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Protozoa in Anaerobic Waste Treatment Systems
Protozoa in Anaerobic Waste Treatment Systems
Faculty of Engineering
Submitted in fulfilment of the requirements for the MSc and the Diploma of Imperial College
London
Declaration:
This submission is my own work. Any quotation from, or description of, the work
of others is acknowledged herein by reference to the sources, whether published
or unpublished.
Signature: ___________________________________
Abstract
Protozoa play an important role in the environment as a link between primary producers and
decomposers, and higher organisms in the ecosystem. They are also common in anaerobic
environments because they have hydrogenosomes that enable them carry out anaerobic
fermentation. It therefore important to understand their physiological behaviour and the roles
they play in different environments including aerobic wastewater treatment and anaerobic
environments like the rumen and sediments and harnessing this to increase CH 4 production
and substrate utilization in anaerobic digestion. Furthermore, it is essential to understand the
role they play in enteric pathogen inactivation in soil.
Protozoa have several behavioural traits that enable them take part both directly and
indirectly in the anaerobic degradation of waste and generation of CH 4. They enhance
bacterial activity by grazing on them and supplying essential growth nutrients as byproducts
of their metabolism thereby increasing substrate utilization. Furthermore, protozoa have the
ability to degrade recalcitrant cellulose mainly intracellularly through phagocytosis as
observed in the rumen. In addition to this, they are directly involved anaerobic fermentation
by ingesting particulate and soluble organic matter. They contribute to the generation of CH 4
by enhancing methanogenic bacterial activity and through their relationship with
endosymbiotic methanogens that utilize the byproducts of their metabolism to generate CH4.
Cellulose is common in sewage sludge and municipal solid waste but is not readily degraded
in anaerobic digesters therefore the use of protozoa as a biological pretreatment of waste for
anaerobic digestion has been proposed because of their efficient degradation of cellulose. In
the soil, protozoa preferentially graze on gram-negative bacteria, which are the major human
gastro intestinal pathogens and therefore play a biotic role in their inactivation. However,
they sometimes internalise pathogens therefore contributing to their continued persistence
and spread. Further research should be carried out to determine combined effect of this
behaviour to establish a scientific approach to land restrictions on the use of biosolids.
Acknowledgments
There are several people I would like to thank for helping me successfully complete my
dissertation.
I am sincerely grateful for the guidance given by Prof. Stephen Smith, my supervisor,
throughout my research. His insightful mentorship was paramount in enabling me conduct
well rounded research. I am also grateful to Dr. Hannah Rigby, my co-supervisor, for her
comments that greatly improved my dissertation.
In addition, I would like to thank my family and friends for their moral support and
encouragement throughout this period.
Last but not least, I thank the Lord Almighty who preserved me in good health throughout.
Table of Contents
1 Introduction...........................................................................................................1
1.1
Background................................................................................................................... 1
1.2
Thesis Structure............................................................................................................2
1.3
1.3.1 Aim.......................................................................................................................... 3
1.3.2 Objectives................................................................................................................3
2 Aerobic Protozoa..................................................................................................4
2.1
2.2
3 Anaerobic Protozoa..............................................................................................7
3.1
Oxygen effects............................................................................................................... 7
Anaerobic Metabolism...................................................................................................8
Symbiosis.................................................................................................................... 12
Anaerobic Digestion.....................................................................................................14
Ammonia toxicity..........................................................................................................18
3.7
Classification............................................................................................................... 24
Nutrition....................................................................................................................... 26
Movement.................................................................................................................... 28
Reproduction............................................................................................................... 30
4.5
5.2
Methane production.....................................................................................................45
5.4
Methane production.....................................................................................................50
6.3
Ammonia in sediments................................................................................................50
6.4
Soil Protozoa............................................................................................................... 51
6.6
7 Protozoa as a pretreatment................................................................................57
7.1
8 Discussion...........................................................................................................64
9 Conclusions.........................................................................................................67
9.1
9.2
Methane production.....................................................................................................68
9.3
9.4
References................................................................................................................70
Appendix A................................................................................................................78
List of Figures
Figure 3.1: Anaerobic respiration with lactic acid fermentation (Fenchel & Finlay, 1995)........9
Figure 3.2: Metabolism in the hydrogenosome of Tritrichomonas foetus (Priya, 2009).........11
Figure 3.3: Metabolism in a hydrgenosome of Daystricha ruminantium (Fenchel & Finlay,
1995)............................................................................................................................ 11
Figure 3.4: Metabolism in cytosol of Giardia lamblia (Fenchel & Finlay, 1995).....................12
Figure 3.5: Endosymbiotic production of CH4 (Fenchel & Finlay, 1995)................................13
Figure 3.6: A schematic of the steps in anaerobic digestion (van Haandel & Van Der Lubbe,
2007)............................................................................................................................ 15
Figure 3.7: Perturbation of electrochemical gradients and pH as NH3 diffuses out of the
mitochondria (Schneider et al., 1996)...........................................................................19
Figure 3.8: Perturbation of electrochemical gradients and pH as NH3 diffuses into the
mitochondria (Schneider et al., 1996)...........................................................................20
Figure 4.1: Degradation of a food particle by phagocytosis (Funke et al., 2004)..................27
Figure 4.2: Amoeboid movement (Zug, 2015)......................................................................29
Figure 4.3: Protozoa in the planktonic food web (Pauli et al., 2001; Porter et al., 1985).......32
Figure 5.1: The ruminant stomach (Schoenian, 2014)..........................................................34
Figure 5.2: A schematic of the reticulorumen compartments (Worfolk, 2015).......................36
Figure 5.3: Schematic structure of ciliate (Jouany & Ushida, 1999)......................................40
Figure 6.1: Schematic representation of the three major layers in sediments (yellow, grey
and black), the redox discontinuity layer, sediment surface, as well as some compounds
and ions in sediments; modified from (Fenchel, 1967)..................................................47
Figure 7.1: A pretreatment hydrolytic plug flow reactor with a mechanical treatment unit with
a solids recycle stream.................................................................................................58
Figure 7.2: A comparison between the degradable substrates concentration in sludge before
and after hydrolysis......................................................................................................63
List of Tables
Table 2:1: The effect of ciliate protozoa on the effluent quality in activated sludge plants
(Pike & Curds, 1971)......................................................................................................5
Table 3:1: A summary of the concentrations at which ammonia is beneficial, inhibitory or
toxic to the AD process (Rajagopal et al., 2013)...........................................................21
Table 4:1: Taxonomic classification of the single phylum Protozoa according to the Report
published by Honigberg et al (1964) (Corliss, 2001).....................................................25
Table 4:2: Classification of the aquatic microbial community by size (Porter et al., 1985).. . .31
Table 5:1: A summary of some of the approximate physical, chemical, and microbiological
conditions in the rumen (Moran, 2005).........................................................................37
Table 5:2: A summary of the protozoa that act on the different plant polysaccharides in the
rumen (Wang & McAllister, 2002).................................................................................42
Table 7:1: Composition of mixed sewage sludge (Haghighatafshar et al., 2014)..................63
Anaerobic Digestion
Adenosine diphosphate
Activated Sludge
Amino acids, sugars and Long chain fatty acids.
Adenosine triphosphate
Biological Oxygen Demand
Carbon to Nitrogen ratio
Coenzyme A
Chemical Oxygen Demand
Continuous Stirred Tank Reactor
Dissolved Organic Matter
Oxidized Ferredoxin
Reduced Ferredoxin
Granular Activated Carbon
Glutamate dehydrogenase
High Pressure Homogenizer
Hydraulic Retention Time
High Solids
Induced Blanket Reactor
Hydrolysis constant
50% lethal concentration
Low Solids
Mixed liquor Suspended Solids
Membrane Micro Aerated Digester
Medium Solids
Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide phosphate
Organic Fraction of Municipal Solid Waste
Organic Loading Rate
Pyruvate: ferredoxin oxidoreductase
Particulate Organic Matter
Redox Potential discontinuity
Substrate Level Phosphorylation
Superoxide dismutase
Solid Retention Time
Total Ammonia Nitrogen
Temperature Phased Anaerobic Digestion
Total Solids
Up flow Anaerobic Sludge Blanket
Volatile Fatty Acid
Volatile Solids
Waste Water Treatment Plant
Degradable particulate organic matter
Hydrolysis rate
1 Introduction
1.1
Background
Human impacts on the climate through fossil fuel consumption for energy generation is the
main cause of the current global warming problem according to most climate scientists
(NASA, 2015; Appels et al., 2011); human beings will need to rely more on renewable
energy sources for the future to offset this problem. Anaerobic digestion (AD) is able to
generate renewable energy in form of biogas from the treatment of waste therefore it is
considered as one of the most important potential renewable energy sources (Appels et al.,
2011). In addition to generation of biogas, AD is advantageous because of its low energy
and space requirements as compared to aerobic digestion. This makes it possible to apply it
on both a large and small scale.
There are a variety of potential feedstock sources for anaerobic digesters such as sewage
sludge, municipal solid waste and agricultural waste. Sludge has the highest biogas
production capacity worldwide at approximately 0.590 m3 kg-1 organic dry solids but its
disposal accounts for up to 50% of the current operational costs of a wastewater treatment
plants (Appels et al., 2011). Optimizing the AD process would greatly cut down on the
operation costs of waste treatment plants by utilizing the biogas produced as a source of
energy for the plant operations and selling off the excess.
AD is a biological process of treating waste and bacteria are the most important group of
microorganisms that have been recognized in the process although other microorganisms
like protozoa potentially have a critical supporting role. Protozoa are a diverse group of
unicellular microorganisms that have the ability to thrive and survive in diverse
environmental conditions. They are unique because of their ability to degrade cellulose that
is common in municipal solid waste and sewage sludge but is not readily degradable in
anaerobic digesters (Noike et al., 1985). They have also been observed in soils where they
preferentially graze on gram-negative bacteria (Perez-Viana, 2010). It is therefore useful to
know the precise role protozoa play in AD waste treatment systems and soil by studying their
behaviour and harnessing this to increase CH 4 production, substrate utilization and enteric
pathogen inactivation.
1.2
Thesis Structure
Chapter 2 examines aerobic protozoa and the role they play in aerobic wastewater treatment
as well as their ability to be used as indicator organisms for activated sludge system
performance.
Chapter 3 focuses on anaerobic protozoa i.e. their metabolism, the effects of oxygen on their
behaviour, their symbiotic relationship with bacteria and their role in anaerobic wastewater
treatment. Furthermore, the dynamics of AD, the different types of digesters and factors that
affect their performance are discussed.
Chapter 4 is a review of the fundamentals of protozoa including classification, nutrition,
reproduction, movement and their role in the aquatic food web.
