concentration limits are given in Table 1.

Table 1: Typical regulatory concentration and load based limits for nutrients.

Receiving

Environment Nitrogen Phosphorus

Sewer NH₃ ≤ 50 mg/L TP ≤ 10 - 20 mg/L

TN ≤ 100 mg/L

River discharge

NH₃ ≤ 1 mg/L TP ≤ 1 - 40 mg/L

TN ≤ 50 - 100 mg/L

(site specific)

(very site specific)

Also typical load based limits

Land irrigation

(soil and crop specific)

TN: 250 - 500 kg/ha/yr TP: 30-40 kg/ha/yr

Load based limits Load based limits

(AMPC, 2017)

1.1 Abattoir wastewater

A high accumulation of waste is projected with steady doubling growth of meat production

until 2050 (Bouwman et al., 2013; Bustillo-Lecompte & Mehrvar, 2015; Mekonnen &

Hoekstra, 2012) . The global consumption of the four major meat categories is estimated

around 335 million tonnes in 2018, virtually matching the production forecast (FAO, 2018).

The slaughtering of animals for commercial meat production is a multistage process, each

stage producing waste with different characteristics (Ruiz, Veiga, de Santiago, & Blázquez,

1997). Wastewater arising from abattoirs are biodegradable and can be characterized by the

presence of high concentrations of whole blood of the slaughtered animals and suspended

particles of semi-digested and undigested foods within the stomach and intestine of

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slaughtered animals (Ahmad, Ejaz, Ali, Qadir Jahangir Durrani, & Khan, 2014; Ramakant &

Vanerkar, 2005; Ruiz et al., 1997).

1.2 Current treatment method

Existing treatment methods are already in place in the meat industry with majority of

Australian abattoirs already establishing biological treatment systems on site such as

anaerobic digestion which treats wastewater by reducing organic content through the use of

microorganisms under oxygen-depleted conditions (Ahmad et al., 2014; Fornarelli et al.,

2017; Park, Jin, Lim, Park, & Lee, 2010). Anaerobic digestion is a widely used technology for

organic waste treatment. This option allows for cleaner production and sustainable

development due to the positive energy balance for the waste treatment (no energy is input,

and captured biogas produced is an energy source) (López & Borzacconi, 2010). In the

treatment of wastewater, biological treatment options appear to be a promising technology to

attain revenue from Certified Emission Reduction (CER) credits, more commonly known as

carbon credits from the Clean Development Mechanism (CDM) as methane gas generated

through anaerobic digestion can be utilized as a source of renewable energy (Chan et al.,

2009).

Although organic and nutrient levels within the effluent are reduced through AD treatment,

there are still high traces of inorganic nutrients such as phosphorus and nitrogen in the

anaerobically treated effluent which can have an adverse effect on the environment

(Fornarelli et al., 2017). Environmental risks arising by the direct discharge of AD effluents

can be associated with greenhouse gas emissions from ammonia volatilization,

eutrophication of soils and surface waters due to nutrient imbalance, and groundwater

nitrogen contamination (Fornarelli et al., 2017).

The AD systems employed in Australia, typically consist of low-rate open anaerobic lagoons,

which are well suited to occupy the vacant land space. Currently, synthetic membrane

covered anaerobic lagoons that can capture the biogas produced and reduce GHG

emissions is highly sought after. As the biogas produced contains a high percentage of

methane (CH4) which is a powerful GHG with a global warming potential (GWP) 21 times

that of carbon dioxide (CO2) (Harris & McCabe, 2015; MTU, 2010). While it has been noted

that anaerobic lagoons are not optimised treatment strategies, they are low-capital

investments which can affect a large degree of organic degradation and methane generation

(Harris & McCabe, 2015; Jensen, Sullivan, Carney, & Batstone, 2014). Major drawbacks of

current management practices are the large land footprint and loss of the environmental and

5

economic value of the wastewater effluent in the form of water and nutrients recovery

(Fornarelli et al., 2017).

A complete degradation of OM and the removal of nutrients present in SWW is not

conceivable using anaerobic treatment alone (Bustillo-Lecompte & Mehrvar, 2015). Due to

the environmental and economic burden associated with current wastewater treatment

practices, innovative cost effective and efficient nutrient removal/recovery systems are

perceived a priority by the red meat processing industry (Fornarelli et al., 2017).

1.3 Gaps in current processes

Several knowledge gaps have been identified in which research is still required to improve

current wastewater treatment methods and reduce the emissions and energy costs of

current wastewater treatment in abattoirs (AMPC, 2014; Harris & McCabe, 2015). One

limitation of anaerobic digestion is that it does not significantly reduce the amount of

inorganic nutrients in the digestate, it favours the accumulation of more bioavailable nitrogen

forms such as ammonium (Franchino, Comino, Bona, & Riggio, 2013). Wastewater treated

through AD processes are still limited by high inorganic nutrient content and generally fail to

meet environmental standards, resulting in AD treated water being channelled into

evaporation ponds. Jensen et al. (2014) found that the treatment of wastewater from cattle

slaughterhouses through conventional anaerobic lagoons alone was inefficient. Cattle yard

waste and paunch waste degrade slowly and require longer treatment times while the

degradability of these streams is also lower and this would lead to accumulation of inert

solids in lagoons, therefore shortening the period between de-sludge events.

Previously, various studies have focused on nitrogen and phosphorus removal mechanism

from municipal wastewater using biological, physical and chemical methods (Blackall,

Crocetti, Saunders, & Bond, 2002; Mallick, 2002; Wang, Yu, Lv, & Yang, 2013). Few studies

have investigated the potential of wet market wastewater and SWW for the production of

microalgae biomass (Maizatul et al., 2017).

