Growth comparison for treating anaerobic digested effluent in an open pond and inclined reactor by

microalgae

4A-108

Report prepared for the

Co-operative Research Centre for High Integrity Australian Pork

By

Authors: Navid Reza Moheimania*, Mohammad Javad Raeisossadatia, Ashiwin

Vadivelooa, David Parlevlietb, Parisa Bahrib, John Pluskec

Affiliations:

a Algae R&D Centre, School of Veterinary and Life Sciences, Murdoch

University, Western Australia 6150, Australia.

b School of Engineering and Information Technology, Murdoch University,

Western Australia 6150, Australia.

c School of Veterinary and Life Sciences, Murdoch University, Western

Australia 6150, Australia.

* Corresponding author: n.moheimani@murdoch.edu.au

May 2017

Executive Summary

There is no doubt that we need to generate more food for future generations. Pig

production is one of the key promising protein sources. However, mass pig production

would result in generating vast amounts of waste. One acceptable method to treat

piggeries waste is anaerobic digestion (AD). AD would convert organics carbon to

methane, which can generate electricity and heat for piggeries. However, one of the

challenges of AD process is the generated effluent. Anaerobic digesatate piggery

effluent (ADPE) has high ammonium content (toxic to most organisms), is very turbid,

and needs to be further treated. Our previous results showed we can use microalgae to

treat ADPE using raceway ponds. However, raceway ponds can only be operated at a

depth greater than 25 cm. This means that light cannot efficiently penetrate to the depth

of the culture. Our previous results also indicated that due to better light diffusion,

helical tubular (Biocoil) photobioreactors can treat ADPE more efficiently than raceway

ponds. However, the overall Capex of closed photobioreactors may become prohibitive

when treating ADPE, especially if the aim is to generate commodity products. In this

study, we compared growth, productivity and nutrient removal rates of inclined pond

and raceway pond. The main advantage of inclined ponds is the very shallow (0.5cm)

operational depth resulting in much better light availability for microalgal

photosynthesis. In this study, we evaluated the outdoor growth of microalgae in ADPE

through a customised inclined pond (operational depth was 0.5cm) compared to a

conventional paddle wheel driven raceway pond (operational depth was 15cm) with the

same surface area. In terms of volumetric productivity (mg. L-1.d-1) (please see

appendix1), the algal biomass productivity and the nutrient removal rates of the inclined

pond was found to be significantly higher than the raceway pond. This was mainly due

to the improved surface area to volume ratio.

However, it is important to note that the total volume of the raceway pond was four

times greater than inclined reactor (greater depth and operational volume). This resulted

in a significantly higher biomass productivity on a per area basis (g m-2 d-1), refer to

appendix A) in the raceway pond when compared to inclined pond. Our results clearly

indicate that based on the tested operational conditions, raceway ponds are better

candidates for treating ADPE than inclined ponds.

Table of Contents

Executive Summary ................................................................................. 2

1- Introduction ........................................................................................ 5

1.1. Microalgae and their Promise .................................................... 6

1.2. Microalgae based Wastewater Treatment ................................ 7

1.3. Microalgae Cultivation Systems ................................................ 8

1.4. Anaerobic Digestion of Piggery Effluent ................................. 15

1.5. Microalgae Cultivation for the Treatment of ADPE ............. 15

1.6. Aim and Rationale of Current Project .................................... 17

2- Methodology ....................................................................................... 17

2.1. Microalgae culture .................................................................... 17

2.2. ADPE and growth media .......................................................... 18

2.3. Experimental setup and cultivation condition ........................ 22

2.4. Analytical methods .................................................................... 26

2.5. Statistical analysis...................................................................... 27

3- Results and discussion ..................................................................... 28

3.1. Culture conditions ..................................................................... 28

3.2. Ammonium removal rates ........................................................ 35

3.3. Economics ................................................................................... 42

4. Conclusion ........................................................................................ 942

5. Acknowledgements ............................................................................ 44

6. References ........................................................................................... 44

Appendix 1 - Notes ................................................................................. 50

Confidential Information ................................................................... 50

Deficient Report .................................................................................. 50

Ownership of Reports ........................................................................ 50

Appendices .............................................................................................. 51

1- Introduction

Australia produces over 350, 000 tons of pork per annum; attributing 0.5% of the total

global production (Maraseni and Maroulis, 2008). The piggery industry not only plays a

significant role in supporting the development of the rural economy but also helps to

provide beneficial employment opportunities to the local community. Nonetheless, the

commercial husbandry of pigs can be of great concern to the environment as poorly

managed piggery waste can contribute to climate change (carbon footprint) through the

emission of greenhouse gases, outbreak of diseases, pollution of soil, surface and ground

waters by nutrient enrichment and leaching (Maraseni and Maroulis, 2008, Diez et al.,

2001).

In Australia, anaerobic digestion systems are currently the primary treatment method

for piggery effluent. Anaerobic digestions are generally composed of covered outdoor

ponds containing wastewater, which is biologically treated by heterotrophic

microorganisms in the absence of oxygen (Ayre, 2013, Buchanan et al., 2013). Such

systems generally allow for the partial degradation of organic matter, the removal of

solids through settling, production and capture of biogas and odour control. (Leitão et al.,

2006). On the other hand, anaerobic digestion systems produce anaerobic digestate

piggery effluent (ADPE). ADPE is rarely seen to have any positive value and generally

brings forward detrimental effects to the environment and economic costs to the society

(Buchanan et al., 2013). ADPE has a high nitrogen and phosphorous content, can release

volatile gasses (methane) from storage pits and can result in the eutrophication of water

bodies if directly released to the environment (Tucker et al., 2010).

Current available technologies have been shown to be ineffective in handling the

nutrient load associated with the ADPE generated from piggeries (Wang et al., 2010,

Ogbonna et al., 2000). Thus, there is great need for innovative technologies to treat ADPE

efficiently for maximum nutrient recovery and potential recycling and reuse of treated

water within the piggery facility.