Chapter 5 discusses the role of protozoa in the rumen highlighting their contribution to
digestion and CH4 production.
Chapter 6 examines marine and terrestrial habitats including sediments and the soil to
consider the role of protozoa within them. Additionally, the effect of protozoa on bacteria and
their role in pathogen inactivation and persistence in soil is discussed.
Chapter 7 discusses how protozoa can be used as a biological pretreatment of waste for AD
by mimicking their behaviour in the rumen.
Chapter 8 is analyses the role protozoa play in the various environments studied and the
potential effect on AD of waste and the inactivation of pathogens.
Finally, Chapter 9 is a summary of the key findings of the thesis and future research and
recommendations are made.
1.3
1.3.1 Aim
To investigate the role of protozoa in anaerobic waste treatment systems, including their
influence on CH4 production, substrate utilization and pathogen inactivation.
1.3.2 Objectives
i.
Provide fundamental and process level understanding of the role and behaviour of
ii.
iii.
iv.
2 Aerobic Protozoa
2.1
ii.
iii.
Ingesting suspended solids and dissolved organic matter and indirectly by the
excretion of mineral nutrients as products of their metabolism; N and P are released
in the form of
NH 4
Additionally, it has been observed that protozoa increase the rate of nitrification
because of their ability to influence bacterial growth (Madoni, 2011).
These effects are shown by the studies of Curds et al (1968) on activated sludge (AS) with
and without ciliates, which found that protozoa in the AS process lead to a reduction in
Biological Oxygen Demand (BOD), suspended solids, Chemical Oxygen Demand (COD),
bacterial numbers and organic nitrogen. This is shown in Table 2.1 where the effects of
ciliates on activated sludge are represented.
Table 2:1: The effect of ciliate protozoa on the effluent quality in activated sludge plants
(Pike & Curds, 1971)
Parameter
BOD (mg/l)
COD (mg/l)
Organic nitrogen (mg/l)
Suspended solids (mg/l)
Optical Density at 620nm
Viable bacterial count
Without ciliates
5370
198250
1421
86118
0.95 1.42
106160
With ciliates
724
124142
7 - 10
2634
0.23 0.34
19
(cfu/ml 106)
2.2
The colonization dynamics of ciliates show determinate temporal successions of groups and
species, from plant starting phases to the stabilization of the AS system. This succession
limits the number of species present in each phase of the biological waste treatment
process; therefore ciliates are considered as potential indicators of plant performance (Mara
& Horan, 2003). Three major phases have been recognized during the succession, as
shown in Figure 2.1. The first phase is characterised by a high number of free-swimming
bacteriophagous species; in the transition phase the free-swimming forms are substituted by
attached and crawling forms (the decomposers) and the maturity phase is characterized by a
stable ciliate community whose composition reflects the stable condition of the biological
plant (Mara & Horan, 2003). Flagellates are not linked to the presence of sludge therefore
they are not considered when looking at indicators or performance although they are present
in the system.
Figure 2.1: Microorganism succession during the colonization of activated sludge (Mara &
Horan, 2003)
Due to the fact that each phase has a specific ciliate community, a fully functioning plant
should not have not host species characteristic of one of the colonization phases. However,
this can occur if dysfunctions in the degree of aeration, the amount of sludge, sewage
retention time or organic loading cause regression in the environmental conditions (Mara &
Horan, 2003).
3 Anaerobic Protozoa
3.1
Oxygen effects
Anaerobic protozoa fall into several categories depending on their response to oxygen
including obligate, facultative and microaerophilic anaerobes. The point at which facultative
anaerobes convert from aerobic respiration to a fermentative metabolism is called the
Pasteur point. It is usually set at 1% atm.sat although it is not a constant as it varies between
species. For example, many anaerobes are inhibited at oxygen tensions < 0.1% atm.sat
while some tolerate normal atmospheric oxygen tension. A partial pressure of oxygen < 0.1%
atm.sat is considered an anaerobic condition and 0.1 5% atm.sat microaerobic (Fenchel &
Finlay, 1995). Organisms are considered anaerobic if they have an energy metabolism
independent of oxygen and can complete their entire life cycle in the absence of oxygen;
facultative if they are capable of oxidative phosphorylation but oxygen is not a requirement
for growth; strict anaerobes if they are inhibited below the detection limit of oxygen and
microaerophilic if they are sensitive to oxygen above a certain threshold value (Fenchel &
Finlay, 1995).
harmfully for example H2O2 or singlet oxygen are produced. The enzymes superoxide
dismutase (SOD) and catalase are used to protect the anaerobes from the toxins. SOD
catalyses the dismutation of
O2 :
2O -2 + 2 H+ O 2 + H2 O2 ( SOD )
Equation 3.1
2 H2 O2 2 H2 O + O 2 (Catalase)
Equation 3.2
On the other hand, oxygen consumption can be beneficial by balancing inwards diffusion so
the cells retain a constant anaerobic interior as observed in Metopus contortus (Fenchel &
Finlay, 1995).
3.2
Anaerobic Metabolism
Glucose
2 NAD+
2 ADP
2 NADH
2 ATP
+ 2 H+
2 Pyruvate
2 NAD+
2 Lactate
Figure 3.1: Anaerobic respiration with lactic acid fermentation (Fenchel & Finlay, 1995).
After pyruvate, the pathway is modified in different fermenting organisms. The lactic acid
fermentation process breaks down glucose to yield 2 mol ATP, but fermentation pathways
are more efficient and yield 4 ATP/glucose, therefore either incorporation of another
substrate level phosphorylation (SLP) occurs or electron transport to an organic electron
acceptor. The additional SLP depends on the production of Acetyl-Coenzyme A (acetyl-CoA)
and a greater variety of end products can be formed for example ethanol, CO 2, H2, butyrate,
propionate etc. (Fenchel & Finlay, 1995). Certain prokaryotes that are found in soil,
sediments and in the digestive tracts of ruminants, such as cows and sheep use other
electron acceptors for example rumen protozoa harbour methanogenic endosymbiotic
bacteria that utilize H2 to reduce CO2 to CH4 (Fenchel & Finlay, 1991). Similarly, sulphatereducing bacteria reduce
SO2-4
have several metabolic pathways that are discussed in Chapter 3.2.1 below.
In Trichomonads, which are anaerobic flagellates shown in Figure 3.2, the enzyme pyruvate:
ferredoxin oxidoreductase (PFOR) catalyzes the oxidative decarboxylation of pyruvate to
acetyl-CoA and CO2; see Equation 3.3. PFOR assumes a reduced state by the transfer of
electrons from pyruvate to the ferredoxin portion of the enzyme (Embley et al., 2003).
2 H + 2e H2
Equation 3.4
Energy resulting from the activity of PFOR is conserved in one molecule of ATP, which is
formed by the enzymes acetate:succinate CoA transferase and succinate thiokinase
(succinyl-CoA synthetase) that catalyze the subsequent metabolism of acetyl CoA into
acetate and ATP (Rogers, 2010), refer to Figure 3.2. Malate, which is produced in the
ctytosol, is oxidatively decarboxylated to pyruvate when it enters the hydrogenosome
(Fenchel & Finlay, 1995).
In Daystricha ruminantium, which is a common rumen ciliate, metabolism occurs in the
hydrogenosome, see Figure 3.3; therefore, its metabolic pathways are similar to
trichomonads. However, unlike the trichomonad it is able to produce short chain fatty acids
10
Fdox
Fdred
CoA
ATP
ADP+ Pi
2H+
Pyruvate
ADP
ATP
CoA
produced from pyruvate in the hydrogenosome. Some of the enzymes e.g. malate
dehydrogenase are located in the cytosol. Additionally, lactate fermentation can occur in both
the cytosol and inside mitochondria (Fenchel & Finlay, 1995).
Figure 3.2: Metabolism in the hydrogenosome of Tritrichomonas foetus (Priya, 2009). Fdox is
oxidized ferredoxin and Fdred is reduced ferredoxin.
11
AcetylCO2
2NADH
Acetyl-CoA
NAD
Aceto-acetyl-CoA
Butryl-CoA
12
NAD(P)H
NAD(P)
NADPH
NADP
Giardia lamblia is an example of an anaerobe that commonly occurs in vertebrate hosts and
whose enzymes of metabolism occur in the cytosol, as represented in Figure 3.4. The basic
fermentation pathway occurs to produce acetate. However, in this case, PFOR is not
coupled to a hydrogenase but rather transfers its reducing power on organic compounds to
produce ethanol and alanine. PFOR is associated with either the plasma or the endoplasmic
reticulum membrane.
Figure 3.4: Metabolism in cytosol of Giardia lamblia (Fenchel & Finlay, 1995). Fdox is
oxidized ferredoxin and Fdred is reduced ferredoxin.
3.3
Symbiosis
Most anaerobic protozoa have other microorganisms either living inside of them or attached
to their external surfaces, although some species like Metopus contortus have both. These
microorganisms are either endosymbiotic purple non-sulphur photosynthetic bacteria and
methanogens; or ectosymbiotic sulphate-reducing bacteria. The endosymbionts are
protected from competition with sulphate reducers in such an environment and from oxygen
inactivation (Fenchel & Finlay, 1995).
13
Alanin
The oligotrich ciliates, which are predominantly found in benthic environments, contain
endosymbionts. Strombidium purpureum is an oligotrich found in the illuminated zone of
anoxic marine sediments and has a purple cytoplasm due to the presence of non-sulphur
bacteria that contain photosynthetic membranes. The bacteria consume H 2 and other
fermentation products as reductants for photosynthesis and transfer a fraction of their
photosynthate to the ciliate. Purple non-sulphur bacteria are sensitive to light and thus able
to carry out oxidative phosphorylation at low oxygen tensions in the dark. This behaviour
relieves the threat of oxygen toxicity (Fenchel & Finlay, 1995).
3.3.2 Methanogens
Anaerobic protozoa sometimes contain methanogens, which release CH4 by using the end
products of metabolism for growth and energy production (Priya, 2009). The endosymbionts
are vertically transmitted: at mitosis, where they are distributed to the daughter cells and
are also retained at encystation (Hackstein, 2010). This accounts for their continued
presence in protozoa. Symbiotic bacteria benefit from protozoa by obtaining growth
substrates (H2, CO2 and acetate) and protection from predators. The most common species
of protozoa that harbour CH4-producing symbionts are the ciliates. The ciliates contain
methanogenic archea for example Metopus contortus, Plagiopyla frontata, Trimyema sp. and
Cyclidium porcatum that utilize H2 to produce energy by the reduction of CO 2 and the
excreted dissolved organics are utilized by the protozoa (Fenchel & Finlay, 1991); see
Equation 3.3 and Figure 3.5 representing the relationship between an endosymbiont and
hydrogenosome in protozoa.