1.4 Microalgae

Microalgae are unicellular photosynthetic micro-organisms. They are a very diverse group of

predominantly aquatic photosynthetic organisms that convert sunlight, water and carbon

dioxide to algal biomass (Ruane, Sonnino, & Agostini, 2010). Algae accounts for almost 50%

of the photosynthesis that takes place on Earth (Blinová, Bartošová, & Gerulová, 2015; Chiu

6

et al., 2015). Algae play a vital role in the global carbon cycle by removing elevated

concentrations of carbon dioxide from the environment (Blinová et al., 2015).

Microalgae play an important role in the self-purification of contaminated environment

waterbodies by naturally assimilating nutrients found in the wastewater (Maizatul et al.,

2017; Ramaraj, Tsai, & Chen, 2015). Microalgae use solar energy to supply oxygen required

for aerobic degradation by bacteria and recycle the nutrients responsible for eutrophication

into potentially valuable biomass, representing an inexpensive alternative to conventional

forms of tertiary wastewater treatment (Craggs et al., 1997; Maizatul et al., 2017; Órpez et

al., 2009). Microalgae are an ideal platform for the large-scale production of biomass

because they are fast-growing, solar-powered ‘biofactories’ with low nutrient requirements

(Masojídek & Torzillo, 2008).

Scenedesmus and Chlorella are both green algae belonging to the division Chlorophyta.

Chlorella sp. is a freshwater, unicellular, green microalgae which is widely distributed around

the world. Most species of this genus are spherical and under 10 µm in diameter (Masojídek

& Torzillo, 2008; Wang et al., 2013). Chlorella is the most commonly cultivated eukaryotic

alga due to its robustness and favourable biochemical composition as it contains proteins

(up to 60% of dry weight), polysaccharides (10–15%), lipids (12–15%), unsaturated fatty

acids, and carotenoids (predominantly lutein), as well as some immunostimulators, vitamins,

and minerals (Masojídek & Torzillo, 2008).

There are many factors that can affect microalgae cultivation such as environmental,

operational and biological parameters. Cultivation conditions of microalgae (temperature,

pH, light, nutrient quantity and quality, salinity and aerating/mixing) are the major factors that

influence the growth and productivity of microalgae (Blinová et al., 2015; Fornarelli et al.,

2017; Harun, Yahya, Chik, Kadir, & Pang, 2014).

Most algal species are typically cultured between the temperature range of 16-27°C, with

temperatures lower than 16 °C negatively affecting their growth, whereas temperature higher

than 35 °C being lethal for a number of species (Blinová et al., 2015; Cormier, 2010).

Different Chlorella strains have shown a high temperature tolerance as some strains can

grow between 15 and 40 °C (Masojídek & Torzillo, 2008). Converti, Casazza, Ortiz, Perego,

and Del Borghi (2009) reported that Chlorella vulgaris at 35 °C exhibit a 17% decrease in its

growth rate when compared to at 30 °C. Similarly, Barghbani, Rezaei, and Javanshir (2012)

obtained the highest biomass yield at 30±2 °C. Scenedesmus sp. also show high growth

tolerance within temperatures ranging between 10 to 40 °C (Maizatul et al., 2017).

7

The acceptable pH range for most algal species is between 7 and 9.5 with an optimum

range of 8.2-8.7 at which the microalgae exhibit a higher efficiency in capturing CO2 in the

atmosphere which then induces the improved production of biomass (Blinová et al., 2015;

Cormier, 2010; Maizatul et al., 2017). Xin, Hong-ying, Ke, and Jia (2010) reported that the

proper pH for Scenedesmus sp. LX1’s growth and nutrient uptake was at pH 7 when

ammonium was the main N source in wastewater. Zhang, Wang, and Hong (2014)

suggested that the optimal initial pH values for Chlorella sp. to produce biofuels, combined

with purification of wastewater, should be controlled between 7.0 and 9.0.

An efficient light source is pre‐requisite for rapid production of microalgae biomass as light is

one of the most important factors for microalgae growth (Chikako Okumura, 2014).

Considering industrial applications and ratio between the cost of energy and the

corresponding biomass productions, 12 to 15 hours duration for the illuminated phase is

generally considered as optimal for algae growth (Harun et al., 2014). Light cycle allows for

lower production costs, an increase in final concentration and allowing cell division to occur

under dark conditions for many unicellular photosynthetic cultures, with some cultures cell

division occurs in both illuminated and dark phase (Blinová et al., 2015; Harun et al., 2014).

Hultberg, Jönsson, Bergstrand, Carlsson, and Sveriges (2014) found higher amount of

biomass was achieved when using yellow, white and red light after 7 days of Chlorella

vulgaris growth.

Mixing is necessary to prevent the sedimentation of algae and homogeneous conditions, and

ensure that all cells of the population are equally exposed to the light and nutrients, to avoid

thermal stratification and the occurrence of nutrient and pH gradients, and improves gas

exchange between the culture medium and the air (Blinová et al., 2015; Craggs et al., 1997).

Overall, sufficient mixing is seen to improve the photosynthetic efficiency and growth of

microalgae cells (Blinová et al., 2015).

1.4.1 Potential of microalgae wastewater treatment

The cultivation of microalgae in wastewater offers the combined advantages of treating the

wastewaters and simultaneously producing algal biomass. This biomass produced can be

further exploited as a source for protein complements and food additives, energy (biogas

and fuels), agriculture (fertilizers and soil conditioners), pharmaceuticals, cosmetics and

other valuable chemicals (Cormier, 2010; Mallick, 2002).

