Microalgae culture to treat piggery anaerobic digestion - Pork CRC

95 Pages·2014·4.36 MB·English

Microalgae culture to treat piggery
anaerobic digestion effluent
A thesis submitted in partial fulfilment for the award of the degree of
Honours
in Biotechnology
Jeremy Ayre
BBiotech
2013
I declare that this thesis is my own account of my research and contains work
which has not been previously submitted for a degree at any tertiary institution.
Jeremy M Ayre
i
“You are capable of more than you know. Choose a goal that seems right for you
and strive to be the best, however hard the path. Aim high. Behave honourably.
Prepare to be alone at times, and to endure failure. Persist! The world needs all
you can give.”
Edward O. Wilson
This work is dedicated to my mother who has always supported me,
my father who has inspired my curiosity for science and my beautiful
wife Cheryl who has encouraged me to follow my dreams.
Jeremy 2013
ii
Abstract
Theuseofmicroalgaetechnologyforthetreatmentofpiggeryanaerobicdigestion
effluent offers attractive advantages over current wastewater treatment systems
used by Australian piggeries. These include recovery of nutrients in the form of
biomass that might be used as pig feed or to enable the production of biofuel,
better recycling of water, improved economic returns and better environmental
outcomes.
Thisstudyutilisedbioprospectingstrategieswhichincorporatedtheselectionand
culture of algae species which were capable of growing on undiluted, untreated
piggery anaerobic digestion effluent for this purpose. The successful isolation of
a Chlorella species using a synthetic medium containing 500 mg NH -N.L 1 and
3 �
the operation of several raceway ponds over a course of 20 weeks with ammonia
concentrationsofupto1,600mgNH -N.L 1 withamixedalgaecultureprovided
3 �
data to support the hypothesis that algae culture is not out of reach for this
application.
The data showed that high pH levels, temperature extremes and variable nutri-
ent composition could be accommodated through the careful management of an
outdoor pond system. It was also found that some aspects of the algae growth
performancesuchaschlorophyllcontentcanbeimprovedbytheadditionofCO
2
to the culture medium.
iii
Contents
Abstract iii
Acknowledgements vi
1 Introduction 1
1.1 Piggery wastewater treatment . . . . . . . . . . . . . . . . . . . . . 1
1.2 Characteristics of piggery anaerobic digestion effluent . . . . . . . . . 5
1.2.1 Ammonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.2.2 Turbidity and Phosphorous . . . . . . . . . . . . . . . . . . 7
1.2.3 Biochemical Oxygen Demand . . . . . . . . . . . . . . . . . 7
1.2.4 Chemical Oxygen Demand . . . . . . . . . . . . . . . . . . . 8
1.3 The potential of microalgae wastewater treatment . . . . . . . . . . . 8
1.3.1 Ammonia reactions and pH dynamics in algae culture . . . . . 10
1.3.2 Turbidity and Phosphorous . . . . . . . . . . . . . . . . . . 13
1.3.3 Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.4 Bioprospecting Strategies. . . . . . . . . . . . . . . . . . . . . . . . 14
1.4.1 Adaptation and Mutation . . . . . . . . . . . . . . . . . . . 15
1.5 Turbidity and Pond Design . . . . . . . . . . . . . . . . . . . . . . . 15
1.6 Summary and Aims of The Experiments . . . . . . . . . . . . . . . . 18
2 Materials and Methods 20
2.1 Chemicals and Solvents. . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2 Algal Culturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2.1 Synthetic Media . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2.2 Sterile Techniques . . . . . . . . . . . . . . . . . . . . . . . 23
2.2.3 Piggery anaerobic digestate based media . . . . . . . . . . . . 24
2.2.4 Setup of Sand Filter . . . . . . . . . . . . . . . . . . . . . . 25
2.3 Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.3.1 Nutrient Characterisation . . . . . . . . . . . . . . . . . . . 28
2.3.2 Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.3.3 Chlorophyll determination . . . . . . . . . . . . . . . . . . . 29
2.3.4 Cell Count . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.3.5 Ammonia determination . . . . . . . . . . . . . . . . . . . . 30
2.3.6 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.3.7 pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.4 Isolation of Algae Strains . . . . . . . . . . . . . . . . . . . . . . . . 30