CO2 + 4H 2 CH 4 + 2H 2 O
14
Equation 3.5
Hydrogenosome
Pyruvate
CO2
Protozoa
ATP
H2
Endosymbiotic
Bacteria
CH4
2-
SO 4
although they have been observed in other habitats like the rumen (Fenchel & Finlay, 1995).
15
They attach to the host by various mechanisms for example: insertion into pits in the cell
membrane observed in the ciliate Parablepharisma pellitum, living in sheaths that attach to
the host cell membrane in Metopus contortus and in Sonderia the ectosymbionts lie
embedded in a thin mucous layer which covers the cell surface. The
SO2-4
reducers utilize
the fermentation products from the ciliates for their metabolism (Fenchel & Finlay, 1995).
3.4
Anaerobic Digestion
Proteins
Carbohydrates
Fats
Hydrolysis
Amino acids, sugar
Fatty acids
Acidogenesis
Intermediates
Propionic acid, butyric acid
Acetogenesis
Acetic acid
Hydrogen
Methanogenesis
16
Methane
Figure 3.6: A schematic of the steps in anaerobic digestion (van Haandel & Van Der Lubbe,
2007).
Acetoclastic methanogens and H2 utilizing methanogens are the two main types of
microorganisms responsible for the formation of CH 4. The acetoclastic methanogens split
acetate into CO2 and CH4; see Equation 3.6 (Griffin, 2012).
2CH3 COOH CH 4 + CO 2
Equation 3.6
The H2 utilizing methanogens generate CH4 by using H2 to reduce CO2; see Equation 3.7
(Mara et al., 2003).
CO2 + 4 H2 CH4 + 2 H2 O
Equation 3.7
The dominant CH4 forming mechanism in AD reactors is the acetoclastic pathway, which
accounts for approximately 70% of the total CH 4 formed, because H2 is limited in AD (Griffin,
2012).
Reactor configuration
The key difference between batch systems and continuous systems is that in batch systems
the reactor is loaded with feedstock after which it is run to completion, emptied and reloaded
while continuous systems have the reactors being continuously fed with feedstock; this
allows for both a steady- state to be achieved in the reactor as well as for a constant gas
17
yield. Batch reactors have low operation costs and are simple to operate although they have
a large footprint and suffer from instabilities in the microbial population. In contrast,
continuous systems have higher operating costs but are able to maintain microorganisms
within the system (Griffin, 2012).
ii.
The total solid content (TS) influences the size of the digester where by the higher the solids
content, the small the reactor will be because of lower water requirements. Low solids (LS) is
10% of TS, Medium solids (MS) is 15 20% and high solids (HS) is from 22-40% (Monnet,
2003).
iii.
Number of Stages
The number of stages can either be single or multiple. In a single stage reactor all the
digestion processes take place simultaneously and this approach generally has the lowest
operating and capital costs. The most common multiple stage reactor is a two-stage reactor
whereby the first vessel is used for hydrolysis and acidogenesis while the second is for
acetogenesis and methanogenesis. The first reactor is limited by the rate of hydrolysis of
cellulose while the second by the rate of microbial growth (Monnet, 2003).
Temperature - There are two main temperature ranges: mesophilic (20-45 C) and
thermophilic (50-65C) (Monnet, 2003). Higher temperatures increase the reaction
rates in a digester resulting in more efficient operation (increased loading rate and
gas production) and lower retention time requirements (Cloete & Muyima, 1997).
ii.
18
if acid accumulates in the system the pH drops to between 4.5 and 5 causing
process inhibition (van Haandel & Van Der Lubbe, 2007).
iii.
Retention time - this is the time needed to achieve complete degradation of organic
matter and it varies with parameters such as temperature or the waste composition.
In a mesophilic digester the retention time is typically 15-30 days while in a
thermophilic it is 12-14 days (Monnet, 2003).
iv.
Mixing - This improves the contact between microorganisms and the substrate,
improves homogeneity of the feedstock and prevents the formation of scum and
development of temperature gradients within the digester. Slow mixing is preferred as
excessively high speeds can disrupt the microorganisms (Monnet, 2003).
v.
vi.
Carbon to Nitrogen ratio (C:N) This is the relationship between carbon and nitrogen
present in organic matter whereby carbon serves as an energy source for
microorganisms while nitrogen enhances microbial growth. Therefore, in situations of
limited nitrogen availability (a high C:N), the microbial populations will be smaller and
take a longer time to decompose the available carbon resulting in a low gas yield.
However, a low C:N causes high NH3 levels because the excess nitrogen is excreted
as NH3 gas. The optimum ratio is 30:1 in an AD digester and can be achieved by
mixing material of low and high C:N ratios (Igoni et al., 2008; Monnet, 2003).
vii.
3.5
Ammonia toxicity
19
NH +4
Ammonia is the end product of digestion of proteins, urea and nucleic acids in the absence
of oxygen. It is present in wastewater in either the ionized
( NH +4 )
or non-ionized form
(NH 3 ) and is the most significant inhibitor of the AD process (Rajagopal et al., 2013). The
toxicity of total TAN is primarily due to NH 3 because of its lipophilic properties which enable it
+
NH 4
NH 3
and
NH 4
with the
NH 3 + H2 O NH4 + OH
Equation 3.8
When the pH is low, the reaction is driven to the right, and when the pH is high, the reaction
is driven to the left (Sawyer, 2015). In addition to pH, temperature is another major factor
influencing of NH3 concentrations. At a constant pH, there will be higher concentrations of
NH3 at higher temperatures. A study by Kayhanian (1999) showed that the concentration of
NH3 at thermophilic (55 C) temperatures is expected to be six times higher than under
mesophilic conditions at the same pH (Rajagopal et al., 2013).
NH3 toxicity can lead to detrimental effects on the growth and reproductive activities of
animals, sometimes leading to death (Xu et al., 2004). It is thought to be brought about in
cells is by perturbation of electrochemical gradients and pH and interaction with enzymes
(Schneider et al., 1996).
20
+
NH 4 diffuses across the cell membrane very slowly, but is actively transported across the
cell by specific transport proteins and by facilitated diffusion (Schneider et al., 1996).
NH +4
ions compete with the transport of K ions into the cytoplasm resulting in an
increased demand in maintenance energy and reduced cell growth (Hauser & Wagner,
2015). On the other hand, diffusion of NH 3 across the cell membrane follows its chemical
potential gradient, which can be approximated by the gradient of its partial pressure. It is
produced in the mitochondria by the action of enzymes and then diffuses out through the
membrane into the cytoplasm pool and environment leading to a drop in the mitochondria
pH as shown in Figure 3.7. This leads to acidic conditions in the mitochondria and alkaline
conditions in the environment and cytoplasm.
Figure 3.7: Perturbation of electrochemical gradients and pH as NH3 diffuses out of the
mitochondria (Schneider et al., 1996).
It will then be actively transported back to the cell as
cytoplasm. This leads acidic conditions in the cytoplasm leading to an increase in pH in the
mitochondria and other organelles when NH3 diffuses into them; see Figure 3.8.
mitochondrion
pH
cytoplasm
Glutamine catabolism
mitochondrion
diffusion
pH
diffusion
diffusion
cytoplasm
active transport
diffusion
active
transport
ammonium
addition
Figure 3.8: Perturbation of electrochemical gradients and pH as NH3 diffuses into the
mitochondria (Schneider et al., 1996).
21
NH4
consuming cycles and continuous changes in pH. An increase in pH of the cell is harmful to
some organelles for example lysosomes with a naturally low internal pH. Additionally, a low
pH results in the inactivation of some key enzymes in glycolysis like of PK-1
(Phosphofructokinase-1) (Hauser & Wagner, 2015).
NH+4
regulatory sites of enzymes. The main source of the NH3 in cell cultures is glutamine and it
has been suggested by Schneider, Marison & von Stockar (1996) that NH3 might drive a
futile cycle of glutaminase and glutamine synthetase thus affecting ATP production.
Glutaminase catalyzes the deamidation of glutamine to yield
NH +4
NH+4
3.6
High NH3 concentrations are one of the main causes of digester failure because of its
inhibitory effects on microbial activity. Inhibition is demonstrated by a decrease in the steady
state rate of CH4 production and toxicity is manifested by a total cessation of methanogenic
activity (Rajagopal et al., 2013). The decrease in CH4 production may happen due to TAN
levels between 15007000 mgL-1. The wide range of inhibiting concentrations exists due to
the differences in nature of substrates, microorganisms, environmental conditions
(temperature, pH) and acclimation periods (Rajagopal et al., 2013). A summary of the
beneficial, inhibitory and toxic levels of NH3 in AD is given in Table 3.1.
Table 3:2: A summary of the concentrations at which ammonia is beneficial, inhibitory or
toxic to the AD process (Rajagopal et al., 2013).
Effect on AD process
22
References
Beneficial
No antagonistic effect
Inhibition (especially at
higher pH values)
Complete inhibition or toxic
50200
2001000
McCarty (1964)
Hobson and Shaw (1976)
15003000
3000
at any pH
3.7
Prochzka et al (2012)
23
An experimental set up using CSTRs was made to determine the effect of protozoa on
methanogenesis and COD removal whereby protozoa were inhibited using cycloheximide
(Priya et al., 2008). Ciliate density and COD removal had a strong positive correlation, with a
correlation coefficient of R2 = 0.974 for particulate oleate and R2 = 0.966 for soluble acetate.
The strong relationship could imply either that ciliates enable COD removal or that COD
removal enables ciliate growth (Priya et al., 2008). Although the difference between oleate
and acetate COD removal was not significant, it was more consistent and stable with the
oleate suspension. Additionally, the reactor fed with particulate COD had a higher number
and diversity of ciliates indicating a possible direct uptake of particulate oleate by ciliates
(Priya et al., 2007).
Changes in the MLSS concentrations in CSTRs in the presence and absence of protozoa
were studied. In the presence of protozoa, the MLSS concentration was observed to decline
as ciliate numbers increased. The correlation coefficient was R2 = 0.87 and MLSS
concentration was 1634% lower in the CSTR with protozoa. This can be interpreted as the
grazing activity of ciliates reducing the sludge biomass while enhancing COD removal
therefore protozoa may play a direct role in anaerobic degradation and production of CH 4
(Priya et al., 2008; 2007).
Batch tests were used to determine the contribution of protozoa in AD by inhibiting the
growth of protozoa and measuring CH4 production and COD removal, which were then
compared with results from a control. The reactor with ciliates had greater than 95% COD
removal while the one without had less than 66% COD removal and CH4 produced was 29 33% lower in the absence of protozoa (Priya et al., 2007).