8

Algae have been shown to thrive in nutrient rich and polluted water, removing nutrients and

sequestering carbon as they grow. Microalgae, in particular, are a diverse group adaptable

to many environments, and many species are well suited to thrive in wastewater conditions

(Cormier, 2010).

Many microalgae species have been used in the phycoremediation process of wastewater.

Chlorella sp. and Scenedesmus sp. are the most common and extensively used microalgae.

Scenedesmus sp. has high potential to tolerate acidic eutrophic water conditions and a wide

range of temperature for growth (Maizatul et al., 2017). Chlorella sp. is one of the most

extensively used microalgae for nutrient removal due to its ability to grow at high

concentration of inorganic nutrients and its suitability for use as crop fertilizer that has shown

to lead to positive effects on the environment in terms of the health of soils and plants

(Fornarelli et al., 2017). Chlorella sp. and Scenedesmus sp. are usually the predominant of

the phytoplanktonic communities in oxidation ponds (Masseret, Amblard, Bourdier, &

Sargos, 2000), and in high-rate algal ponds (Canovas et al., 1996; Pittman, Dean, &

Osundeko, 2011). Among several species of microalgae of Chlorophyceae, Chlorella sp.

and Scenedesmus sp., are common in the production of animal feed supplements, fertilizers,

health skin products, and other applications (Maizatul et al., 2017; Pulz & Gross, 2004).

Microalgae consortium tend to be favoured over monocultures as they tend to be more

effective than the pure cultures in biological treatment systems as different species have the

ability to consume different pollutants in the wastewater (Fornarelli et al., 2017; Taskan,

2016; Ye, Ohtake, & Toda, 1988). Hernández et al. (2016) reported microalgal–bacterial

consortia of raw diluted piggery SWW achieved high capacity to remove organic matter and

nutrients. Studies have been carried out using mostly pure or mixed algae cultures for the

treatment of nutrients from wastewaters (Asmare, Demessie, & Murthy, 2014; Yang et al.,

2011). Including recent studies conducted at Murdoch University demonstrating that

Chlorella sp., Scenedesmus sp. and pennate diatom can grow efficiently on undiluted AD

piggery effluents (Ayre, 2013; Fornarelli et al., 2017). Wang et al. (2013) found that the use

of Chlorella sp. culture in wastewater to reduce nutrients and produce microalgae biomass is

a promising approach for the production of renewable energy as an additional benefit from

wastewater treatment. Numerous studies have focused on Chlorella sp. which can remove

various phosphorus and nitrogen compounds and can also be easily cultivated (Taskan,

2016; Yan, Luo, & Zheng, 2013; Yang et al., 2011).

Figure 1 illustrates the proposed integration of microalgae cultivation within the current

wastewater treatment process of abattoirs. It highlights the current primary processes

9

employed in abattoirs (i.e. anaerobic digestion) and the potential integration of microalgae

cultivation as a subsequent treatment step post AD. The process below shows the natural

capture and assimilation of CO2 produced through AD by the microalgae through

photosynthesis. In addition, the major advantage of the proposed system is that the algae

biomass produced can be used to generate additional revenue streams through use as an

animal feed, energy source as biogas, fertilizer and high-value pigments (Fornarelli et al.,

2017). The treated nutrient depleted water post algal cultivation can then be reused on site

providing it meets standards or safely disposed.

Figure 1: Current and proposed microalgae integration treatment within abattoirs.

(Fornarelli et al., 2017)

1.4.2 Benefits of microalgae wastewater treatment

Microalgae are photosynthetic microorganisms that are widely employed in the treatment of

wastewater due to their inherent ability to assimilate nutrients efficiently for the production of

valuable biomass (waste-to-profit) (Choi & Lee, 2015; Pittman et al., 2011; Wang et al.,

2013). Microalgae cultivation is a natural method for removing nutrients and provides an

alternative treatment option for wastewater with numerous advantages, mainly depuration,

recovery of nutrients and exploitation of the produced biomass (Hernández et al., 2016).

The cultivation of microalgae on AD effluents is seen as a positive impact on the operation

costs, as these inorganic nutrients in wastewaters are suitable and cost effective alternatives

to synthetic nutrients (Fornarelli et al., 2017; Wang et al., 2013). Biomass production from

algae seems promising because it can be used for animal feed, pharmaceutical and energy

purposes, such as biodiesel. Although the OM and nutrient content of SWW are suitable

sources for the generation of algae, the number of studies on the treatment of SWW so far

10

has been limited (Taskan, 2016). One major attraction of microalgae as a biofuel feedstock

is that they can be effectively grown in conditions which require minimal to no freshwater

input unlike many plant-based biofuel crops and does not compete for arable land. It can

utilise land which is otherwise non-productive to plant crops, thus making the process

potentially sustainable with regard to preserving freshwater resources (Pittman et al., 2011).

Microalgae also offer an economic and environmental advantage due to its rapid growth rate

and favourable biochemical content (i.e. lipid and protein) and the fact that its cultivation

does not compete with food crops for land and freshwater (Blinová et al., 2015; Cai, Park, &

Li, 2013; Cormier, 2010; Fornarelli et al., 2017; Wang et al., 2013; Wijffels & Barbosa, 2010).

Research indicated that microalgae can be used for simultaneous biogas upgrading and

biogas slurry nutrient reduction (Yan, Muñoz, Zhu, & Wang, 2016). Microalgae have

potential to play an important remediation role particularly during the final (tertiary) treatment

phase of wastewater (Pittman et al., 2011).