1.1. Microalgae and their Promise

Microalgae are photoautotrophs, which are unicellular in size (ranging from 0.2 µm up

to 100 µm) and are classified to various different Phyla (major taxonomic groups)

(Borowitzka, 2001). Microalgae are generally found in aquatic or moist habitats such as

fresh, marine and hyper saline waters. They can also grow well in various soils (especially

when moist), and in drastic environmental conditions such as salt lakes, hot springs, ice

and snow (Borowitzka, 2001). Owing to their fast growth rates and the fact that they

produce more than 50% of atmospheric oxygen content, microalgae are reputed to be

nature's most successful photosynthetic organism in harnessing available sunlight (Sayre,

2010, Chapman, 2013). Moreover, when compared to higher plants, microalgae are

distinguished with enhanced photosynthetic efficiency (PE) due to multiple different

factors, such as the reduction in a number of internally competitive physiological

functions, fast reproduction rates, limited nutrient requirements, and their ability to adapt

to a wider range of spectral irradiances (Gordon and Polle, 2007, Carvalho et al., 2011).

The distinctive advantages of microalgae in not competing with regular food crops over

the need for arable land and their enhanced potential in scavenging and utilising nutrients

such as nitrogen and phosphorus from waste effluents makes them as excellent candidates

for the field of bioremediation (Munoz and Guieysse, 2006, Suresh and Ravishankar,

2004).

1.2. Microalgae based Wastewater Treatment

The successful use of microalgae for the bioremediation of various different types of

wastewater has been reported for over half a century (Oswald and Gotass, 1957, Abdel-

Raouf et al., 2012, Delrue et al., 2016). Due to their inherent ability of stripping and

utilizing organic and inorganic nutrients, algal phytoremediation is an environmentally

favourable solution for the treatment of wastewater (An et al., 2003, Olguín et al., 2003).

Due to this natural potential, microalgae hold an enormous potential for the later steps of

wastewater treatment, especially for the reduction of nitrogen (NH3 and NH4+),

phosphorus and chemical of oxygen demand (COD) (Abdel-Raouf et al., 2012).

The cultivation of microalgae on wastewater holds great promise as it combines both

the treatment of a waste stream with the production of biomass which can be further

converted into various commodities such as aquaculture and animal feed, biofertilisers,

biofuels and bioactive products (An et al., 2003, Rawat et al., 2011, Delrue et al., 2016).

Using waste effluent as a culture medium represents an ideal solution in reducing the high

overall cost commonly associated with the mass microalgae cultivation, especially if the

aim is producing commodity products (Delrue et al., 2016). Moreover, the added ability

of microalgae to grow via different nutritional conditions such as photoautotrophic,

mixotrophic and heterotrophic enhances its capabilities in removing various different

types of pollutants and chemical from aqueous matrices. For example, microalgae cells

have also been found to be efficient in degrading and acquiring complex molecules such

heavy metals, hydrocarbons and antibiotics from waste streams (Delrue et al., 2016).

Further, microalgae growth is not inversely limited by the decreasing concentration of

pollutant (nutrients) as observed with strictly heterotrophic microorganisms (Delrue et

al., 2016). The ability of microalgae in sequestrating carbon (CO2) allows for mitigation

of greenhouse gases (GHG) emissions. The synchronised algal-bacteria relationship

established is also ideally synergetic for the bioremediation of wastewater (Munoz and

Guieysse, 2006). Through photosynthesis, microalgae provide oxygen required by

aerobic bacteria for the mineralisation of organic matter as well as the oxidation of NH4+

(Munoz and Guieysse, 2006). In return, the bacteria supply carbon dioxide for the growth

of microalgae, significantly reducing the amount of oxygen required for the overall

wastewater treatment process (Delrue et al., 2016).

1.3. Microalgae Cultivation Systems

In order to fulfil the global demand for various products and applications, successful

commercial scale cultivation systems of microalgae are required (Spolaore et al. 2006).

Currently the mass cultivation of microalgae can be classified into open cultivation

systems in which cultures are directly exposed to the environment and closed cultivation

systems where the culture is enclosed in a transparent vessel. Nonetheless, both these

systems have their own pros and cons (Borowitzka, 1999).

1. Open Cultivation Systems

Due to their larger capacity and significantly lower overall cost-associated capital cost,

open cultivation systems are the simplest and most commonly used method for mass

commercial cultivation of microalgae (Borowitzka and Moheimani, 2013). Open

cultivation systems can either be naturally forming complexes such as shallow lagoons

and ponds, or fabricated structures such as inclined, circular and raceway ponds

(Borowitzka and Moheimani, 2013). Open ponds are subjected to incident sunlight as

their source of illumination. The major advantages associated with the use of open

systems for the cultivation of microalgae are a) their low capital and operating cost; b)

simple design; and c) their relative ease of operation (Borowitzka and Moheimani, 2013).

Nonetheless, despite being a cost effective method for the cultivation of microalgae, open

based systems are limited by low productivity and the high risk of biotic contamination

such as form other heterotrophic algae, bacteria and protozoans which can significantly

outcompete and diminish overall biomass productivity of the desired microalgae

(Borowitzka and Moheimani, 2013). In addition, due to their direct exposure to the

environment, open ponds are subject to robust changes in culture parameters such as pH,

temperature and light resulting in lower productivity of the cultivated microalgae

(Oswald, 1988, Chen et al., 2011).

Lagoons: Lagoons are naturally forming lakes, which have been used for the

cultivation of microalgae for thousands of years. Currently, the largest commercial

microalgae pond in the world is located in Hutt lagoon (28.1621° S, 114.2450° E),

Western Australia (Borowitzka and Borowitzka, 1990). Hutt lagoon plant is currently

owned by BASF for the production of β -carotene from the green alga Dunaliella salina

(Figure 1). The inherent high salinity of such lagoons helps reduce cross-contamination

of other organisms while maintaining the propagation of the desired species.

Figure 1. BASF owned Hutt lagoon plant (http://www.bsb.murdoch.edu.au/groups/beam/BEAM-

Appl0.html).

In general, shallow and deep lagoons allow for low cost mass cultivation of microalgae.

Nevertheless, they are severely limited by various factors such as poor mixing (through

wind flow and convection) and many other uncontrolled environmental variables (i.e.

temperature, pH and salinity) resulting in significantly low areal biomass productivity

(Borowitzka, 2005). Such open cultivation systems have been previously used for the

simple treatment of wastewater at a later stage, but have been found to be inefficient

(Buchanan et al., 2013).