Bacteria suppression was achieved by the addition of 1ml of antibiotic solution to the
reactors. Direct utilization of soluble and colloidal COD by flagellates and ciliates was
observed. In the reactor with particulate sodium oleate, ciliates thrived while flagellate
species were dominant in the culture with soluble acetate (Priya et al., 2007). The results
indicate the direct consumption of colloidal substrates by ciliates. Furthermore, filtered COD
removal was 69% in the case of oleate and 65% in the case of acetate. These results
confirm the direct participation of protozoa in anaerobic degradation (Priya et al., 2007).
Biagini et al (1998) studied the behaviour of bacteria feeding on plant material in anaerobic
microcosms by monitoring the CH4 and sulphide amounts produced. The ciliate Metopus
palaeformis was introduced into the environment leading to a reduction in bacteria numbers.
However, bacteria activity increased resulting in an increase in bacteria CH 4 and the rate of
24
activity was positively correlated with ciliate numbers. The contribution of the endosymbiontic
methanogens from M. palaeformis was not significant at 3-8% of the total CH 4 generated.
The increase in CH4 production was mainly attributed to the excretions of organic acids by
protozoa like acetate and propionate that stimulate bacteria activity. It was also due to
protozoa grazing that resulted in nutrient cycling.
25
4.1
Classification
There are several classifications of protozoa however the classification system of Honigberg
et al (1964) is widely accepted and divides the phylum Protozoa into four subphyla:
Sarcomastigophora, Sporozoa, Cnidospora and Ciliophora as shown in Table 4.1.
Superclass Mastigophora These are protozoa in which the organs of motion and
food capture in the adult are the flagella for example euglena (Minchin, 2003). The
body is covered by a pellicle and they undergo asexual reproduction by binary
fission. It has two classes: Phytomastigophorea and Zoomastigophorea.
ii.
iii.
Superclass Sarcodina - These protozoa move and feed by the use of pseudopodia.
The protoplasmic layer is non-corticate hence the body tends to be spherical in
26
floating forms or continually changing in the creeping species (Minchin, 2003). It has
three major classes: Class Actinopodea, Piroplasmea and Rhizopodea.
Table 4:3: Taxonomic classification of the single phylum Protozoa according to the Report
published by Honigberg et al (1964) (Corliss, 2001).
Subphylum I. Sarcomastigophora
Superclass 1. Mastigophora
Class 1.
Class 1. Myxosporidea
Phytomastigophorea
Class 2. Microsporidea
Class 2. Zoomastigophorea
Superclass 2. Opalinata
Superclass 3. Sarcodina
Class 1. Ciliatea
Class 1. Rhizopodea
Class 2. Piroplasmea
(2) Peritrichia
Class 3. Actinopodea
(3) Suctoria
(4) Spirotrichia
Class 1. Teleosporea
Class 2. Toxoplasmea
Class 3. Haplosporea
27
i.
Holotrichia free-swimming protozoa, which have cilia uniformly arranged over their
whole bodies (Mara & Horan, 2003).
ii.
Spirotrichia these have a flattened body with cilia found mainly on the lower surface
for locomotion (Mara & Horan, 2003).
iii.
Petitrichia these have inverted, funnel or bell-shaped bodies which are mounted on
a stalk. The wide end of the bell acts as an oral aperture and they have cilia arranged
around this to aid feeding (Mara & Horan, 2003).
iv.
Suctoria these have cilia when young that enable them disperse from their parents,
after which they lose their cilia and develop a stalk and feeding tentacles (Mara &
Horan, 2003).
4.2
Nutrition
28
Protozoa exhibit diverse forms of nutrition, four of which are represented in wastewater
treatment systems (Mara & Horan, 2003). The ability to utilize organic carbon for growth by
these organisms is important in the treatment of waste because they are responsible for
biochemical oxygen demand (BOD) removal. Chemoheterotrophs demonstrate three major
forms of substrate intake:
i.
29
iii.
Filter feeding This involves the transport of water through a filter created by cilia or
pseudopodial tentacles and microvilli (Boenigk & Arndt, 2002). A feeding current is
created which is then passed to a membranelle that sieves out solid particles based
the size of its spaces (Mara & Horan, 2003). Filter feeding is responsible for the clear
effluent produced when ciliates are present in activated sludge systems because the
sieving process typically retains particle sizes between 0.3 and 1.5 m.
ii.
Diffusion feeding This relies on the motility of the prey and not the predator and is
practiced by the sarcodines (Rnn et al., 2012; Mara & Horan, 2003). In the
suctorians, which are common in AS and mainly feed on ciliates, the swimming prey
collides with their sticky tentacles through which the cell contents are withdrawn
(Mara & Horan, 2003).
iii.
Direct interception This is the interception of particles carried along the flow line
due to motility of the predator (Boenigk & Arndt, 2002). Each particle is captured
separately so protozoa are able to discriminate what is ingested based on prey size
or type. It mainly occurs in small flagellates and amoeba (Mara & Horan, 2003).
Protozoa are able to preferentially choose between different bacterial species for
example gram-negative bacteria that are preferentially selected in soil (van-Elsas et
al., 2006).
4.3
Movement
There are three major organs of locomotion utilized by protozoa whereby the Sarcodina
utilizes pseudopodia, ciliates utilize cilia and the flagellated protozoa use flagella (Mara &
30
Horan, 2003).
endoplasmic sol begins flowing towards the area has changed from gel to sol causing the
cell wall to expand and the pseudopodium to extend forward. Upon reaching the tip, the
endoplasmic sol extends laterally and is transformed into a gel (Zug, 2015).
Figure 4.10: Amoeboid movement (Zug, 2015)
31
4.4
Reproduction
The growth curve of the free-living protozoa involves an increase in cell size followed by
some form of asexual reproduction (Mara & Horan, 2003). Asexual reproduction occurs
under favourable conditions, the most common form of which is binary fission where the
organism divides into two equal sized daughter cells. Multiple fission, plasmogamy and
budding (demonstrated by the Suctoria) also occur (Priya, 2009). Sexual reproduction,
mainly through syngamy and conjugation occurs during periods of environmental stress
(Priya, 2009; Mara & Horan, 2003).
4.5
Protozoa act as a link between the classical food chain and the microbial food web and
phagocytosis underpins their role in the latter (Priya et al., 2007). They have two major roles
in the planktonic food web:
i.
They are a link between the smaller phytoplankton and bacteria and the larger
zooplankton that cannot effectively feed on small zooplankton (Porter et al., 1985).
Protozoa provide particulate organic matter (POM) to higher trophic levels by
repacking the organic material into edible portions and thus making it available to
crustaceans, rotatoria, and other metazoans (Pauli et al., 2001).
32
ii.
They are involved in nutrient cycling of carbon compounds and mineral elements (N
and P) by the direct and indirect effects of their metabolism (Porter et al., 1985).
Plankton is composed of the phytoplankton (the plants of the sea) and zooplankton, which
are typically the tiny animals found near the surface in aquatic environments. The
zooplanktonic community is categorized by size of organisms, whereby the size classes
relate to the pore size of the filters used in feeding to collect and separate the components.
The classification is shown in Table 4.2.
Table 4:4: Classification of the aquatic microbial community by size (Porter et al., 1985).
Size Class
Picoplankton
Heterotrophs
Bacteria
Autotrophs
Cyanobacteria
0.2 2.0m
Microflagellates
Chemolithotrophic bacteria
Nanoplankton
Microflagellates
Eukaryotic algae
Phytoflagellates
2 20m
Naked ciliates
Non-flagellate algae
Microplankton
Naked Ciliates
Smaller diatoms
Larger diatoms
20 200m
Tintinnids
Larger dinoflagellates
Larger dinoflagellates
Amoeboid protozoa
Copepod nauplii
Rotifers
Other metazoa
33
Autotrophic Algae
Macrozooplankton
DOM/POM
Microzooplankton
Myxotrophic Algae
34
Bacteria
(autotrophic pikoplankton-cyanobacteria)
Figure 4.11: Protozoa in the planktonic food web (Pauli et al., 2001; Porter et al., 1985)
The colourless microflagellate protozoa have been proposed as the predominant grazers of
bacteria and autotrophic pikoplankton (Porter et al., 1985). Small ciliates, which form part of
the nano plankton, feed on bacteria and pico and nano autotrophs. Myxotrophic algae graze
on picoplankton and utilize DOM and POM for energy in addition to being photosynthetic
The nanoplankton are fed on by the microzooplankton, for instance large ciliates graze on
nanoplanktonic algae and are in turn fed on by the macrozooplankton, that are 20-200 mm
in size. Flagellates graze on bacteria and inhabit a central position in the transfer of organic
carbon to higher trophic levels due to their high turnover rates (Pauli et al., 2001). This food
web is graphically represented in Figure 4.3.
Protozoa nurture the growth of bacteria by grazing on them thus keeping them in the log
growth phase and preventing ageing cells from accumulating therefore minimizing the
possibility of substrate limitation and by providing dissolved organic carbon and minerals
through their activities (Porter et al., 1985). In addition to this, the P excreted by protozoa is
beneficial to phytoplankton (Pauli et al., 2001). Protozoa are decomposers associated with
the decay of organic matter and enhance the decomposition process because they increase
the turnover of the bacterial population (Priya et al., 2007).
The contribution of different groups of organisms is seasonally variable and the presence or
absence of a single species can modify the resource transfer pathways of the food web
(Pauli et al., 2001), therefore the links in Figure 4.3 are simply a representation of the
various possible pathways.
35
i.
The Rumen this is the largest compartment, and it harbours the microorganisms
responsible for fermentation. A stable pH is maintained by the absorption of the acidic
36
end products of fermentation via the rumen wall and bicarbonate from saliva. A
ruminant produces approximately 150L of saliva per day, which contains Na and K
that buffer against acidity (Moran, 2005). The walls of the rumen continuously move,
churning and mixing the ingested food with rumen fluids and microbes. The finer food
passes to the omasum, while the rest is regurgitated to the mouth for further chewing
to break it down into smaller particles, which increases the surface area available for
enzymes and microorganisms, hence increased fermentation. The cuticle layer on
plants and legumes, which is resistant to microbial and enzyme action, is destroyed
through repetitive masticating (Wang & McAllister, 2002).
ii.
Reticulum this allows the animal to regurgitate and reprocess its food (Moran,
2005).
iii.
Omasum this lies in between the reticulum and abomasum. The material entering
the omasum is 90 to 95% water, so its primary function is to absorb some of the
water and further break down feed that is then directed to the abomasum (Moran,
2005). It is responsible for absorption of fatty acids and bicarbonate. The removal of
fermentation products such as VFAs is important for an efficient activity of cellulolytic
microorganisms (Bayan & Guiot, 2011).
iv.