Overall the cultivation of microalgae on abattoir effluent reduces the environmental impact of

ADAE through the reduction of GHG emissions and eutrophication outcomes whilst

producing valuable biomass and nutrient depleted water that can be recycled on site

(Fornarelli et al., 2017).

1.5 Economics

Microalgae can serve as an appropriate alternative feedstock for biofuel production, the high

microalgal cultivation cost has been a major reason why algal-based wastewater treatment

is not used extensively by the wastewater industry (Pittman et al., 2011; Wang et al., 2016).

One of the feasible solutions for cost reduction is to couple microalgal biomass production

system with wastewater treatment (phycoremediation) (Maizatul et al., 2017; Wang et al.,

2016).

Characteristics such as the low energy consumption and production of a valuable biomass

are of significant importance in order to achieve energy-neutral balance and self-sufficiency

in wastewater treatment plants (Díez-Montero, Solimeno, Uggetti, García-Galán, & García,

2018). The operating costs of algal based systems are low because wastewater inherently

contains inorganic nutrients that are available to the microalgae. The treatment of

wastewater with microalgae is not associated with any additional pollution. This method can

offer an ecologically safer, cheaper and more efficient way of removing nutrients from

wastewaters (Choi & Lee, 2015). Microalgae may represent carbon neutral fuel feedstock,

which does not add to total carbon emissions as it offsets carbon dioxide emitted through

11

burning of biomass and absorbs the same amount of carbon dioxide during its growth phase

through photosynthesis (Hossain, Salleh, Nasrulhaq Boyce, Partha, & Husri, 2008; Prakash,

Gautom, & Sharma, 2015).

There have been numerous assessments as to the economic viability of algal biofuels.

Pittman et al. (2011) argued that dual-use microalgae cultivation for wastewater treatment

coupled with biofuel generation is an attractive option and shows both economic and

environmental sustainability. Cormier (2010) produced a STELLA model and conducted an

economic analysis under a best, average, and worst-case scenario and determined that

algal production can be profitable as a wastewater treatment, depending on the market

conditions and cost of energy. Fornarelli et al. (2017) provides techno-economic feasibility

report that demonstrates microalgae cultivation as a cost-effective treatment technology, that

capture nutrient and harvest algae biomass whilst producing a nutrient-depleted water

effluent. Díez-Montero et al. (2018) assessed the energy balance of hypothetical microalgae-

based wastewater treatment plant of 13 different climate conditions and found that electrical

energy balance was not only neutral but positive in all locations and the feasibility of the

positive energy balance can be achieved.

1.6 Aim and objective

The aim of this study was to evaluate and compare the efficiency of locally isolated

microalgae for treating undiluted ADAE. The biomass generated from the process can be a

source of revenue for the targeted abattoir. The cultivation of mono and mixed culture

(Chlorella sp. and Scenedesmus sp.) on undiluted ADAE was assessed in this study with

emphasis on growth rate, biomass productivity and nutrient removal rates.

The objective for the study is to identify and establish the growth of local microalgae strains

in undiluted ADAE for the bioremediation and biomass production of the waste stream. The

ideal microalgae strain capable of growing in ADAE should have the following

characteristics:

• Tolerance to raw ADAE,

• Cell growth rate,

• Biomass production, and

• Reduction in nutrient concentration of ADAE.

No current literature is available on such work whereby, if the tested species are found to

perform in these conditions, it will be the first to our knowledge. Additional research with the

12

potential of scale up experiment would need to be performed for possible future integration

of microalgae production within abattoirs, providing an environmental, economic and

sustainable waste management system.

2. Methods

The different freshwater microalgae (Chlorella sp. and Scenedesmus sp.) species used in

this study were obtained from the culture collection of the Algae Research & Development

Centre at Murdoch University. These microalgae strains were previously isolated locally and

found to grow and successfully treat high strength piggery effluent (Ayre, 2013). The ADAE

used in this study was sourced from a local beef abattoir within Western Australia. Due to a

confidentiality agreement between the local abattoir and Murdoch University, it cannot be

named and hereafter will be known as Site A.

The ADAE samples were collected from an open anaerobic pond located at Site A and

transported to Murdoch University Algae Research & Development Centre in storage

containers. The effluent was stored in dark and cool conditions to avoid changes to its

biochemical composition. For the experiment, a 2:1 ratio consisting of 200mL ADAE and

100mL of each microalgae culture was prepared. A total of 12, 500mL Erlenmeyer flasks

were used which consisted of four replicates for each microalgae species that consisted of

Chlorella sp., Scenedesmus sp. and a mixed consortium culture (50mL Chlorella sp. + 50mL

Scenedesmus sp.). The nutrient concentration for all treatment samples were analysed for

COD, ammonium, phosphate, nitrite and nitrate. The mixing of cultures was brought forward

using magnetic stirrers at 240 rpm to maintain homogeneous conditions. The experiment

was conducted under laboratory controlled conditions at Murdoch University, Algae

Research & Development Centre over a period of 15 days.

2.1 Nutrient composition of ADAE and removal rates

The concentration of different nutrients in the wastewater was characterized using a Hanna

photometer HI 83099, Hanna COD reader HI 839800 and Hanna reagents according to the

methods provided by the manufacturer.