Raceways Ponds: Raceway ponds are the most preferred open system for growing

microalgae. They have reasonably high production efficacy and require low

construction and maintenance cost (Jorquera et al., 2010). Raceway ponds are composed

of artificial shallow ponds (0.2-0.6m) that are typically constructed of concrete with an

inbuilt lining of plastic sheets or fiberglass liner (Borowitzka, 2005). As shown in

Figure 2, paddle wheel driven raceway ponds are normally designed as closed loops,

oval in shape with baffles or recirculation channels and have a relatively large surface

area to volume ratio for improving light penetration and distribution (Borowitzka,

1999). Cultures in open raceway ponds are mechanically mixed by paddle wheels, water

jets, pumps or propellers to prevent the sedimentation of algal cells at the bottom and to

maximize light availability to cells (Borowitzka and Moheimani, 2013).

Figure 2. Earthrise paddle wheel driven raceway ponds used for culturing Spirulina platensis

(http://www.bsb.murdoch.edu.au/groups/beam/BEAM-Appl4a.html)

Inclined open ponds: In inclined ponds the algae culture flows down an inclined

surface and then is collected at the bottom and pumped back to the top of the incline

(Borowitzka and Moheimani 2013). Microalgal inclined open cultivation systems

(Figure 3) were developed due to their added advantages such as high turbulent flow

rates and thin culture layers, resulting in higher cell densities and improved surface area

to volume ratio when compared to raceway ponds (Setlik et al., 1970, Doucha and

Livansky, 1995). In such a system, the microalgae culture travels down an inclined

surface, is collected at the bottom and is recirculated to the top of the incline surface by

a submerged pump (Setlik et al., 1970). Culture depth is such systems can be as low as

0.5 cm. Inclined systems were initially developed and used in the Czech Republic where

cultures were distributed and continuously recirculated down a 3% inclined glass

surface during the day through pumping and at night were stored in large mixed and

aerated vessels (Setlik et al., 1970, Doucha and Livansky, 1995). Such an operation was

found to reduce the overall cost of pumping and reduces the need of cooling the culture

at night. In Dongara Western Australia (29.2500° S, 114.9300° E), a 0.5 ha pilot scale

inclined system with a greater slope was constructed using a plastic lined shallow pond

on an inclined hillside. This pond was used to cultivate Chlorella with an annual

average productivity of around 20 g m-2 d-1 (Borowitzka, 1999).

Figure 3. Sloping shallow cascade system at Trebon, Czech Republic with baffles installed to

increase turbulence (image is the courtesy of Borowitzka & Moheimani 2013).

2. Closed Systems:

The recent exploitation of microalgae for the production of various high value

products such as nutraceuticals and fine chemicals have brought forward the need for

developing closed photobioreactors which are free of contamination and have

reasonably higher microalgae productivity rates compared with open ponds (Ugwu et

al., 2008, Borowitzka, 1999). Closed cultivation systems (i.e. photobioreactors - Figure

4) are typically made up of either plastic or glass for either microalgae to be grown

photoautotrophically or mixotrophically outdoors using natural sunlight or indoors

using artificial lighting (Ugwu et al., 2008, Borowitzka, 1999). Mixing in closed

photobioreactors is ensured by the recirculation of the entire culture using external

pumps or airlifts, maximising light exposure to cells and allowing for better gas

exchange (Ugwu et al., 2008, Borowitzka, 1999). Closed photobioreactors offer better

control of growth conditions when compared, to open systems leading to higher

biomass productivity and the clean proliferation of monocultures free of contamination

(Ugwu et al., 2008). However, due to their significant high capital and operational costs,

closed photobioreactors are currently only used for the production of high value

products (Ugwu et al., 2008).

Figure 4. Different closed photobioreactor designs (a) a stirred and aerated carboy, (b) bag photobioreactor,

(c) 1000 L outdoor airlift driven tubular photobioreactor (Biocoil) and (d) inclined plate photobioreactor

(Moheimani 2005).

Recent innovation in the design and operation of closed systems has brought forward

multiple types of photobioreactors with various added advantages and commercial

applications. Nevertheless, many of these new designs are based on two basic outlines.

Flat plate photobioreactors (Figure 4d) consisting of a transparent rectangular container

(Tredici and Zitelli, 1997, Pulz, 1994, Hu et al., 1996); or tubular photobioreactors

(Figure 4c) composed of solar collector tubing made from either glass or plastic

(polyethylene) in which microalgae culture is recirculated by aeration or external pumps

(Torzillo, 1997, Richmond et al., 1993, Miyamoto et al., 1988)..

1.4. Anaerobic Digestion of Piggery Effluent

Anaerobic digestion (AD) is the one of the most common treatment options for treating

activated sludge from sewage treatment facilities, as well as for wastewaters abundant

with dissolved organic matter. The product arising from this treatment is biogas (i.e.

methane) and a liquid effluent rich in inorganic nitrogen (in the form NH4 +), phosphorus

(in the form PO4 3- ), pathogen load, and biological oxygen demand (Delrue et al., 2016).

Currently, more than 80% of Australian piggeries utilise anaerobic or a sequence of

facultative ponds for the treatment of their piggery wastewater (Piazza Research 2010).

However, due to their elevated nutrient and organic content, anaerobic digitate piggery

effluent (ADPE) cannot be discharged unless it has been treated (Tucker et al., 2010).

Currently, the most common disposal method for anaerobically treated solid and liquid

piggery waste is by sprinkling these on land as a fertiliser (Piazza Research 2010). In

addition, ADPE is also either reused within the shed and/or as a wash down water as well

as flushing water in piggeries (Piazza Research 2010).