Abomasum this chamber is the true stomach with an acidic pH of about 2, therefore
microbes are destroyed here. Additionally, pepsin is released that carries out the
initial digestion of protein (Moran, 2005).
5.1
The reticulorumen, represented in Figure 5.2, is composed of the reticulum and the rumen.
Rumen fermentation combines both homogenization and stratification of the digesta, due to
its compartmentalization. Flow of digesta through a ruminant can be classified into three
major stages:
i.
Mastication
Digestion in ruminants begins with mechanical grinding and moistening of the ingested
material in the mouth after which it enters the rumen (Bayan & Guiot, 2011).
37
ii.
Mixing
Digesta is inoculated with microorganisms in the reticulorumen (Bayan & Guiot, 2011).
Upon entering the rumen, fiber floats on the surface of the liquid of the rumen content. Flow
starts with contraction of the reticulum, which directs liquid flow to the cranial sac and fiber
flow to the dorsal rumen. Contraction of the cranial sac and cranial pillar directs flow into the
dorsal sac. The dorsal sac then contracts to direct flow into the ventral sac. Finally,
contraction of the ventral sac moves the rumen content forward over a relaxed cranial pillar
toward the reticulum. The contraction cycles are initiated approximately every minute
(Gookin et al., 2011). Feed particle sizes are reduced and their densities increased during
the digestion process. The smaller, more dense particles produced during this process are
able to settle and reach the ventral sac after which they are returned to the cranial sac where
they are aspirated through the rericulo-omasal orifice into the omasum (Bayan & Guiot,
2011). Refer to Figure 5.2 for a schematic of the reticulorumen compartments.
iii.
38
The speed of digestion in the rumen is influenced by type and number of microorganisms,
feed composition, pH and growth limiting nutrients. The diverse microbial community
consists of anaerobic protozoa, bacteria and fungi and a summary of some of the
approximate physical, chemical, and microbiological conditions is provided in Table 5.1.
(Moran, 2005).
Table 5:5: A summary of some of the approximate physical, chemical, and microbiological
conditions in the rumen (Moran, 2005)
Characteristic
Physical
pH
Temperature
Dry matter
Property
5.5 6.9 (mean 6.4)
38 - 41C
10 18%
Chemical
Gas phase (%)
Volatile Fatty Acids (mmol L-1)
Always present
Always present
Always present
High Na; generally good supply
Always present; good supply of B vitamins
Microbiological
Bacteria
Ciliate protozoa
Anaerobic fungi
39
5.2
Protozoa are often found in large numbers in the rumen of ruminants resulting in more stable
fermentation, higher levels of NH3, reduced number of bacteria, higher ruminal and tract
digestion of organic matter and cellulose (Veira, 1986). Protozoa are 104 106 per g in the
rumen as shown in Table 5.1, and represent approximately 50% by mass of the microbial
biomass (Jouany & Ushida, 1999). There are more than 100 species that have been
identified although no more than 15 to 20 species are found in an individual ruminant
(Hobson & Stewart, 1997). The predominant group of protozoa in the rumen are the ciliates
although
some
flagellates
have
been
identified
including
Trichomonas
spp.,
Monoceromonas sp. and Chilomastix sp. from the Class Zoomastigophorea. The two
principal ciliate groups in the rumen are Entodiniomorphida and Holotrichs under subclass
Trichostomatia are although their roles differ significantly because of their different metabolic
behaviour (Veira, 1986).
Metadinium,
Epidinium,
Enoploplastron,
Ophryoscolex,
Epiplastron,
40
(Williams, 1986). The three principal species are Isotricha intestinalis, Isotricha protosoma
and Dasytricha ruminantium. There is a diurnal variation in the number of Isotricha,
Dasytricha, and Buetschlia spp. whereby the numbers increase before feeding and decline
after feeding, which is attributed to post feed increase in rumen outflow rates, protozoal
settlement, disintegration and sequestration on food particles or the reticulum wall where
there are higher oxygen levels.
Holotrich protozoa are aerotolerant anaerobes and commonly survive longer than other
ciliates when oxygen is present. Oxygen gradients along the gastrointestinal tract may
stimulate protozoan activity and in turn improves their hydrolytic capacity (Bayan & Guiot,
2011). The major organelle of carbon metabolism is the hydrogenosome, which produces H 2
under anaerobic conditions but in presence of oxygen acts as a respiratory organelle
(Williams, 1986). Oxygen affects the proportion of metabolites formed and the effects are
dependent on its concentration. Atmospheric levels are toxic while low oxygen tensions have
pronounced energetic benefits. The metabolic processes in the hydrogenosomes of
holotrichs are influenced by physiological levels of oxygen which inhibit H 2 production,
increase acetate production, reduce butyrate synthesis, increase glucose uptake and in the
absence of glucose, increase extracellular lactate consumption (Hobson & Stewart, 1997).
41
i.
Ciliate protozoa are responsible for 30-40% of fiber degradation in the rumen (Bayan &
Guiot, 2011). There are two forms of protozoa are found in the rumen: the free-swimming
species associated with ruminal fluid, which preferentially digest soluble feed components,
and those that are attached either loosely or firmly to the substrate (Wang & McAllister,
2002). Protozoa have generation times of 5 to 14 h so it is important for them to attach to the
substrate to be retained in the rumen for them to successfully degrade plant material (Wang
& McAllister, 2002). The Entodiniomorphida are responsible for the larger fraction of plant
cell breakdown (Jouany & Ushida, 1999). Ingestion of plant particles is achieved by various
mechanisms, which are species dependent. Small particles are wafted into the oesophagus
by ciliary action while large flexible particles are surrounded by the vestibule, which opens
up around the plant material. Additionally, large rigid particles are grasped by the vestibular
lips that adhere strongly to the ingested material, detaching some of the plant debris from
large plant material (Jouany & Ushida, 1999). Refer to Figure 5.1 for a schematic description
of the ciliate structure.
42
Protozoa are efficient at hydrolysing cellulose because they carry out phagocytosis and can
localise substrates within internal food vacuoles, thus minimizing wastage of enzymes. In
addition, some ciliates excrete cellulases into the surrounding medium for partial degradation
of plant fragments to make them easier to digest e.g. Epidinium caudatum (Fenchel &
Finlay, 1995). In Epidinium caudatum, the partially degraded cellulosic material is enclosed
in a primary vacuole immediately after ingestion, which then migrates from the pharyngeal
cytoplasm to the general cell endoplasm. Bacteria are also ingested with plant particles
contained in the primary vacuole. Pseudopodial tongues emerging from the pharyngeal
cytoplasm separate the bacteria from the plant material leading to the formation of
secondary vacuoles containing the separated the bacteria and food particles. Protozoa
enzymes are believed to be concentrated in the cytoplasm and are released into the vesicles
to degrade plant fragments (Jouany & Ushida, 1999). The enzymes involved in cellulose
digestion are cellulases while the hemicellulase enzymes break down hemicellulose and
pectinases degrade the pectin (Mackie et al., 2001).
Products from substrate degradation are found in vesicles that move from the endoplasm to
the ectoplasm and undigested cellulose and bacteria are released and discharged from the
cell through the cytopract. In some species like Polyplastron and Eudiplodinium, which have
a similar method of digestion, the digestion products are passed from the vacuoles to the
adjacent cytoplasm (Jouany & Ushida, 1999). In vitro studies by Castillo-Gonzlez et al
(2014) show that Polyplastron and Eudiplodinium are the major degraders of cellulose.
All rumen entodiniomorphid protozoa except the small Entodinium spp. contain the enzyme
cellulase, with the highest activities occurring in Eudiplodinium maggii, Epidinium ecaudatum
caudatum and Ostrcodinium obtusum bilobum (Hobson & Stewart, 1997). Holotrichs do not
contain cellulases but they have hemicellulolytic and pectolytic activities that enable them to
weaken plant cell walls and release the more readily fermentable carbohydrates (Hobson &
Stewart, 1997). A summary of the protozoa that act on the different plant polysaccharides is
given in Table 5.2.
ii.
Synergies
43
anaerobes for example methanogens that grow at a redox potential lower than -0.3 V (Kim &
Gadd, 2008). Other synergies include the release of valuable nutrients and bacterial gazing,
which further increases bacterial activity.
Table 5:6: A summary of the protozoa that act on the different plant polysaccharides in the
rumen (Wang & McAllister, 2002).
Protozoa
Cellulolytic
Eudiplodinium maggii
Ostracodinium dilobum
Epidinium caudatum
Metadinium affine
Eudiplodinium bovis
Orphryoscolex caudatus
Polyplastron multivesiculatum
Diplodinium pentacanthum
Endoploplastron triloricatum
Orphyroscolex tricoronatus
Ostracodinium gracile
Entodinium caudatum
Isotricha intestinalis
Isotricha prostoma
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Hemicellulolyti
Pectinolyti
c
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
44
amylopectin inside the protozoa. Ingested starch is degraded in the endoplasm where about
50% is released in the medium in form of maltose and glucose. The other part is stored in
granules or in the skeletal plates inside the cells as amylopectin-like polysaccharide (Jouany
& Ushida, 1999).
Holotrichs are attracted by chemotaxis to sources of soluble sugar, which they assimilate
non-selectively. They convert the sugars to starch and ferment them to acetic, butyric and
lactic acid. The rate of sugar uptake is dependent on the nature and concentration of the
sugars, temperature and pH. Glucose uptake is inhibited by lactic acid but is stimulated by
ambient concentrations of oxygen (Hobson & Stewart, 1997). The range of saccharides
fermented is genus dependent whereby Isotricha spp. utilizes starch of a suitable grain size
and D. ruminantium readily ferments sucrose, fructose, D-glucose, raffinose and inulin.
The rate of carbohydrate utilization and nature of products formed is dependent on the
substrate (Williams, 1986). Protozoa contain a wide variety of carbohydrase enzymes and
polysaccharide degrading enzymes. Isotricha and Daystricha spp. have amylolytic enzymes,
which are inhibited by short chain oligomeric degradation products. They reduce the rate of
fermentation by uptake of readily fermentable sugars and starch thereby removing them
from immediate fermentation by bacteria, coupled with the reduced numbers of bacteria in
the rumen of faunated animals. This is important because it stabilizes ruminal fermentation
and prevents excessive amounts of lactate by increasing the number of lactate utilizing
bacteria. This enables the rumen system to successfully adapt to a change in feed
composition (Veira, 1986).