An initial characterisation of the raw ADAE sample was analysed before inoculation and at

the start of the experiment (Day 0). The nutrient composition of raw ADAE collected from

Site A is characterized in Table 2. The nutrient composition of the cultures after inoculation

was measured every fourth day by filtering 10mL of each culture through Vacuum filtration

manifold Milipore 1225, collecting and freezing the filtrate for nutrient measurement to

13

characterise nutrient removal/increase rate.

Table 2: Nutrient Composition of Raw ADAE before inoculation.

2.2 Cell count

Cell count for each culture was conducted to evaluate the progress and growth of the

different microalgae species in ADAE over time. Using the same protocol outlined in

Moheimani, Borowitzka, Isdepsky, and Sing (2013), cell counts were conducted on each

replicate treatment every second day. Cell counts were performed using a modified

Neubauer haemocytometer and LEICA ICC50 microscope. Contamination of cultures by

other microorganisms (e.g. bacteria and protozoans) was monitored through this method

throughout the experimental period.

The culture was stirred before extraction to homogenise samples, 1mL of algae culture was

placed on the Neubauer Haemocytometer and placed under LEICA ICC50 microscope,

allowing a few minutes for the culture to settle. The cells in 5 squares were counted for each

replicate then multiplied by 5 to obtain the number of cells per central counting area. The cell

number is calculated using Equation 1.

Equation 1: Cell number mL -1 calculation

𝐶𝑒𝑙𝑙 𝑁𝑢𝑚𝑏𝑒𝑡 (𝑐𝑒𝑙𝑙𝑠 𝑚𝑙−1) = 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 x 1000

Growth and specific growth rates of the cultures were calculated using Equation 2 and

Equation 3.

Equation 2: Growth rate

𝑘′ =Ln(𝐶𝑒𝑙𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑖𝑜𝑛 𝑎𝑡 𝑡ℎ𝑒 𝑒𝑛𝑑 𝑜𝑓 𝑒𝑥𝑝𝑜𝑛𝑒𝑛𝑡𝑖𝑎𝑙 𝑝ℎ𝑎𝑠𝑒 𝐼𝑛𝑖𝑡𝑖𝑎𝑙⁄ 𝑐𝑒𝑙𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑎𝑡 𝑑𝑎𝑦 0)

𝐿𝑎𝑠𝑡 𝑑𝑎𝑦 𝑜𝑓 𝑒𝑥𝑝𝑜𝑛𝑒𝑛𝑡𝑖𝑎𝑙 𝑝ℎ𝑎𝑠𝑒 − 𝐷𝑎𝑦 0

Equation 3: Specific growth rate

𝜇 = 𝐾′/ 𝐿𝑛 2

Characterisation COD 𝑁𝐻3

− 𝑁 𝑁𝐻3 𝑁𝐻4

+ 𝑃𝑂43− 𝑃 𝑃2𝑂5 𝑁𝑂2

− 𝑁𝑂2

−

− 𝑁 𝑁𝑎𝑁𝑂2

𝑁𝑂3−

− 𝑁 𝑁𝑂3

−

Raw ADAE

(mgL-1) 302.5 221.5 269.5 286 89.5 29.5 67 0 0 0 0 0

14

2.3 Biomass yield

Biomass yield was measured for each culture every fourth day using methods described in

Moheimani et al. (2013). Five pre-combusted Whattman 25mm GF-C fibre glass filters were

used for each replicate where 2 filters were used to measure biomass yield while 3

remaining filters were used to concentrate samples for protein, chlorophyll and any other

biochemical compound analysis. For the biomass analysis, the filters were first weighted to 5

decimal places on an analytical balance. The weighed filters were subsequently placed into

a vacuum Millipore 1225 filtration manifold where 5mL of algae culture per filter were added

and filtered using vacuum filtration unit. The filters are then folded in half with three filters

placed in foil and frozen, the two remaining filters are placed in crucibles then into 90 °C

oven for at least 6 hours. The filters are then placed into a desiccator over KOH pellets for at

least one hour to cool. After that, the filter plus algae weight was measured using an analytic

balance to 5 decimal places. The dry weight was calculated using Equation 4.

Equation 4: Dry weight

𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 (𝐷𝑊) = (𝑤𝑒𝑖𝑔ℎ𝑡 𝑓𝑖𝑙𝑡𝑒𝑟 𝑝𝑙𝑢𝑠 𝑎𝑙𝑔𝑎𝑒) − (𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑓𝑖𝑙𝑡𝑒𝑟)

Subsequently, the filters were then placed into a 450°C furnace for another 6 hours and later

placed into a desiccator over KOH pellets to cool for at least one hour. The filters are then

weighed to obtain the weight after ashing. The ash free dry weight (AFDW) in gL-1 is

calculated using Equation 5.

Equation 5: Ash free dry weight

𝐴𝑠ℎ 𝐹𝑟𝑒𝑒 𝐷𝑟𝑦 𝑊𝑒𝑖𝑔ℎ𝑡 (𝐴𝐹𝐷𝑊) = 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 – 𝑊𝑒𝑖𝑔ℎ𝑡 𝑎𝑓𝑡𝑒𝑟 𝑎𝑠ℎ𝑖𝑛𝑔

𝑚𝑙 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 x1000

Biomass growth is measured by calculating the productivity of algae cultures and comparing

the different algae. When dealing with wastewater, AFDW is preferred measure for biomass

productivity as this gravimetric measurement of ash-free (organic) dry weight is the most

reliable method (Moheimani et al., 2013). The biomass productivity rate is calculated using

Equation 6.