1.5. Microalgae Cultivation for the Treatment of ADPE

The combination of microalgae cultivation with the treatment of piggery wastewater

has been studied over several decades due to the enormous potential benefits from such

integrated systems. However, the majority of these studies have only been able to grow

microalgae on diluted ADPE as un-diluted ADPE can inhibit algal growth due to its very

high turbidity (colour), high ammonia content as well as high pH (Chung et al., 1978,

Chiu et al., 1980, Fallowfield and Barret, 1985, Hu, 2013). Moreover, various pre-

treatment steps such as filtration and autoclaving of ADPE (Park et al., 2010),

electroflocculation to remove suspended particles (Aguirre et al., 2011), and the addition

of chemicals such as ferric chloride and freshwater to reduce turbidity (Barlow et al.,

1975) have been shown to be required before introduction of microalgae to the ADPE.

Diluting ADPE is impractical under most Australian climatic conditions where fresh

water is very limited. Therefore, there is great need for the bioprospecting of microalgal

species capable of treating undiluted ADPE. Through our previously funded Pork CRC

project 4A-106: “Growth development and use of algae on piggeries untreated anaerobic

digestion effluent”, Ayre et al. (2017) isolated and identified multiple strains of

microalgae capable of growing on undiluted ADPE with up to 1600 mg L-1 NH4+ in

paddle wheel driven raceway ponds. This proof-of-concept study clearly illustrated the

potential of culturing locally isolated strains of microalgae in undiluted ADPE with high

ammonium content for the bioremediation of the effluent. However, the high turbidity

(dark colour) of ADPE was found to restrict the growth and productivity of the isolated

strains of microalgae. Irrespective of the cultivation system, light (quantity and quality)

is by far the main limiting factor of any nutrient replete microalgae culture where

photosynthesis is seen to correlate with an increase in light irradiance until maximum

algal growth is achieved at the light saturation point (Richmond, 2004, Vadiveloo et al.,

2017, Bouterfas et al., 2002).

To date, the main cultivation systems used for treating ADPE is open paddle wheel

driven raceway ponds. The raceway ponds require minimum 30-50cm depth for

operation. When cultivated in undiluted ADPE, a combination of the effluent turbidity

and the depth of the algal pond significantly reduce the availability of light to the algal

cells, negatively affecting their growth and productivity. Thus, there is great need for

innovative options to maximise the availability of light for algal cells when cultivated

directly in undiluted ADPE.

1.6. Aim and Rationale of Current Project

Identifying these shortcomings, in this study we investigated the potential use of a

customised inclined thin layer open cultivation system for treating undiluted ADPE under

the outdoor climatic conditions of Perth, Western Australia. Inclined open systems can

be operated at less than 1cm in depth. Such low depth would allow for improved light

distribution in the pond and to the microalgae culture potentially resulting in higher

biomass productivity. Moreover, thin layer cultivation can reduce the negative effect of

ADPE turbidity on microalgae growth by creating a shorter light path (2-5 cm).

Therefore, this study specifically evaluated the potential of a customised inclined open

system when compared to typical paddle wheel driven raceway ponds for 1) microalgal

cultivation and 2) nutrient removal rates and bioremediation of ADPE. We also tested the

lipid productivity of grown biomass.

The use of innovative cultivation systems such as the inclined open pond represents a

sustainable production system for both the enhanced treatment of ADPE and the

improved cultivation of algae biomass. The improved cultivation of algae on piggery

effluent allows for the potential of an income from what would otherwise be a waste

stream. Converting piggery effluent to high-value crops increases the productivity and

profitability of piggeries as well as reducing their carbon footprint.

2- Methodology

2.1. Microalgae culture

We previously isolated microalgal consortium used in this study (Ayre et al 2017). The

consortium was a mixture of Chlorella sp. and Scenedesmus sp (Figure 5).

Figure 5. Dominated Scenedesmus (a) and Chlorella (b) found in inclined and raceway ponds for

the duration of cultivation.

2.2. ADPE and growth media

Anaerobic digestate piggery effluent (ADPE) used in this study was collected from the

Medina Research Station located in Kwinana, Western Australia (32.2376° S, 115.8285°

E). The Medina research station employs a biological anaerobic digestion pond to treat

its wastewater, which includes a covered anaerobic digestion pond (Figure 6).

Figure 6. Photograph of the covered anaerobic digestion pond at Medina Research Station

(photographed by Ayre).

Despite the anaerobic treatment process, the ADPE still contains very high inorganic

nutrient (e.g. nitrogen and phosphorous) load at the point of discharge to the evaporation

pond. The ADPE was sand-filtered (Figure 7) and used for cultivation of microalgae

without any further pre-treatment. Physiochemical properties of the sand-filtered ADPE

were characterised using standard protocols.

Table 1. Chemical composition of untreated and undiluted ADPE used for the growth of the

microalgae).

Parameter Value

Ammonia (mg L-1 NH4+-N) 960-1000

Total Phosphate, (mg L-1 PO4-P) 25.0 – 26.5

Nitrite (µg L-1 NO2-N) 8.0 – 8.5

Magnesium (Mg L-1 mg) 165 - 175

Potassium (mg L-1 K) 530 - 545

Total Iron (mg L-1 Fe) 8.5 -9.5

Nitrate (mg L-1 NO3-N) 14.0 14.5

Chemical Oxygen Demand, COD (mg L-1) 1200 - 1350

Total nitrogen (mg L-1 N) 1050 - 1101

Figure 7. The setup of the sand filter. The materials layered into the drum from bottom to top include:

a) Perforated pipes, b) Coarse Gravel, c) Medium Gravel, d) Coarse Sand, and e) Non-absorbent cotton

wool placed as the top layer in the drum (Ayre 2013).

2.3. Experimental setup and cultivation condition

We used two cultivation systems for microalgal cultivation a) a raceway pond and b)

an inclined reactor both with 11m2 surface area. The raceway pond was made of fiberglass

and operated at a depth of 14-15 cm (Figure 8). A single paddle wheel (4 blades) was

used to generate mixing (approximately 30 cm.s-1).

The inclined pond was built based on Doucha and Lívanský (2014) design. The inclined

pond (Figure 8) consisted of five major parts: 1) structural frame (chassis); 2) incline

Figure 8. 11-m2raceway pond used in this study to treat ADPE.

frame; 3) fluid cultivation surface; 4) sump collection tank; and 5) drainage transfer

plumbing (pipe system and gutter).

Figure 9. Inclined pond overall design (courtesy of Mr. Nugroho Sasongko).