5.2.5 Protein
Protozoa do not have the ability to synthesize amino acids de novo from NH3 (Jouany &
Ushida, 1999), therefore they rely on bacteria and plant proteins. Digestion of engulfed
protein and bacteria occurs intracellularly. Protozoa release exo and endo-peptidases into
the medium, and produce peptides that become available to the bacteria and themselves
(Jouany & Ushida, 1999).
i.
Bacterial Protein
All ciliates ingest bacteria as their principal source of protein amino acids. Some protozoa
species feed on all types of bacteria present for example Entodinium caudatum while others
45
selectively ingest bacteria. The rate of uptake of bacteria is pH sensitive with an optimum pH
of 6 because uptake ceases at pH 5, and is reduced to 75% at pH 7 and 30% at pH 8
(Hobson & Stewart, 1997). Small entodina have been shown to be the major contributor to
bacterial protein turnover in the rumen (Jouany, 1996). Protozoa actively graze on bacteria
and a single protozoan can ingest 10 2 104 bacteria per hour, therefore, by applying a value
of 109 bacteria/mL to the rumen, predation could almost renew the entire bacteria biomass
every 12 hours in a rumen harbouring a high concentration of protozoa (10 5-106/mL)
(Jouany, 1996).
Bacteria are degraded within protozoa into small peptides, then to free amino acids that are
incorporated into protozoal protein without further interconversion. Parts of the peptides are
excreted from the protozoa as amino acids into the ruminal juice. Release of small molecular
weight nitrogenous products may account for as much as 50% of the bacterial protein
ingested by protozoa (Jouany, 1996). Protozoa deaminate amino acids intracellularly and
released NH3 is excreted into the rumen medium (Jouany & Ushida, 1999).
Ciliate protozoa ingest rumen bacteria resulting in increased recycling of microbial N in the
rumen (Jouany, 1996). Constituents of bacterial nucleic acid are incorporated into the nucleic
acid of the protozoa (Hobson & Stewart, 1997). Holotrichs obtain the nucleotide precursors
for nucleic acid synthesis by assimilation and digestion of ingested bacterial nucleic acid
material (Williams & Coleman, 1992).
ii.
Plant protein
Protozoa play a major role in the ingestion both particulate and soluble protein (Mackie et al.,
2001). All entodiniomorphid protozoa contain proteolytic enzymes and protease activity is
present in several holotrichs including the D. ruminantium, Isotricha spp. etc. (Hobson &
Stewart, 1997). Holotrichs degrade soluble proteins easily while the Entodiniomorphida do
not metabolize soluble proteins and do not grow unless insoluble proteins are present
(Jouany & Ushida, 1999). According to the studies by Jouany et al (1992, 1993), all ciliates
have leucine aminopeptidase (a proteolytic enzyme that hydrolyzes the peptide bond
adjacent to a free amino group) and the enzyme exopeptidase (it releases a single amino
acid or dipeptide), which were found in mixed ciliate protozoa especially in entodina. The
active proteolytic enzymes from the ciliates and their ability to engulf feed particles are
factors that contribute to increased dietary protein breakdown (Veira, 1986).
46
5.3
Methane production
5.4
47
6.1
Marine sediments are reducing environments covered only by a thin oxic surface layer
(Kristensen, 2000). POM provides a food source for bacteria and it accumulates in the
sediments by the effect of gravity. Protozoa are attracted to these bacteria and graze upon
them (Fenchel, 1974a). Sediments are anoxic at a certain depth beneath the surface
because the high consumption rate of oxygen (as it is the most favourable electron acceptor
for microbial respiration) combined with its low solubility in water usually prevents deep
penetration of oxygen into sediments (Kristensen, 2000). Additionally, when the food content
(organic material) present is higher than the oxygen input needed for its oxidation, anaerobic
conditions will form; therefore the anoxic depth depends on the food to oxygen flow into
interstices (Fenchel & Riedl, 1970). The penetration depth of oxygen is controlled by the
balance between downward transport of oxygen from above and by consumption processes
of all benthic organisms and their metabolic products within the sediment (Kristensen, 2000).
This is dependent on external parameters including the influx of organic matter, the
mechanical properties of the sediment, water turbulence, light, temperature and bioturbation
(Kristensen, 2000; Fenchel, 1974a).
Marine sediments are composed of three distinct recognizable layers as shown in Figure
6.1. The layer closest to the surface is yellow usually due to the presence of Fe 3+ and
contains free O2, which declines with increasing depth. The grey layer has O 2 and reduced
compounds like H2S in small amounts while the black zone is totally devoid of O 2 and has
H2S in large amounts (Fenchel & Riedl, 1970). A sulphide system is established under the
cover of oxidized marine sediments and the boundary is definable by a redox-potentialdiscontinuity (RPD) (Fenchel & Riedl, 1970); represented in Figure 6.1 i.e. that depth below
the sediment-water interface marking the transition from chemically oxidative to reducing
processes. In the black layer found below the RPD, the initial degradation of organic matter
is due to fermenting bacteria, which generate H 2 and low molecular weight organic
compounds (Fenchel, 1974a). Further decomposition may take place by the activity of
certain bacteria that utilize inorganic compounds other than O2 as H2 acceptors. Sulphate
reduction dominates in marine sediments although
48
H2 acceptors and the end products of the processes are reduced compounds such as H 2S,
NH3, and CH4 (Fenchel & Riedl, 1970).
Sediment Surface
Aerobic Decomposition
Yellow
Oxidized Compounds:
Grey
Redox Discontinuity Layer
Reduced Compounds:
Simple Inorganic compounds
Black
Anaerobic Decomposition
Figure 6.15: Schematic representation of the three major layers in sediments (yellow, grey
and black), the redox discontinuity layer, sediment surface, as well as some compounds and
ions in sediments; modified from (Fenchel, 1967).
H2S is present in different forms depending on the pH of the water but predominantly as HS (Fenchel, 1974a). The inorganic end products may be oxidised as they diffuse through the
sediments by free O2, chemoautotrophic bacteria or anaerobically by photoautotrophic
bacteria thus utilizing H2S as a H2 donor for the reduction of CO2 to organics (Fenchel &
Riedl, 1970). Sulphureta have the highest concentrations of animals i.e. more than 10 7
ciliates per m2. Sulphur bacteria may constitute 25-50% of the food for ciliates for example
immediately beneath the chemocline in sediments, where photosynthetic purple and green
sulfur bacteria that are predominantly consumed by protozoa are active (Fenchel, 1974a).
Diurnal vertical migration of these chemical zones and the associated microorganisms is
observed due to external parameters (Fenchel, 1974a), i.e. influx of organic matter, the
mechanical properties of the sediment, water turbulence, light, temperature and bioturbation.
Reduced permeability brings the RPD closer to the surface, higher temperature causes
higher position of RPD, mixing by winter storms lowers RPD level, whereas, input of organic
matters causes it to rise again (Fenchel & Riedl, 1970). All these processes, as well as the
spatial and temporal patterns which arise from them, allow for the great diversity of protozoa
associated with marine sediments (Fenchel, 1974a).
49
50
PO 3-4
and N2 as NH3 or
NH +4
as a by-
product of their metabolic activities (Pogue & Gilbride, 2007). Furthermore, the contribution
of protozoa to benthic energetics is disproportional to their biomass because of a higher
turnover of smaller cells (Fenchel, 1974b); making them very important in nutrient cycling in
sediments. This is shown in the studies by Hamels et al. (2004) who studied protozoa
behaviour in 2 sandy and 2 silty sediment stations to determine their relative contribution to
metabolism. Protozoan biomass (ciliates and nanoheteretrophs) exceeded metazoan
biomass at one of the sandy stations. However, at the rest of the stations the protozoan
biomass was less than 5% of the combined biomass of meio- and macro- benthos. The
biomass-metabolic rate equation R = a W -0.249 was applied to calculate the relative metabolic
rates for ciliates, nanoheterotrophs, and metazoan; where the metabolic rate per unit weight
(R) increases with decreasing body weight (W) (Fenchel, 1974b). The results showed that
protozoa contributed more to sediment respiration than the metazoa especially at the sandy
stations, with the nanoheteretrophs contributing the largest proportion. This shows that their
metabolic contribution is disproptional to biomass but related to individual microorganism
weight. Protozoa represented 29 to 96% of the combined metabolic rate of benthic
consumers in the sandy stations observed in late spring/early autumn (Hamels et al., 2004).
51
trophic role of reduced low molecular end products of anaerobic decomposition (Fenchel,
1969).
Methane production
6.2
2-
SO 4
step accounting for 90 99% of activity. However, some CH 4 is produced because of the
existence of non-competitive substrates for methanogens like methanol, the existence of
2-
SO 4
free micro niches inside detrital particles and protozoa with endosymbionts.
SO 4
competition with
acetate. In marine sediments the contribution of anaerobic ciliates to the CH4 production (1590%) is greater than the freshwater sediments (maximum of 5%) where it is generally
marginal throughout the year (Priya, 2009; Van Hoek et al., 2006; Fenchel & Finlay, 1995).
In a microbial succession based on the accumulation of organic matter, there is a sequential
depletion of O2,
mineralization process. The anoxic layers in marine sediments can eventually become
depleted of
2-
SO 4
2-
SO 4
6.3
Ammonia in sediments
Microbial reduction of
NO-3
(NO-3 )
is reduced to
(NO-2 ) , which is then further reduced to N 2O or N2 while in the latter, the dissimilatory
pathway,
(NO-3 ) is reduced to
52
supply of organic substrate and dominates nitrate and nitrite reduction in anaerobic
environments like marine sediments (Bothe et al., 2007). Denitrifying and nitrate
ammonification bacteria are the key microorganisms responsible for nitrate reduction
(Vymazal & Krpfelov, 2008). Protozoa in the sediments graze on bacteria thus stimulating
their activity and release
NH 4
2007).
6.4
Soil Protozoa
Protozoa are 103 104 cell g-1 of soil (Hoorman & Islam, 2010), and live in the soil water films
and water-filled pores of soil aggregates. Most of the activity occurs in the rhizosphere next
to roots, which typically contains 1,000 to 2,000 times more microorganisms than the typical
soil without roots (Hoorman, 2011b). The lifecycle of soil protozoa consists of an active
phase that involves feeding and multiplication and a cyst stage where the cell produces a
thick coating during times of environmental distress. The cyst stage enables the cell to
survive for many years during harsh environmental conditions after which it reverts to the
active phase when environmental conditions improve (Hoorman, 2011b). The cysts have a
high dispersal potential, and are easily spread by aerosolization; therefore protozoa can
colonize new substrates rapidly (Rnn et al., 2012). The majority of the protozoa in soil are
bacteriovorus while only one amoebae group, the vampyrellids, feed on fungi. Fungaldominated soils tend to have more testate amoebae and ciliates compared to other protozoa
types while the bacterial-dominated soils predominantly have flagellates and naked
amoebae (Hoorman, 2011b).