Equation 6: Biomass productivity

𝐵𝑖𝑜𝑚𝑎𝑠𝑠 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑔𝐿−1𝑑−1) = ( 𝑝𝑙𝑎𝑡𝑒𝑎𝑢 𝑝𝑒𝑟𝑖𝑜𝑑 – 𝑑𝑎𝑦 0 𝑏𝑖𝑜𝑚𝑎𝑠𝑠)

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑎𝑦𝑠

15

2.4 Quantum yield

During the cultivation period, the physiological condition of the microalgae cultures were

evaluated every second day through the measurement of the effective quantum yield

(Fqʹ/Fmʹ) of photochemistry at photosystem II (PSII). 2mL algae culture sample was collected

from each flask and used to measure the Fqʹ/Fmʹ values using a Chlorophyll a fluorescence

fluorometer (Potable AquaPen-C, Photon Systems Instruments, Czech Republic). The

fluorometer was set to the maximum irradiance which was at 3000 µmol photons m-2 s-1

saturation pulse intensity of red light (620 nm) and measurements were carried out on the

cultures sourced taken directly from the cultivation systems.

2.5 Temperature

The culture room is under controlled temperature of 25±2°C and the temperature was

monitored and recorded every day over the growth period.

2.6 Irradiance

The light received by each culture was provided by cool white LED lamps on a 12 hour

light/dark cycle at an irradiance 149±3 µmol photons m-2 s-1.

2.7 pH

The pH was measured on day 0 and 15 using pH probe and Mettler Toledo Seven Go Duo

pro SG98.

2.8 Statistical analysis

All experimental analyses were performed and analyzed by a One-way analysis of variance

(ANOVA) followed by a post-hoc multiple comparison test, Holm-Sidak method. The

significant difference between treatments is declared at P < 0.05. The SigmaPlot v14.0

program was used for all statistical analyses and measurements reported as means ±

standard error.

3. Results

3.1 Cell density and biomass

Figure 2 illustrates the cellular density and microscopic observation of the different

microalgae cultures over the cultivation period. The cellular density of the monoculture

Chlorella sp. and the mixed culture consisting of both Chlorella sp. and Scenedesmus sp.

continuously increased and reached a maximum concertation on day 7 with a subsequent

reduction in cell concentration observed at day 15. However, the Scenedesmus sp. culture

16

declined from the start of the experiment (Day 0) and was naturally overtaken by cells of

Chlorella by day 7 to become the dominant species within the culture. These naturally

growing cells of Chlorella in the Scenedesmus sp. culture was found to be larger in size

when compared to the mono species of Chlorella sp. and the Chlorella sp. cultivated in the

mixed culture.

The microalgae present in the mixed culture will be referred to as mixed Chlorella sp. and

mixed Scenedesmus sp.. The growth curve of the different microalgae cultures in ADAE is

illustrated in Figure 3. The growth curve of all three cultures exhibited the typical pattern of

microalgae growth consisting of an initial lag phase of one day, brought forward due to the

acclimatization to the new environment by the microalgae. Exponential growth up to day 7 is

demonstrated in Chlorella sp. and mixed Chlorella sp., with Scenedesmus sp. and mixed

Scenedesmus sp. both reaching stationary phase by day 3. The growth curve of the

microalgae cultures was used to identify sampling period for nutrient analysis, day 0 in the

lag phase, day 3 for exponential growth phase and day 7 for stationary phase using 10mL

frozen filtrate sampled. Clumping of cells were common as shown in Figure 2, although

additional processes and efforts were taken to disperse and homogenise samples.

Figure 2: Cell comparison of all cultures at 40x magnification.

17

Figure 3: Growth curve showing cell concentration during cultivation period.

Figure 4 shows there was an increase in the biomass yield for Chlorella sp. and mixed

culture up to day 7, after which it began to plateau while the same was observed for

Scenedesmus sp. until day 11.

18

Figure 4: Biomass yield of all algae cultures on ADAE.

The specific growth rates of each culture are highlighted in Figure 5 showing the proportion

increase in cell number per day. The specific growth rate of the mixed Chlorella sp.

(0.294±0.024 d-1) was significantly higher than Scenedesmus sp. at 0.062±0.011 d-1 and

mixed Scenedesmus sp. at 0.168±0.027 d-1. Whilst mono Chlorella sp. at 0.260±0.047 d-1

was significantly higher than Scenedesmus sp.. There was no significant difference in

specific growth rate between Chlorella sp. and mixed Chlorella sp. whilst Scenedesmus sp.

was significantly lower.

19

Figure 5: Specific growth rate of each culture within growth phase. Each of the same letter

represents no significant difference. Each different letter showing significant difference.

Figure 6 reports the biomass productivity for all treatments showing the rate of biomass

generation per day up to plateau period (see Figure 4). There was no significant difference in

the biomass productivity (Figure 6) of Chlorella sp. (0.032±0.001 gL-1 d-1) and mixed

(0.030±0.001 gL-1 d-1) while Scenedesmus sp. (0.024±0.001 gL-1 d-1) was significantly lower.

20

Figure 6: Biomass productivity of each culture up to plateau period. Each of the same letter

represents no significant difference. Each different letter showing significant difference.

3.2 Effective quantum yield, pH and temperature

The effective quantum yield (Fq’/Fm’) in Figure 7 shows the immediate physiological condition

of the microalgae cultures during the cultivation period. The Fq’/Fm’ values for

Scenedesmus sp. trended similarly between day 0-3 with a visible decline in the

photophysiology observed after day 3. Chlorella sp. and mixed culture are both showing

initial low signs of stress, with a gradual decline in photophysiology due to increase in stress

but recovered subsequently indicating adaption to the condition. As the microalgae isn’t

growing in artificial medium thus doesn’t have all the required nutrients at the appropriate

concentration that the algae needs for optimum growth.