The structural frame was made primarily of pine timber beams that had been varnished

and painted for durability (Figure 10a). The frame was assembled using a specialised

wood adhesion; assorted galvanised nuts and bolts, as well as metal, galvanised joiners

and support brackets (Figure 10a) . The incline frame was constructed using steel that is

typically used in pool fencing, with the deduction of central bars to reduce the weight of

the structure. The fluid cultivation surface consisted of two sheets constructed from two

pieces of PVC Polycarbonate (Figure 10b). Sheets were bound together with silicone.

Walls were set on the long side using strips of clear Perspex glued onto PVC

Polycarbonate sheets sidewalls (Figure 10b). A rectangular 400L PVC black tub was a

used as a sump collection tank (Figure 9). Drainage transfer plumbing consisted of PVC

plumbing pipes connected to a gutter drain with a down drainage pipe connector leading

under the incline bioreactor to the sump. Culture transfer from the sump to the inclined

flow panel was set using hoses and submersible pumps leading from the sump tank to the

flow surface area. The fluid cultivation surface area was created by joining two pieces of

PVC Polycarbonate using silicon as a bonding agent. This formed one full length 11.67m

× 1.045m with the total area of 12.13m2. However, we set up water transfer hoses so that

only 11m2 of inclined surface area was used for algal cultivation (same surface as raceway

pond). Further, sidewalls were created using clear Perspex 6mm x 65mm x 1167mm and

secured to the walls of the sheeting using silicon.

Figure 10. Inclined pond frame (a), operational pond (b) and inoculated pond (c).

We used sand filtered undiluted ADPE in this study. Microalgae consortium was first

cultivated in the 11-m2 raceway open pond. The culture established at 110±10 mg N

NH3.L-1. To inoculate the inclined pond, some of the raceway pond culture was harvested

at stationary phase using a bucket centrifuge. The concentrated algal culture was then

used as inoculum for the inclined pond. The working volume of the raceway pond and

inclined reactor were 1500L and 350L, respectively, (both operated at the same surface

area of 11m2). To examine the growth and nutrient removal rates of both cultivation

systems, microalgae were first grown in three batch cultures (Batch 1 = 7 days, Batch 2

= 5 days and Batch 3 = 7 days) in both cultivation systems. The batch 3 was then followed

by five semicontinuous harvests in both cultivation systems. The harvest interval was

between two and five days depending on the growth of microalgae. The inclined pond

was used as determinant for culture harvest on attainment of maximum cell density and

minimum ammonia concentration. It should be noted that the cultures were topped up

with freshwater every day to compensate evaporative water losses in hot days.

2.4. Analytical methods

Samples were collected for determination of initial and final nitrogen concentration (N-

NH3 and N-NO3-), phosphate concentration, COD, and turbidity measurement at 11am

every second day during the batch and semicontinuous culture. Microalgae biomass

concentration as ash-free dry weight (AFDW), biochemical composition (total lipids),

and the biomass were analysed in both cultures during growth period. The AFDW was

determined according to our standard (Moheimani et al. 2013). Volumetric aerial biomass

productivities were determined based on the changes of AFDW over time (see

Moheimani et al. 2013 for detailed methodology).

Total lipid was determined using the method of Kates and Volcani (1966) as adapted

by Mercz (1994). 10mL of algal culture was filtered using Whatman 2.5 cm GF/C filters,

rinsed with isotonic 0.65M ammonium formate solution and stored at -80◦C for later lipid

extraction. Before extraction the filters were thawed at room temperature, homogenized

in a glass mortar with 5mL of methanol: chloroform: deionised water (2:1:0.8 v:v:). The

extracts were then transferred to 10mL glass stoppered centrifuged tubes and centrifuged

at 1008 g for 10 min at room temperature. The supernatant was then carefully transferred

to glass stoppered graduated glass centrifuge tubes. The extract was made up to 5.7mL

with fresh methanol: chloroform: deionised water and 1.5mL of chloroform was added,

followed by 1.5mL of deionised water and stirred. After partial phase separation had

occurred, the samples were centrifuged for 5 min at 1008 g to complete phase separation.

The green chloroform bottom layer was then transferred to a dry, pre-weighed 4mL glass

vial and a few drops of toluene added to remove any aqueous phase unintentionally

transferred, and the extract was dried withN2. The vials were stored over KOH pellets

overnight in a vacuum desiccator before weighing.

The water analytical analyses (e.g. ammonium and nitrate concentration etc.) were

carried out using a Hana HI 83099 COD and multiparameter photometer. The temperature

and pH of the cultures in both ponds were measured and logged using specific probes and

data loggers.

2.5. Statistical analysis

The difference between growth and nutrient removal rates during growth of microalgae

in raceway and inclined ponds were statistically analysed using a tTest. All measures

were expressed in means ± standard error (SE) over the experimental duration and

significant differences were declared at 5% probability level.

3- Results and discussion

3.1. Culture conditions

The daily solar radiation ranged from 41 Wm-2 at 6am to 1016 Wm-2 at 2 pm and then

dropped to 40 Wm-2 at 7pm with nearly all days of the experiment (Figure 11). Air

temperature varied in a day from 16°C at 6am to 33°C at 2pm and then 22.4°C at 6pm

(Figure 11). It is to be noted that the daylight solar irradiance and temperature in some

days were as high as 1441 Wm-2 and 41°C (Figure 11). This was unavoidable as the

experiments were carried out in Austral summer. These very high air temperatures did

not result in loss of the culture.

The temperature of the culture in both ponds was almost the same, having a range of

18-30°C (Figure 12). However, on very hot days both cultures experienced an average

temperature as high as 35°C in the middle of the day (Figure 12).

Figure 11. Average solar radiation, and air temperatures variation during growth of microalgae

consortium over the experimental period.

Figure 12. Changes in culture temperatures of (A) inclined reactor and (B) open pond throughout

the experimental period.