53
The C:N ratio for protozoa is 10:1 or more, while that of the bacteria on which they graze lies
within the range of 3:1 up to 10:1. This implies that the protozoa take in more much more N
than is necessary for their carbon needs when they graze on bacteria (Hoorman, 2011b).
The excess N is released in form of
other microorganism needs in the soil. Their contribution to total net nitrogen mineralization
is 12 - 30% (Hoorman, 2011b).
i.
The soil texture determines the habitable soil pore space and the water retention capacity.
Coarse soils like sand have larger pore spaces than finer soils like clay therefore the latter
contain a higher number of smaller protozoa (flagellates and naked amoebae), while coarser
textured soils contain more large flagellates, amoebae, and ciliates (Hoorman, 2011b).
Pore size limits the grazing activities of soil protozoa whereby small flagellates and naked
amoebae can only access bacteria in water-filled pores with openings of a minimum size 2-3
m (Rnn et al., 2012). This protects bacteria from predation (Perez-Viana, 2010). In a study
by Rutherford & Juma (1992), bacteria numbers in soils with protozoa were reduced by 68%,
50% and 75% in the silty clay, clay loam and sandy loam respectively compared. Coarse
soils drain water faster than the fine soils and as soil drains, the size of the water filled pores
decreases (Rnn et al., 2012). At the wilting point water filled pores measure approximately
0.3 m therefore excluding protozoan microbial activity (Perez-Viana, 2010).
ii.
Moisture content
Protozoa live in the thin films of water in the soil pores and are not active when confined to
the colloidal film (Hoorman, 2011b). At typical temperate ambient temperatures, a water
content of 30-50% supports the activity of soil protozoa. However, protozoa are inhibited at
lower soil moisture contents and water contents of 20% only support bacterial growth
(Fenchel, 1994).
54
iii.
Substrate supply
Protozoa are found in greatest abundance near the surface of the soil, particularly in the
upper 15 cm (Hoorman, 2011b), and decrease with soil depth from approximately 10 6 g-1 at
0.1m to 10 g-1 below 1m. This is due to decreasing levels of organic matter in the soil profile
(Lal, 2006).
iv.
pH
Protozoa have the ability to survive in a wide pH range but their prey do not. Acidic soil pH
reduces bacterial populations and the substrate supply for growth thus indirectly affecting
protozoa (Perez-Viana, 2010).
v.
Temperature
High temperatures favour metabolism and reduce doubling times, leading to an increase in
protozoan activity. However, the upper temperature limit that they can safely withstand is
30C with 35 40C causing death. They are able to survive extremely high and low
temperatures by encysting (Perez-Viana, 2010).
6.5
Protozoa ingest bacteria by phagocytosis and influence the species composition in soil
because of their high consumption rates and feeding preferences. They select bacteria
based on the following factors:
i.
55
Cell size
Selection by size is typical for filter feeding ciliates and interception feeding flagellates, which
feed most efficiently on intermediate sized bacteria (Rnn et al., 2012). Small and large
filamentous cells may escape predation and the study carried out by (Ronn et al., 2002),
found that the protozoa preferentially fed on cells larger than 0.5 m.
iii.
Bacteria vary in their nutritional value and protozoan growth rates strongly depend on the
type of bacteria they feed on; therefore they will preferentially graze on bacteria with higher
nutritional value (Rnn et al., 2012). Many gram-negative bacteria are good food sources for
protozoa as they produce high growth yields. This is shown in the studies by Ronn et al
(2002) to determine the impact of protozoa grazing on bacteria in soil whereby protozoa
showed preference for bacteria that had an affiliation with species of Enterobacteriaceae,
which produce high protozoan growth yields.
56
iv.
Protozoa prefer to graze on bacteria that have high growth rates because they are able to
replace the cells lost to predation (Ronn et al., 2002).
v.
Motility
High bacterial motility reduces protozoan feeding efficiency by reducing the chance of being
consumed by protozoa (Rnn et al., 2012).
vi.
Biochemical processes
Certain bacteria release toxic chemicals that kill protozoan cells therefore it is beneficial for
protozoa to avoid grazing on them. Furthermore, some bacteria form dense micro-colonies
through the excretion of polysaccharides thereby reducing the susceptibility to grazing by
protozoa (Rnn et al., 2012).
6.6
Free-living soil amoebae like Acanthamoeba, Naegleria and Hartmannella can act as
reservoirs and dispersal agents for pathogenic bacteria like Legionella, Chlamydia,
Salmonella etc. (van-Elsas et al., 2006). This is because when soil protozoa graze on
bacteria, some of the bacteria are not digested but rather internalised within the cell. The
fate of the internalised bacteria falls into three categories: bacteria that multiply and cause
lysis of the protozoan cell, bacteria that multiply and do not cause lysis of the protozoan cell
and bacteria that survive without multiplication (Barker & Brown, 1994).
Although protozoa preferentially feed on gram-negative bacteria, some survive protozoa
grazing either because the protozoa are unable to take them up and digest them or because
they have defensive mechanisms like outer membrane structures, production of toxic
chemicals etc. Certain bacteria are able to form an endosymbiotic relationship with the
protozoa as a survival mechanism in harsh environments (Barker & Brown, 1994). Protozoa
egest vesicles as part of their digestion process through which pathogenic bacteria can be
dispersed (van-Elsas et al., 2006).
57
58
7 Protozoa as a pretreatment
Pretreatment systems can be used to enhance the efficiency of the AD process resulting in
improved digestate quality, increased biogas yield and faster digestion (Montgomery &
Gnther, 2014; WRAP, 2012). Biological processes using specific microbial species offer
advantages such as low capital cost, low energy, no chemical requirement, mild
environmental conditions, and no inhibitory compounds formation (Cesaro & Belgiorno,
2014).
Protozoa are able to degrade cellulose intracellularly, excrete nutrients as by-products of
their metabolism and enhance beacterial activity by grazing on them. Additionally, they
maintain a stable rumen environment by engulfing starch, making it unavailable to rapidly
fermenting bacteria therefore slowing the production of VFAs as discussed in the previous
Chapters. These behavioural traits make them useful as a pretreatment for wastes with a
high cellulolytic content like sewage sludge and municipal solid waste.
The physiological and environmental conditions of the rumen are mimicked in the design of
the reactor because the rumen harbours a large population of protozoa. Protozoa
populations are 104 106 per g in the rumen and they represent approximately 50% by mass
of the microbial biomass (Jouany & Ushida, 1999). Furthermore, ruminants consume forage
as part of their diet that is rich in cellulose. Protozoa are shown to contribute to 43% of the
ruminal cellulase activity in a buffalo (Kuhad & Singh, 2007).
7.1
59
Paddle shaft
Dewatering
unit
Mechanical
pretreatment unit
Effluent
to digester
Feed
Dry AD is commonly used to treat a wide range of inputs but mainly commercial and
municipal solid wastes. A plug flow system allows for more process control because the
material moves predictably and can be tracked. Horizontal pug flow systems have a
minimum retention time of 14-20 days and operate within a 25-40% DS range. The largest
horizontal modules are able to process 20,000-25,000 tonnes per annum, therefore a largescale project will require more vessels arranged in parallel (ADBA, 2013).
The material is pre-blended with recycled digestate being fed into the reactor resulting in
rapid inoculation at the right temperature (ADBA, 2013). An internal paddle mixer system is
used to homogenize the waste and to ensure maximum contact between the waste and
microorganisms; it also ensures a uniform digester temperature and that the less dense
floating layers are blended back into the mix while the denser particles do not settle as the
waste is moved along the vessel (Evans, 2001).
60
7.1.2 Reactor pH
The pH of the system will be kept near neutral (6.5 7), to enhance hydrolysis and prevent
process inhibition due to acidogenesis. In the rumen, cellulolytic activity occurs between pH
6 and 6.9 whereby a pH below 6 leads to a drastic decrease in fibre digestion (Mehra et al.,
2012; Rode, 2000). The entodiniomorphid protozoa and holotrichs are the main rumen
ciliates and the former are observed to degrade cellulose at a pH between 6.5 and 7 while
the latter have maximum productivity at a pH from 6.8 to 7 (Williams & Coleman, 1992). An
anaerobic digester has the ability to buffer its environment therefore providing resistance to
change in pH. Monitoring the ratio of bicarbonate alkalinity to VFAs is used as a control for
the pH whereby the result is used to regulate the feed rate of the digester (ADBA, 2013).
61
subjected to impact and shear stress and a pressure drop across the valve leading to cell
disruption; sonication liquefies the biomass by destroying part of the cells using ultrasonic
frequencies; while maceration technologies grind food waste into a pulp that can be
pumped.
62
Porous diffusers: These diffusers come in various shapes and sizes, such as discs,
tubes, domes, and plates and have a relatively high oxygen transfer efficiency (EPA,
1999; ASCE, 1988). They are mounted or screwed onto the diffuser pipe.
ii.
Nonporous diffusers: The common types of nonporous diffusers are fixed orifices
which are pipes with holes, valved orifices that contain a check valve to prevent the
backflow of waste into the orifice when aeration stops and static tubes which contain
interference devices that create bubbles by breaking up the air flow. The bubble size
of these diffusers is larger than the porous diffusers therefore they do not clog easily
(EPA, 1999; ASCE, 1988).
iii.
Other diffusion devices: These include jet aerators that discharge a mixture of air and
liquid through a nozzle near the bottom of the tank; aspirators mounted at the basin
surface to supply a mixture of air and water; and U tubes that discharge compressed
air into the down leg of a deep vertical shaft (EPA, 1999).
The effects of limited aeration on AD were tested by (Juanga et al., 2005). A batch test was
carried out with a pre-stage (hydrolytic and acidogenic) and a CH 4 producing stage that
occurred consecutively in each digester. One of the three reactors used had a partially
aerated pre-stage and it showed a considerably improved methanogenic activity. It produced
a higher biogas volume and reached the active CH 4 phase (50% CH4 in gas) quicker than
the rest of the reactors. Increased CH 4 production was attributed to better acidification in first
63
phase. This is an indication of the positive effect that partially aerated pretreatment can pose
on anaerobic digester performance (Botheju & Bakke, 2011; Juanga et al., 2005).