21

Figure 7: Effective quantum yield of each culture measured every second day.

Figure 8 illustrates the changes in pH of the different microalgae cultures grown in ADAE

over the cultivation period. Both Chlorella sp. and mixed culture showed minor variation in

pH between day 0 and day 15, whilst there was an increase in the pH of Scenedesmus sp.

culture over this period.

Figure 8: pH at day 0 and day 15 of cultivation.

22

Figure 9 shows the recorded daily temperature during the experiment period with a mean

temperature of 25.1±0.1°C.

Figure 9: Culture room temperature recorded daily.

3.3 Nutrient concentration

The concentration of different nutrient present in the ADAE during the algae growth period is

shown in Figure 10. There was an increase in the COD of the mixed culture when compared

to the other treatments. The highest ammonium removal is seen between day 0 and day 3

for the Chlorella sp. and mixed culture. It is important to note that, there was initially no nitrite

and nitrate present at the start of the experiment in the Scenedesmus sp. cultures and it

remained 0 throughout the experimental period. Nitrate shows a steady increase in the

mixed culture throughout algae growth period this is also seen with Chlorella sp. from day 3.

23

Figure 10: Nutrient concentration during algae growth period of seven days in ADAE.

24

There was no significant difference in COD removal rates found amongst treatments.

Chlorella sp. was found to have significantly higher ammonium removal rate (11.93±1.14

mgLˉ¹dˉ¹) when compared to the mixed culture at 3.75±0.54 mgLˉ¹dˉ¹ and Scenedesmus sp.

at 3.67±0.93 mgLˉ¹dˉ¹ which were significantly lower (Figure 11a). Figure 11(b) shows the

Scenedesmus sp. monoculture had the highest phosphate mean removal rate of 4.25±0.53

mgLˉ¹dˉ¹ compared to Chlorella sp. (-0.10±1.37 mgLˉ¹dˉ¹) which was significantly lower.

Figure 11: (a) Ammonium removal rate and (b) Phosphate removal rate. Each of the same

letter represents no significant difference. Each different letter showing significant difference.

The changes in nitrite and nitrate concentration is reported as increase rates in Table 3 as

there was initially no nitrite and nitrate present within the ADAE before inoculation. There

was, however, nitrite and nitrate present within the microalgae inoculum culture before

inoculation. Table 3 highlights nitrite and nitrate nutrient concentration after inoculation at

day 0 and day 7 with the calculated increase rate during the cultivation period for Chlorella

sp. and mixed culture. Scenedesmus sp. rates aren’t given as 0 nitrite and 0 nitrate was

recorded throughout cultivation period. There was no significant difference in increase rate

amongst nitrite or nitrate cultures due to nitrification taking place.

Table 3: Nitrite and nitrate nutrient concentration and increase rates from day 0 after

inoculation to day 7.

Characterisation

Day 0 Day 7 Increase rate (d ˉ¹) Day 0 Day 7 Increase rate (d ˉ¹)

Chlorella sp. 80.0±5.4 101.3±13.3 3.0 ±2.6 558.0±67.5 1030.5±162.8 67.5±22.4

Mixed 140.0±12.6 110.0±25.0 -4.3 ±3.3 318.6±48.3 1405.2±208.4 155.2±35.1

(a) (b)

𝐍𝟎𝟐−(mgLˉ¹) 𝐍𝟎𝟑

−(mgLˉ¹)

25

4. Discussion

This study examined the growth of microalgae on untreated and undiluted ADAE. The

monocultures of Scenedesmus sp. by day 7 were overtaken and contained only naturally

growing Chlorella cells with a larger cellular morphology. A possible explanation for the

larger sized Chlorella cells found naturally in the Scenedesmus sp. cultures could be due to

the inability of the Chlorella cells to divide. The increase in weight of Chlorella cells could

have contributed to the increase in the biomass productivity within the monocultures of

Scenedesmus sp.. Mixed culture and Chlorella sp. showed highest cell concentration at day

7 by observation, cell concentration and biomass yield.

Chlorella sp. was found to be the most dominant species amongst all different treatments.

The growth rate of mixed Chlorella sp. (0.294±0.024 d-1) and Chlorella sp. (0.260±0.047 d-1)

was significantly higher than Scenedesmus sp.. Ayre et al. (2017) also found that Chlorella

sp. was the dominant species in comparison to other microalgae cultures grown on AD

piggery effluent.

Scenedesmus sp. pH increased from 7 to 10. Su et al. (2012) found similar results with pH

values of Chlorella vulgaris and Scenedesmus rubescens from initial pH of 7 to around 10

after 7 days of cultivation. Chlorella sp. and mixed culture remained around pH 7 at the start

and end of the experiment Zhang et al. (2014) found the pH values showed a change

tendency towards neutrality and ended in a range of 6–9. Generally, photosynthesis could

cause pH to rise with cultivation time. Although slight pH differences in wastewater have not

been shown to completely inhibit algae growth Zhang et al. (2014) found Chlorella sp. could

maintain relatively high growth rate in a wide range of initial pH values. Nutrient removal can

be affected by high pH in wastewater. González, Marciniak, Villaverde, García-Encina, and

Muñoz (2008) found ammonia removal by algae cultured in swine wastewater was inhibited

by pH levels above 9. Prakash et al. (2015) found that the maximum production of the

biomass from Chlorella sp. was recorded at pH 7, while the lowest production was noted at

pH 8. Therefore, the pH of wastewater during the phycoremediation process needs to be

within the optimal pH range (Su et al., 2012).