Culture cell density was monitored throughout the batch and semicontinuous growth

(Figure 13). Scenedesmus sp was the dominant species in both inclined and raceway

ponds cultures (Figure 13). Chlorella sp. and Scenedesmus sp. cell density were almost

the same in the beginning of the culture in both systems but Scenedesmus sp. became

dominant especially during the semicontinuous cultivation (Figure 13). As can be seen in

Figure 13, Chlorella sp. concentration was approximately the same in both open

cultivation systems. However, Scenedesmus sp. cell density was significantly higher in

inclined pond compared to the raceway pond (tTest P<0.05). Results clearly indicate that

when algal cultures were operated semi continuously, Scenedesums became the most

dominant species in both systems.

Figure 13. Microalgal cell density in raceway and inclined ponds during batch and semi continuous

growth.

In-situ culture pH of both ponds was also monitored during the growth period (Error!

Reference source not found.). The pH of the algal culture in the inclined pond was in

the range of pH = 8-10. The algal culture pH in raceway pond (pH = 6-8) was significantly

lower than inclined pond. The higher culture media pH in inclined reactor is directly

related to higher algal growth in this cultivation system when compared with the raceway

pond (see Figure 13 and Error! Reference source not found.). The main reason for such

a difference in pH between the two cultivation systems is the higher growth and yield of

microalgae in the inclined pond. Algal cells require CO2 for their photosynthesis. CO2

uptake results in an increase in culture media pH during the day (Moheimani &

Borowitzka 2011). It should be noted that almost all microalgae mass cultures are carbon

limited and CO2 addition could generally result in up to 100% more biomass productivity

(Moheimani 2016). Higher pH in the inclined pond indicate that the algal culture was

more active in absorbing CO2. To increase biomass productivity and to improve culture

pH in the inclined pond, the addition of CO2 is recommended.

The algal biomass productivities of inclined and open ponds during both batch and

semicontinuous growth is summarised in Table 2. Overall, biomass productivity in both

ponds were calculated through multiple operational parameters to help identify the most

efficient cultivation system. The biomass yield (g.L-1) represents the amount of algae in

a water body. The algal biomass yield in the inclined pond (0.84 g AFDW l-1) was found

to be 2.5 times higher than the biomass yield of the culture grown in the raceway pond

(tTest, P< 0.05).

On the other hand, volumetric productivity (g L-1 d-1, see Appendix A for more

information) illustrates the expected yield of algae in a suspension on a daily basis. No

significant difference was found between the volumetric microalgal biomass productivity

of both the inclined and raceway ponds (tTest, P> 0.05).

However,] when biomass productivity was calculated based on unit of culture area (g.m-

2.d-1, see Appendix A for more information), the raceway pond culture showed four times

greater productivity than the inclined ponds (tTest, P< 0.05). This raceway pond higher

areal productivity was the result of larger operational culture volume of this pond when

compared with inclined pond..

Furthermore, the overall lipid content of biomass grown in raceway pond was 1.6

fold higher than the lipid content of the microalgal consortium grown in the inclined pond

(tTest, P<0.05). Areal and volumetric lipid productivities of microalgal cultures in

raceway and inclined ponds are summarised in Table 2. Such a difference in lipid content

as well as higher aerial microalgal biomass productivity achieved in the raceway, resulted

in 2.7 times higher areal lipid productivity of algae grown in the raceway pond compared

to the inclined pond (tTest, P<0.05). These results clearly indicate that under operational

conditions tested in this study, raceway ponds are better cultivation systems for treating

ADPE,

Table 2. Max volumetric and areal Productivity of Inclined and Raceway pond in three batches and semicontinuous runs from ash free dry weights

Volumetric productivities (mg.L-1.d-1) Areal productivities (g.m-2.d-1)

Inclined pond Raceway pond Inclined pond Raceway pond

Biomass lipid Biomass Lipid Biomass Lipid Biomass Lipid

Batch growth:

1st 0 0 68 35.5 0 0 9.34 4.9

2nd 95 31.8 51 26.6 3.02 1.0 7.022 3.7

3rd 30 10.1 47 24.5 0.954 0.3 6.44 3.4

Semicontinuous

growth:

1st 7.8 2.6 12 6.3 0.248 0.1 1.63 0.8

2nd 15.5 5.2 14 7.3 0.493 0.2 2.011 1.0

3rd 10.8 3.6 26 13.7 0.343 0.1 3.61 1.9

4th 30 10.1 10 5.2 0.954 0.3 1.43 0.7

3.2. Ammonium removal rates

Microalgal nutrient removal rates of both ponds are summarised in Table 3. The

average initial N-NH3 concentrations in ponds was 108.02-±21.11 gL-1. When compared

based on volumetric removal rates, microalgal culture in the inclined pond was more

effective in removing ammonium and phosphate compared to the raceway pond but no

difference was found in COD removal rate of ADPE between the two ponds (Table 3).

On the other hand, due to the 4.2 times higher volume of the culture in raceway pond

compared to the inclined pond, higher ammonium and COD removal rates were observed

in the raceway pond (Table 3). Aerial phosphate removal rates of microalgal culture in

both ponds were the same (Table 3). The nitrogen removal rate from piggery effluent

obtained in this study was significantly higher than that in the literature. Chevalier et al,

(2000) achieved a nitrogen removal rate of 4.0 mgL−1d−1 for Phormidium tenue in 100ml

aerated flask with initial N concentration of 35 mgL-1.

The efficiency of microalgal culture ammonium removal rates in the raceway and

inclined ponds for the duration of the semi continuous growth (Figure 13) is summarised

in Table 4. The microalgal culture in the inclined pond was shown to be 1.4 times more

efficient in removing ammonium compared to the raceway pond. This is generally due to

shallower depth and better light availability to the cells in inclined ponds. Garcia et al

(2000) also showed 49% total inorganic N removal in a high rate algal pond with the

surface area of 1.54 m2 and working volume of 450 L and initial total N concentration of

51mg N L-1. Our raceway pond growth and nutrient removal rates are comparable to the

observation of De Godos et al, (2011). They recorded a removal efficiency of 76% with

nitrogen loading rates of 3gN m2 d-1 in a 456 L raceway pond with surface area of 1.54m-

2. In other words, in our study, the raceway pond could remove 19.7 kg N.NH3 ha-1d-1.

Craggs et al (2011) found a removal rate of 24 kgNha-1d-1 when treating domestic

wastewater to produce biofuels. In this study, COD removal efficiency of algal culture

grown on ADPE in the inclined pond was significantly higher than the raceway pond. De

Godos et al, (2009) found COD removal efficiencies of 57% when using raceway pond

to treat piggery wastewater (initial COD concentration = 526mgl-1).