7.1.6 Inoculation
Anaerobic microorganisms exhibit growth rates, which are much slower than those of
aerobes therefore it is important to adequately seed the reactor with beneficial
microorganisms at start up (Stronach et al., 2012). Facultative and obligate anaerobes are
needed for the hydrolysis of particulate and colloidal compounds (Gerardi, 2003). In
ruminants, newborn animals are inoculated by licking their mothers and other members of
the herd (Husvth, 2011). Digesters are usually seeded with microorganisms from actively
digesting sludge, a municipal digester, a well-rotted manure pit or using cow dung slurry. In a
continuous or semi-continuous operation, the seed material should be at least twice the
volume of the fresh feed during the start up phase, with gradual reductions in seed over a 3
weeks period (NRC, 1977).
7.2
The hydrolysis rate (hyd) is determined using the first order model with respect to substrate;
it a product of the hydrolysis rate constant (k H) and the fraction of degradable POM
expressed as COD (Xs) (Siegrist et al., 2002); see Equation 7.1
hyd =
k H Xs
Equation 7.9
Several kH values have been proposed depending on temperature, waste composition, HRT
etc. Siegrist et al (2002) states that the k H for sewage sludge is 0.25 d-1 under mesophilic
conditions; Stamatelatou et al (2005) established kH as 0.35, 0.2 and 0.063 day1 for
carbohydrates, proteins and lipids respectively under mesophilic and thermophilic conditions
(Fezzani & Cheikh, 2008); Noike et al (1985) determined that cellulose has a k H of 0.1 d-1 at
35C (Vavilin et al., 1996) and Haghighatafshar et al (2014) proposed a value of 0.32 d-1 at
35C for a 15 days HRT. The values used for the analysis of the plug flow digester
performance are based on those from Stamatelatou et al (2005) and (Vavilin et al., 1996).
Mixed sewage sludge values from Haghighatafshar et al (2014) are used for the Xs values.
Carbohydrates, fats and lipids represented as 37%, 32% and 30% of the COD fraction refer
to Table 7.1.
64
Mixed Sludge
Total composition
COD fraction
Fat
(g/L) (%)
5.9
17.1
30
Protein
(g/L) (%)
13
35.4
32
Carbohydrates
(g/L)
(%)
18.2
47.5
37
Inert
(g/L) (%)
13.5 26.5
-
TS
(g/L)
50.5
Hunter & Henkelekian (1965) state that 60 to 80% of the total carbohydrate in sewage
sludge is composed of cellulose (Knapp & Howell, 1978); therefore based on that an
assumption is made that the cellulose content in this sludge is 60 % of the carbohydrates
while starch is the remaining 40%. Applying the model by Siegrist et al (2002) to this sludge
and considering a plug flow retention time of 14 days, cellulose will be 31% hydrolyzed,
protein 89.6%, fats 26.5%, and starch 72.5% to produce amino acids, sugars and long chain
fatty acids; see Figure7.2. The inert fraction of is non-biodegradable and therefore leaves the
system unchanged. Refer to Appendix A for the calculation.
A comparison between the degradable substrates concentration before and after hydrolysis in g COD/L
32.41
Cellulose
Starch
Protein
Fat
8.74
19.50
9.03
2.40
2.03
17.11
12.58
Before hydrolysis
After Hydrolysis
65
8 Discussion
Protozoa demonstrate numerous behavioural traits that are beneficial for the anaerobic
treatment of waste and wastewater treatment as observed by studying the roles they play in
different environments. They play a key role in stimulating bacterial activity by excreting
beneficial nutrients and grazing on them in both the aerobic wastewater treatment and
anaerobic environments (rumen, sediments and soil) studied. Furthermore, in the food chain
protozoa graze on bacteria and provide POM for higher organisms (Pauli et al., 2001),
showing that feeding on bacteria (smaller cell size) is characteristic of their behaviour in
different habitats. Grazing on bacteria is beneficial because they are kept in their log growth
phase and ageing cells are prevented from accumulating by renewing the bacterial
populations (Porter et al., 1985), hence increasing bacteria activity. The mineral nutrients
such P as
PO 3-4
and N as NH3 or
NH +4
metabolic activities as well as organic acids like acetate (Biagini et al., 1998; Porter et al.,
1985). Bacteria utilize these nutrients resulting in an accelerated degradation of organic
matter. Protozoa therefore play an important supporting role in AD to increase the turnover
rate of waste.
In addition to stimulating bacterial activity, protozoa directly consume particulates and take
up dissolved solids therefore directly taking part in fermentation. This behaviour is noticed in
both the aerobic wastewater treatment and anaerobic environments for example in the
rumen where the entodiniomorphid protozoa predominantly engulf particulate matter and the
holotrichs take up mainly soluble sugars (Jouany & Ushida, 1999; Jouany, 1996).
Furthermore, in a study on the role of protozoa in anaerobic wastewater by Priya et al (2007)
there was a decrease in MLSS and COD in the presence of ciliates. This is an indication of
the direct participation of protozoa in substrate utilization in anaerobic digesters. The direct
uptake of POM makes it unavailable to rapidly fermenting bacteria leading to a more stable
fermentation in the rumen by reducing the rate of VFAs formation showing that protozoa play
a role in regulating the environment in AD. This shows that protozoa contribute directly to the
AD process.
However, increased NH3 concentrations are observed in the presence of protozoa in the
rumen as well as in sediments where they contribute to NH 3 generation by stimulating
bacteria activity (Pogue & Gilbride, 2007; Males & Purser, 1970). This is disadvantageous
66
67
2-
SO 4
reduction to
produce CH4 instead therefore reducing the impact of competitive microbial reduction
processes (Fenchel & Finlay, 1995). H2S is a major problem in wastewater treatment as it is
malodorous, toxic and corrosive. The major problem associated with its toxicity is that it
although it is initially pungent; it quickly deadens the sense of smell so detection by sense of
smell is difficult. Furthermore, it weakens the structural integrity of the collection system by
corrosion because it forms sulfuric acid when it interacts with moisture (Carus, 2015).
Protozoa containing endosymbionts in anaerobic digesters can lead to a reduction in H 2S
produced based on these findings but further research needs to be done to determine their
role and the effects of enhancing their activity.
Protozoa can be used as indicator organisms of plant performance because different
species are present during different phases of plant operation (Mara & Horan, 2003). This
colonisation pattern is observed in both the aerobic wastewater treatment process and
anaerobic environments. In a study by Priya et al (2007) on the role of protozoa in anaerobic
wastewater treatment systems, specific protozoan species were observed during the startup
and stabilization phases of the reactors. This behaviour can be used as an empiric tool for
expert system development for anaerobic plants or as an indication of system failure.
68
9 Conclusions
Anaerobic waste treatment systems have the potential to generate biogas as a biorenewable energy source. This dissertation highlights the important role protozoa play in the
AD of waste by increasing substrate utilization and cellulose degradation to enhance CH 4
production, as well as their role in the inactivation of soil pathogen population.
9.1
digestion
Protozoa contribute both directly and indirectly to the degradation of organic matter with
ciliates being the predominant protozoan group responsible for their activity in the rumen,
sediments and anaerobic wastewater treatment.
Protozoa enhance bacterial activity by grazing on them thus keeping the populations
al., 1998).
The concentration of NH3 is increased in the presence of protozoa in the rumen
(Males & Purser, 1970). Protozoa also stimulate bacterial activity in sediments
69
Methane production
9.2
Increased CH4 production is observed in the presence of protozoa in the rumen and they
contribute significantly to CH4 amounts in anaerobic marine sediments. Furthermore, CH4
amounts are observed to increase with increasing ciliate populations in AD (Priya et al.,
2007). Anaerobic protozoa possess hydrogenosomes that are used for fermentation to
produce ATP and H2 as an end product (Fenchel & Finlay, 1995). They contribute to
increased CH4 generation through the following mechanisms:
9.3
In the soil, protozoa play a biotic role in the inactivation of enteric pathogens but also
contribute to their spread therefore influencing the pathogen dynamics in the following ways:
in soil.
In some cases, the pathogens are not digested but are internalised within the
protozoan cell which leads to their survival and spread in soil (van-Elsas et al., 2006).
9.4
70
Basing on the discoveries from this research, future research areas and recommendations in
relation to increasing anaerobic digestion performance and the inactivation of pathogens
have been made.
71
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Appendix A
Hydrolysis Calculations for the plug flow digester
Table A.1: A table showing the substrate composition for mixed sewage sludge from
Haghighatafshar et al (2014).
Mixed Sludge
Total composition
COD fraction
Fat
(g/L) (%)
5.9
17.1
30
Protein
(g/L) (%)
13
35.4
32
Carbohydrates
(g/L)
(%)
18.2
47.5
37
Inert
(g/L) (%)
13.5 26.5
-
TS
(g/L)
50.5
The COD fraction is obtained based on the average chemical composition of each of the
substrates with fat 2.9g COD/g of fat, protein 1.4g COD/g of protein and carbohydrates 1.2g
COD/ g of carbohydrate. 1 % of the VSS as COD is assumed to be inert (Haghighatafshar et
al., 2014). The proceeding calculations are based on the values in TableA1.1.
Table A.2: A table showing the degradable portion of the sewage sludge, the hydrolysis rate
constants, the hydrolysis rate per day and the % hydrolysed after 14 days.
(g/L)
5.9
13
7.28
10.92
13.5
Fats
Protein
Starch
Cellulose
Inert
g COD/g
2.9
1.5
1.2
1.2
-
g COD/L
17.1
19.5
8.7
13.1
-
Cellulose is assumed to be 60% of the carbohydrate content and starch 40%. The inert
fraction of waste is non-biodegradable and therefore not hydrolysed.
Table A.3: A table showing the hydrolysis rate constants, the hydrolysis rate per day and the
% hydrolysed after 14 days.
Fats
Protein
Starch
Cellulose
Xs (%)
30
32
14.8
22.2
KH(1/d)
0.063
0.2
0.35
0.1
hyd (%/d)
1.89
6.4
5.18
2.22
80
Retention time
14
14
14
14
% Hydrolysed
26.46
89.6
72.52
31.08
The kH values used are from literature by Stamatelatou et al (2005) for carbohydrates,
protein and lipids and Vavilin et al (1996) for cellulose. hyd is the hydrolysis rate, Xs is the
biodegradable fraction of substrate in terms of COD and KH is the hydrolysis rate constant.
hyd = k H Xs
The % Hydrolysed is obtained by multiplying hyd by the 14 days plug flow retention time.
Table A.4: A table showing the concentrations of the hydrolysis end products and the
amounts hydrolysed.
% Hydrolysed
Initial (g COD/L)
Amount hydrolysed (g COD/L)
After hydrolysis (g COD/L)
Fat
26.46
17.1
4.53
12.58
Protein
89.6
19.5
17.47
2.03
Starch
72.52
8.7
6.34
2.40
Cellulose
31.08
13.1
4.07
9.03
ASL
32.41
81