2.4.1 Sources of Samples . . . . . . . . . . . . . . . . . . . . . . . 30
2.4.2 Bioprospecting Process . . . . . . . . . . . . . . . . . . . . . 33
2.4.3 Bioprospecting Culture Tanks . . . . . . . . . . . . . . . . . 34
2.5 Single species isolation on Agar plates . . . . . . . . . . . . . . . . . 38
2.6 Paddle wheel driven raceway ponds . . . . . . . . . . . . . . . . . . 38
2.6.1 Location and climate . . . . . . . . . . . . . . . . . . . . . . 38
2.6.2 Operating conditions . . . . . . . . . . . . . . . . . . . . . . 38
iv
2.6.3 Culture establishment phase . . . . . . . . . . . . . . . . . . 39
2.6.4 Batch and semicontinuous phase with high ammonia and CO
2
addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3 Microalgae bioprospecting 42
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4 Reliable long term outdoor cultivation 55
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.2.1 Natural ammonia decrease in raceway ponds . . . . . . . . . . 58
4.2.2 Batch phase growth . . . . . . . . . . . . . . . . . . . . . . 59
4.2.3 Semicontinuous phase . . . . . . . . . . . . . . . . . . . . . 67
4.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
5 General conclusions 77
5.1 Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Bibliography 81
v
Acknowledgements
Firstly I must thank my primary supervisor Dr Navid Moheimani for his un-
wavering support and encouragement throughout this honours project and my
secondary supervisor Professor Michael Borowitzka. The advice and wisdom
they have both shared with me has been at times challenging and at other times
inspirational throughout 2013.
I also thank Josh Sweeny, Hugh Payne and the other staff who have been helpful
and accommodating on my visits to the Medina Research Station and the stu-
dents at the Algae R & D Lab who have been welcoming assistants, tutors and
conversationalists throughout the year.
My most humble thanks also to my wife Cheryl who has been an amazing sup-
porter, advisor and always will be my best friend.
I owe a lot to all of you.
vi
Chapter 1
Introduction
1.1 Piggery wastewater treatment
The environmental impacts of intensive pig production can be significant. A
poorly managed piggery may risk wastewater pollution to local waterways, pro-
duce offensive odour emissions and outlet greenhouse gases into the atmosphere
(Maraseni and Maroulis, 2008). In contrast, a well managed piggery handles and
reuses wastewater appropriately, maintains control of odour emissions and out-
putsverylittlegreenhousegas(MaraseniandMaroulis,2008;APL,2011;O’Neill
and Phillips, 1991; Vanotti et al., 2008). It has been claimed that production
of pig meat has the potential to be one of the most emission friendly sources of
animal protein for the human diet (APL, 2010c).
RecentfiguresasofJuly2009indicatethat91%ofpigsatAustralianpiggeriesre-
side on farms with large herds of more than 1000 animals (Pink, 2012). Wastew-
ater generated through high intensity pig production is high in ammonia and
phosphorous as well as has high chemical and biological oxygen demands (COD
and BOD) (Olguin et al., 2003; Boursier et al., 2005; APL, 2011). High phos-
phorous levels have been shown to correlate to high turbidity levels giving the
effluent a dark colour (Ong et al., 2006).
HighCODandBODpresentsathreattowaterreceivingoutflowsintermsofpo-
tential oxygen depletion (Fallowfield and Garrett, 1985). Such excessive levels of
pollutants can cause eutrophication in waterways, compromising a resource that
supports wildlife and human populations (Carpenter et al., 1998; Woltemade,
2000). In severe cases, blooms of cynobacteria as a result of high nutrient levels
inwastewaterproducetoxinsthatarelethaltolivestockandhumans(Ongetal.,
1
60 A. USHIKUBO ET AL.
manure to the immediate area of its production (Young, 1980). On-site re-
cycling alternatives include: animal bedding, animal rations, combustible fuel
or biogas generation (Shaffer and van der Meulen, 1987; Japan Environmen-
tal Agency, 1988).
OXIDATION DITCH TREATMENT SYSTEMS
The simplest oxidation ditch treatment systems are modifications of the
activated sludge process. The most common of these is a single closed-loop
channel, 1.2-1.5 m deep, ~ 3 m wide and 15 m long with one or more surface
rotor type aerators (Barnes et al., 1983 ). The rotors serve two functions. They