Table 3. Algal nutrient removal rates (data are mean ± se, n = 5)

Volumetric (mg L-1 d-1) Aerial (g m-2 d-1)

N-NH3 N-NO3- PO4

3- COD N-NH3 N-NO3- PO4

3- COD

Pond

Raceway: 13.13±2.14A 5.9±10.99A 0.03±0.187A 38.87±9.14A 1.97±0.32A 0.88±1.64A 0.004±0.028A 5.83±1.37A

Inclined: 19.23±3.22a -0.19±3.69a 0.75±0.36a 41.17±13.57A 0.67±0.11a -0.007±0.13a 0.003±0.01A 1.44±045a

* lower and upper case letters indicate the significance in data in each column (tTest, P<0.05)

Table 4. Ammonium and COD removal rate efficiency of algal culture grown on ADPE (data are mean ± se, n = 5)

Inclined pond Raceway pond

Removal rate efficiency

Ammonium 98.06±1.13% A 68.86±4.01% a

COD 44.39±6.49% A 39.28±3.71 a

* lower and upper case letters indicate the significance in data in each row (tTest, P<0.05)

Based on the nitrogen (NH3 and NO3-) removal rates and algal biomass yield (g L-1),

the nitrogen mass balance was calculated (Table 5). Nitrogen assimilation into the

biomass was three times higher in the inclined reactor compared with the raceway pond

(Table 5). Further, the total inlet nitrogen assimilated into N-NO3- through nitrification

process in the inclined pond was 1.4 times less than the raceway pond (Table 5). Our

data presented here are significantly higher than those reported previously by De Godos

et al, (2009). They found 27% and 10% of N removed in biomass and oxidised in NO-3,

in a raceway pond culture with a 40-day growth period.

Table 5. Nitrogen mass balance.

Input

N*

(mg L-1)

Left over

N*

(mg L-1)

Produced

biomass**

(g L-1)

N oxidised to

NO3- *

(%)

N removed in

biomass ***

% mgl-1

Pond

Inclined 863.6 385.33 2.95 45 51 442

Raceway 1423 1048 1.63 62 17 245

* calculated based on the total N in NH3 and NO3- for the duration of growth. ** calculated based on the total biomass productivity for the duration of semicontinuous growth (Table 2).

*** Calculated in line with Redfield ratio (Redfield 1958; Stumm and Morgan 1996) the stoichiometric formula for algae is estimated

to be C106H263O110N16P1 (N:C = 0.14 and P:C = 0.02).

The biomass concentrations achieved in our study (Table 2) are also higher than

results reported in the literature regarding wastewater treatment. Jiang et al (2011)

found a maximum biomass concentration of 0.212 gl-1 for Nannochloropsis sp.

cultivated in a 1L flask in seawater containing 50% wastewater with N-NH3

concentration of 46mg l-1. The corresponding lipid content was also 27.8%, which is

less than what we achieved (See table 2). Woertz et al (2009) mix culture of green algae

in municipal wastewater containing 39 mgl-1 N-NH3 in a 40L flat plate photobioreactors

resulted in a maximum lipid content of 29%. The overall microalgal biomass

productivity in various open cultivation systems are summarised in Table 6. It can be

noted that there is very little outdoor data available on mass culturing alga using

inclined ponds.

For microalgae to be suitable for an integrated piggery effluent management plan,

such algae must be robust to achieve efficient ammonium removal and tolerate the

wastewater conditions. The results of the current study showed that microalgae cultures

in both raceway and inclined ponds have a high potential to uptake a significant amount

of ammonia to produce algal biomass. Integrating microalgal culture to piggery

management strategies for nutrient removal with methods for biomass removal can add

economic incentives for producers. The harvested biomass can potentially have

commercial functions such as bio-fertilisers, feed, and/or bioenergy feedstock (Cavallo

et al., 2006; Nwoba et al., 2016). Our current productivity results in both cultivation

systems are very low when compared to other studies using clean water (Table 6).

Addition of CO2 and optimising other growth variables can certainly increase the

biomass productivity and nutrient removal rate. Further growth optimisation and longer

growth trials are necessary before any commercialisation can take place.

Table 6. Reported biomass productivities of microalgae in outdoor inclined and raceway ponds culture (data are From Borowitzka & Moheimani 2013

including current results [in bold]).

Cultivation

System

Culture volume

(L)

Culture period

(months)

Productivity range Species Location

Areal

(g dry weight .m-2.d-)1

Volumetric

(g dry weight.L-1.d-1)

Racewaya 300 +3 9.4-23.5 ≈ 0.031-0.078 Anabaena sp. Spain

Raceway 600 12 5 - 40 0.008-0.060 Tetraselmis sp Japan

Racewaya ? +3 1.6-3.5 ? Dunaliella salina Spain

Racewaya 110 12 20-37 0.22-0.34 Dunaliella salina Perth, Australia

Raceway 200 -3 14.7-18.1 0.183-0.226 Gloeotrichia natans Israel

Raceway 100 0.67 12.9±0.16 0.129 Muriellopsis sp Italy

Racewaya 160-200 12 16-33.5 0.11-0.21 Pleurochrysis

carterae

Perth. Australia

Racewaya 750 +3 15-27 0.06-0.18 Spirulina platensis Israel

Raceway 12a 8.2 Spirulina platensis USA (California)

Raceway 13,200 – 19,800 12 14.5 (5.8-24.2)b 0.03-0.12 Spirulina platensis Antofagasta, Chile