Chlorella sp. removed up to 96% of ammonium during algae growth period. Franchino et al.

(2013); Li et al. (2011); Wang, Xiong, Hui, and Zeng (2012) all found similar results showing

Chlorella removed 90-99% of ammonium. With Scenedesmus sp. the highest ammonium

removal occurs after day 3, this may be due to Chlorella becoming the dominant species

within the culture. With nitrate increasing this is likely due to nitrification taking place with

bacteria conversion of ammonium to nitrate. Studies by Ogbonna, Yoshizawa, and Tanaka

26

(2000); Su et al. (2012); Xin et al. (2010) all found that when both ammonia and nitrate were

present many species prefer ammonia for growth although nitrate is typically the most

available form of nitrogen. Ogbonna et al. (2000) results showed nitrate removal started

once the ammonia was no longer available from the wastewater and it was not inhibited

even up to nitrate concentrations of 700 mg/L. González et al. (2008) found the main

removal of ammonium was through assimilation of biomass and nitrification. On the other

hand, nitrogen limiting environments can trigger carbohydrate accumulation in cells, which

could lead to biomass loss in cyclically cultivated (light/dark) Chlorella cells (Ogbonna &

Tanaka, 1996). Prakash et al. (2015) found that Chlorella sp. showed highest biomass

production when exposed to 24hr light. Biomass productivity maybe affected when

ammonium is favoured uptake over nitrate as Xin et al. (2010) reported that the algal

biomass concentration was significantly lower when feeding with ammonium than that with

nitrate.

Scenedesmus sp. showed the highest removal of phosphate among all the treatments which

could be potentially related to the naturally growing larger Chlorella cells present in the

culture. Xin et al. (2010) reported with ammonium as the nitrogen source, the maximum algal

density was relatively low, and the nitrogen and phosphorus removal efficiencies were low.

Godos, Blanco, García-Encina, Becares, and Muñoz (2009) found phosphate concentrations

remained rather stable within piggery wastewater with phosphorous removal efficiencies

lower than 10%. More studies in the efficient removal of phosphate must be examined to

elucidate the effect of phosphate metabolism in an algal wastewater treatment system (Lee

& Lee, 2001).

5. Conclusion

Microalgae bioremediation offer great potential for coupling the treatment of various

wastewater with the production of valuable microalgae biomass, minimizing land and water

footprint requirement while simultaneously allowing for waste-to-profit conversion outcome.

The experiment showed Chlorella sp. and the mixed Chlorella sp. were both significantly

higher to Scenedesmus sp. in specific growth. Chlorella sp. and mixed culture was

significantly higher to Scenedesmus sp. in biomass productivity with variation amongst the

removal of nutrients. Chlorella sp. was the most effective treatment being significantly higher

in ammonium removal of up to 11.93±1.14 mgL-1 d-1 and biomass productivity of 31.52±0.81

mgL-1 d-1. The study has shown that adequate nutrients are available for the growth of

microalgae and biomass production on undiluted ADAE. It has highlighted the importance for

further analysis with the use of Chlorella sp. As population and energy demand rise and

water resources become scarcer it is imperative to harness and recycle resources. In

27

conclusion Chlorella sp. can efficiently grow on undiluted ADAE producing valuable biomass

and reducing the harmful ammonium nutrient from ADAE.

To the best of my knowledge, to date, this is the first study to demonstrate growth of

microalgae using undiluted ADAE. This study is adding value to research, for potential of

microalgae integration with wastewater for converting waste-to-profit. Further studies are

required to fully explore the potential of this emerging technology.

6. Recommendations

This experiment was the first step to identifying whether microalgae could grow on undiluted

ADAE. Literature has shown that, in general, consortium of mixed algal culture is favoured

as it can consume different pollutants within the wastewater. A consortium of Chlorella sp.

and other microalgal species that can efficiently grow on undiluted ADAE would further

reduce nutrient concentration within the ADAE.

Previous studies indicate a significant increase in algal biomass productivity when CO2 is

added to the culture. Makareviciene, Andruleviciute, Skorupskaite, and Kasperoviciene

(2011) reported that the biomass yield of Chlorella sp. and Scenedesmus sp. increased

simultaneously with the increase of CO₂ concentrations. With the supplementation of CO₂ to

the proposed process here, microalgal growth could be enhanced.

Implementing optimum cultivation conditions, can maximise removal of wastewater nutrients

to biomass. Choi, Lim, Lee, and Lee (2013) found red LEDs with aeration are optimal for

cultivation of Chlorella sp..

As stationary phase is reached at day 7, semi continuous cultivation is recommended at day

7 or just before. Ayre et al. (2017) states that batch mode isn’t the most efficient for biomass

production in comparison to semicontinuous cultivation.

Further, the use of generated biomass must be tested. Finally, the economics of the

proposed project must be tested prior to any large-scale operation.

28

7. References

Ahmad, M. I., Ejaz, O., Ali, A., Qadir Jahangir Durrani, M. A., & Khan, I. A. (2014). Anaerobic

digestion of waste from a slaughterhouse. Journal of Environmental Chemical

Engineering, 2(3), 1317-1320. doi:10.1016/j.jece.2014.04.001

AMPC. (2014). Evaluation of greenhouse gas mitigation activities undertaken by the red

meat processing industry. Retrieved from

https://www.ampc.com.au/uploads/cgblog/id6/Evaluation-of-Greenhouse-Gas-

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