bring sufficient oxygen into the liquid to support the microbial population,
200a6n)d. Gthreoyu nmdiwxa taenrdc amnoavlseo tbhee astorliisdks.o Afc doniatagmraimna toifo na fsriommpmlei sopxlaidcaedtiopnig gcehraynnel
efflsuyesnttem(S viso bporedsae,n1t9e9d5 ;inK Fraigp.a c1.e t al., 2002).
The original design of circular channel systems with surface aerators to aer-
A watied,e mvaixri eatnydo fpwroapsteelw tahtee rtrteraeatetdm ewnatsmteewthaotedrs waraes adveavileablolepethda itn ctahne b1e9u2s0esd and
to r1e9d3u0cse. tThheiasv maileatbhloedn uhtarsie unntdloeardgoinneth seeveffleruael nmtoodutifpiuctaftrioomnsp siigngceer itehs.e Tfihrsets efully
sysotepmersatmioanyailn oclxuiddeataiocno mdibtcinhaetsi owneoref ainertroobdicuctreeda ttmo etnreta,ta mnauenroicbiipcadli wgeasstitoenw,ater
faciunl tTathiev eNpeotnhdesrlaannddse vianp tohrea tliaotne p1o9n5d0ss (aAnhdl btheerg UannidteBdo yKkion,g1d9o7m2; iFne n1l9o6n3a n(Fdors-
ter, 1983; Johnstone et al., 1983).
Mills, 1980; APL, 2010b; Buchanan et al., 2013).
With the advent of new plastics during the late 1960s, rotating biological
contactors (RBC) were developed that retain many advantages of the old
rock trickling filters without some of their disadvantages. RBC systems were
Figure 1.1: A basic design of an aerobic treatment system. In this case an
extensively employed in the mid and late 1970s. However, use of these sys-
oxidation ditch system is integrated with extended treatment and disposal options
(tUesmhisk udbeocleitnaeld., i1n9 9t1h)e. 1980s primarily because of structural problems related
\~ ~. receivingw aters
oxidation pond or I// FinalD isposal Options
~ / ....
aerated lagoon
~/.~- ~/I / landa pplication
I1'11
~ ~
rotor
return sludge excess sludge
Fig. 1. Oxidation ditch system integrated with extended treatment and disposal options.
One treatment system which is gaining acceptance in Australian piggeries is
anaerobic digestion ponds. These systems typically consist of a covered pond
containing wastewater which is biologically treated by heterotrophic microor-
ganisms in the absence of oxygen (APL, 2010a). The covered digesters allow the
production and capture of biogas including methane (CH ) and carbon dioxide
4
(CO ) (Sowers, 2009; Buchanan et al., 2013). The benefits obtained in these
2
ponds are, the removal of solids through settling, capture of biogas for use as a
biofuel and the reduction of odour emissions (Leitão et al., 2006; APL, 2010b).
The utilisation of methane as a fuel source allows the ability to generate heat
and other forms of energy for localised use (Lim and Headberry-Partners-P/L,
2004; Dimpl, 2010). This can effectively reduce dependence on energy sources
from outside the piggery such as the electric grid or fossil fuel. However, as CH
4
2
is utilised, CO , another green house gas (GHG) is produced. Ideally CO will
2 2
be captured and reused within the piggery, if a model incorporating CO uptake
2
such as algae culture were to be adopted.
In recent decades awareness of the importance of piggery wastewater treatment
systems has increased along with the use of anaerobic digestion ponds. Cur-
rently around 83% of Australian piggeries use anaerobic digestion wastewater
treatment ponds as part of their wastewater treatment system (APL, 2010a)
cited in (Buchanan et al., 2013).
Figure1.2: SchematicofeffluentmanagementatMedinaresearchstationshowing
Schematic of effluent management at Medina RS showing effluent flow through covered
effluent flow through covered anaerobic pond (CAP). (Image provided by Hugh
anaerobic pond (CAP)
Payne, WA Department of Agriculture and Food).

2
CAP
Pond 3 CAP Pond 1(disused)
3 1

Solar powered flare
1. Holding Sump A
2. Run-down screen
Condensation trap 3. Holding Sump B
Gas pipeline to flare and blower fan
Outlet from CAP
through water Effluent Pathway
level control a) Effluen t drains from sheds into Tank 1
sump to Pond 2 b) Pumped from Tank 1 over rundown screen and
drained into Tank 3
c) Pumped from Tank 3 into CAP as necessary
d) Treated effluent displaced from CAP as effluent
Pond 2 enters
e) Effluent drains from CAP into sump which
controls water level in CAP
f) Drains from Sump into Pond 2

Gas Pathway
i) Gas passes through collection ring
ii) Gas drawn along pipeline to condensation trap
iii) Blown through flare by 24volt fan
iv) Biogas ignited at top of flare.

3