Racewaya 282 +3 14.47±0.16 0.183±0.02 Spirulina platensis Italy

Racewaya 135,000 +3 2-17 0.006-0.07 Spirulina sp. Spain

Racewaya ? +3 9-13 ? Spirulina. sp. Mexico

Raceway 300 - 600 6 5-26 0.01 – 0.05 Tetraselmis suecica Italy

Raceway 120 12 5.0-24.0c 0.04-0.20 Porphyridium

cruentum

Israel

Raceway 1500 -3 4-6 0.03-0.04 Consortium of

Chlorella and

Scenedesmus

Perth, Western

Australia

Inclined thin

layer pond

1,000 +3 10-30 1-3 Chlorella sp Czech Republic

and Spain

Inclined thin

layer pond

2,500 7 19 - Scenedesmus

obliquus

Rupite, Bulgaria

Inclined thin

layer pond

2,500 +2 12 - Scenedesmus sp. Tylitz, Poland

Inclined thin

layer pond

350 -3 0.5-1 0.03-0.04 Consortium of

Chlorella and

Scenedesmus

Perth, Western

Australia

a This figure is the annual average productivity at Earthrise farms, but the growth season is only 8 months.

b data are the annual mean and the summer and winter mean in brackets. Culture depth was 12 cm in winter and 18 cm in summer

c data shown are the highest productivity achieved in winter and summer

3.3. Economics

In general, our results indicated that raceway ponds are more effective than inclined

ponds in treating ADPE due to their larger operational volume. (Table 6). Capital cost

(CAPEX) of large-scale paddle wheel driven raceway ponds are summarised in Table 7.

There is almost no literature available on the CAPEX or large-scale incline ponds.

However, our estimate is that inclined pond Capex will be similar or greater than the

raceway ponds. If we assume the same Opex in both systems, raceway ponds will be a

better system for treating ADPE.

4- Conclusion

Based on the overall volumetric results (productivity and nutrient removal rates), the

inclined reactor was found to perform better than the raceway pond for treating ADPE.

On the other hand, based on the aerial growth, productivity and nutrient removal rates,

our results indicated that algae grown in a raceway pond can treat ADPE significantly

more effectively than inclined pond grown algae. We operated our inclined pond at the

0.5 cm depth with operational volume of 350L. This is the main reason for the overall

lower areal productivity of inclined pond when compared to the raceway pond, which

was operated at 1500L culture volume. As highlighted earlier, ADPE is very dark and

the main reason for operating the inclined pond at such a low depth is to increase light

penetration to the cells. Higher yields achieved in the inclined pond compared to the

raceway pond indicates that this method was successful. However, this resulted in much

lower aerial productivity in inclined pond compared to raceway pond. Previous studies

showed that in some cases inclined ponds can be operated in depth of up to 2 cm.

Further studies on operating the inclined pond with various depths using ADPE will

allow us to estimate the suitability of this design when compared with raceway ponds.

Table 7. Capital cost of large-scale raceway ponds.

Benemann et al. 1982 Benemann and Oswald 1996 Weisman & Goebel 1987

1982 1994 1983 1994 1987 1994

Unit area for calculation 809 Ha 100 Ha 192 Ha

Capital Cost ($/Ha)

Growth Ponds:

1. Grading, earth works 2270 3205 1996 2759 9885 12060

2. Walls(perimeter, central) 4118 5692 7176 8755

3. CO2 Sumps 2780 3925 1378 1681

4. Mixing System 2471 3489 969 1339 4919 6001

Subtotal 19382 27364 13527 18696 37000 45140

System-wide Cost

5. Carbonation System See 9,10 2083 2879 1830 2233

6. Instrumentation (Misc) 500 610

7. Water Supply System 618 872 455 629 3473 4237

8. Water distribution (pipe & channel) See 7 2985 4125 984 1200

9. CO2 delivery system to module 4510 8367 155 214

10. CO2 distribution system to ponds 1587 2241 5620 7768 260 317

11. Nutrient supply to system 371 524 781 953

12. Building. Roads, Drainage 3460 4885 184 254 1094 1335

13. Electrical distribution and supply 3364 4749 640 884 1922 2345

14. Machinery 10562 14598 417 509

Subtotal 17617 24871 20601 28473 20219 24667

Other capital cost factor

Engineering (10% of total capital cost) 3700 5223 3413 4717 5722 6981

Start-up cost (5% of PFI) 1850 2612 1706 2358 2861 3490

Contingency (15% of total capital cost) 6382 9010 5887 8137 9441 11518

Subtotal 11932 16846 11006 15212 18024 21989

CAPEX total 48931 69080 45135 62380 75243 91796

2017 CAPEX including inflation rate of 1.3% 72051 90529 65852 81749 105716 120299

Average CAPEX ± standard error 89366±8438

5- Acknowledgements

Authors would like to thank Mr Chia Lee and Mr David Juszkiewicz for their great

effort and help in constructing the inclined pond and also for their help with the initial

stage of the project. We would also like to thank Mr Jack Weatherhead for his great

help with harvesting both ponds for the duration of the experiment. Invaluable

assistance was also provided by the Department of Agriculture and Food, Western

Australia (DAFWA) and the staff of the Medina Research Station. Many thanks to

station manager Gavin d’Adhemar and DAFWA employees Josh Sweeny and Hugh

Payne for their assistance during this project.

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Appendix 1 - Notes

Confidential Information

If a Final Report contains Confidential Information:

NA

Deficient Report

If the Pork CRC reasonably forms the view that the Final Report does not

adequately set out matters referred to, it must notify the Researcher of the extent

to which it believes the Final Report is deficient.

Ownership of Reports

The Researcher will own copyright in all Reports, but not the Project Outcomes

described in the Reports. The Researcher grants to the Corporation a perpetual,

irrevocable, fully paid, royalty-free, worldwide licence to use the Reports and the

information disclosed in them and any other copyright material provided with the

Reports for the Corporation’s purposes, including reporting to its stakeholders,

including the government.

Appendices

Appendix A:

Biomass productivity

Biomass productivity of microalgae is commonly reported in the following

ways:

Areal productivity is reported as grams per square meter per day or,

tonnes per hectare per year.

Volumetric productivity of algae biomass is commonly reported as

grams per litre per day.

In general. Investigators studying closed photobioreactors use volumetric

productivity (e.g., Moheimani 2005), whereas open pond productivities are

usually compared based on areal productivity (e.g., Moheimani and Borowitzka

2006), although this measure does not take account of pond depth. The volume

of a culture is easily and unambiguously determined irrespective of the culture

system used, however the culture area – which is easy to determine for open

ponds – can be more difficult to determine for closed photobioreactors.