EOT - Applied Micro ALL WEEKS

CO2 fixation - Photosynthesis

Use sunlight to convert carbon dioxide + water → glucose + oxygen (photosynthesis)

CO2 fixation - Chemosynthesis

organisms use energy from oxidising inorganic molecules to reduce CO₂ into organic compounds for growth and energy

electron donors = hydrogen, sulfur, nitrifying and iron bacteria

Decomposition

organic compounds broken down to CO2 by microorganisms called degraders via fermentation or respiration (chemoorganotrophs)

Fermentation

Anaerobic break down of organic compounds, no additional electron acceptor for energy generation

Respiration

Aerobic or anaerobic breakdown of organic compounds to CO2 where ATP generation is by oxidative phosphorylation

AEROBIC Respiration

oxygen serves as terminal electron acceptor in oxidation of organic compounds by microorganisms

ANAEROBIC Respiration

Non-oxygen compound is terminal electron acceptor in oxidation of organic compounds by microorganisms e.g. sulfate/sulfur reducing bacteria

Methanogens (methanogenesis)

group of obligate anaerobic archaea, reduce CO2 → CH4 with H2 as an electron donor to produce methane in anoxic habitats

Methylotrophs

microorganisms capable of utilising C1 compounds as energy and carbon source

Methanotrophs

Aerobic oxidisation of methane to CO2, oxic and microaerophilic conditions, help reduce methane

Acetogenesis

Acetogens produce acetate in anoxic habitats with H2 as an electron donor, compete with methanogenesis

Greenhouse gases

Carbon Dioxide/CO2 (81%), Methane/CH4 (11%), Nitrus Oxide/N2O (6%)

Non-greenhouse gases

Nitrogen, Oxygen

Bioethanol

Sugars, polymers, starch wastes or cheap starchy crops converted to ethanol via carbohydrate fermentation, used as fuel additive or industrial solvent, clean renewable resource

Methane (biogas)

Mixture of methane and carbon dioxide produced from anaerobic digestion of organic matter by methanogens, fermenters, acetogens,

Biohydrogen

Hydrogen as a fermentation end product via dark fermentation and photo fermentation (purple and green bacteria)

Aerobic biodegradation

Organic contaminant is electron donor in aerobic respiration and oxidised, insertion of an oxygen molecule into compound is rate limiting step

Anaerobic biodegradation

1. Anaerobic respiration = contaminant is electron donor and oxidised, electron acceptor is non oxygen compound

2. Reductive dehalogenation = contaminant is electron acceptor and reduced, anoxic biodegradation by removal of halogen group 

Nitrogen cycle - Ammonification

Conversion of organic nitrogen → ammonia via biodegradation and decomposition. Proteins → amino acids → Ammonia, breakdown of amino acids to ammonia is deamination step

Nitrogen cycle - Nitrification

Aerobic oxidation of ammonia → nitrate, nitrifying bacteria = chemolithotrophs and obtain energy from the process

Nitrogen cycle - Denitrification

Reduction of nitrate → nitrogen gas or ammonia

Nitrogen cycle - Nitrogen fixation

Conversion of atmospheric N2 → ammonia

Desulfurylation

Putrefaction (decay) = organic sulfur → H2S, organic sulfur decomposed by microorganisms to release H2S and related degradation products

Sulfur reduction

SO → H2S. Elemental sulfur is used as a terminal electron acceptor, mainly chemolithotrophs

Sulfate reduction

Assimilatory sulfate reduction = SO4 → S2- → organic sulfur compounds

Dissimilatory sulfate reduction = SO4 → H2S

Sulfur and sulfide oxidation

H2S → S0 → SO4

1. abiological oxidation

2. anoxygenic oxidation (green and purple sulfur)

3. aerobic oxidation (neutral pH = chemolithotropic micoraerophilic microorganisms) 

4. aerobic oxidation by Acidithiobacillius (acidic pH)

Iron Oxidation

Fe 2+ → Fe 3+ , non acideophiles in neutral conditions, or acidophiles

Iron Reduction

Fe 3+ → Fe 2+ , Fe3+ can be terminal electron acceptor in anaerobic respiration

Waterborne diseases - Pathogenic Bacteria

E. coli, shigella, salmonella, vibrio, legionella

Waterborne diseases - Pathogenic Protozoa

Cryptosporidium parvum, Giardia lamblia

Waterborne diseases - Pathogenic Viruses

Adenovirus, Enteroviruses (poliovirus, enterovirus), hepatitis viruses, viral gastroenteritis 

Conventional water treatment system

1. Sedimentation to remove particles 2. coagulation + flocculation form additional aggregates which settle out 3. Filtration 4. Disinfection = removes pathogens over a range of physical and chemical conditions (chlorine gas, chloramine, UV)

Australian Drinking Water Guidelines

Provides information on pathogenic waterborne microorganisms, guideline valuesf or physical properties and chemicals that can affect water quality, advice on the operation of water supply systems

Drinking Water Monitoring

• accounts for high proportion of cost of drinking water production

• detect changes in raw water quality

• make sure plant is working properly

• check distribution system is in good condition

• ensure water meets drinking water guidelines

Drinking Water Tests

Tests carried out for :

• colour, taste, odour

• turbidity + suspended solids

• salinity + total dissolved solids

• pH

• organic and inorganic chemicals as appropriate

• indicator organisms and total heterotrophic bacteria 

Indicator Organisms

Not pathogenic, indicator of faecal contamination and therefore possible pathogen presence

bioremediation

stimulating microorganisms applied to clean up toxic pollutants from soil or water by providing limiting nutrients, biodegrading organic pollutants or removing metals

Oil Recovery

1. primary recovery (10-20%) - recovery of oil and gas under reservoir pressure or pumping

2. secondary recovery (30%) - waterflooding

3. tertiary (enhanced) recovery - physical and chemical methods e.g., hot water, gases, or microbial treatment

common contaminants of bioremediation

1. petroleum hydrocarbons 

2. polycyclic aromatic hydrocarbons 

3. volatile organic compounds 

4. xenobiotics 

5. other organic wastes 

6. metals

Microbial Treatments

Types - inoculation & nutrient addition

Results - selective plugging & biopolymer production

Problems - souring of oil fields and degradation of oils

Manganese Cycle

Oxidation: Mn2+ —> MnO2

Reduction: MnO2 —> Mn2+ (dissmilatory)

Bioleaching 

solubilisation and recovery of metals from solid minerals or ores using microorganisms 

chemolithotrophic - autotrophic process

1. Direct - MS + O2 —> MSO4 (microbial oxidation

2. Indirect - FeSO4 + 1/2O2 + H2SO4 —> Fe2(SO4)3 + H2O (microbial)

E.coli

• gram -ve rod, enterotoxigenic, enteropathogenic, enteroinvasive, enterohaemorrhagic, feacal/ oral route of transmission, travellers diarrhea

Shigella

• shigellosis (dysentery), human pathogen, low infective dose, enterotoxin 

salmonella

mild gastroenteritis to severe illness and death, s. typhi, s. enteritidis, food-borne

vibrio

V. cholera, central and south America, Asia, Africa, severe vomitting, diarrhea, dehydration

Legionella

pneumonia and resp failure, aerosol; aquatic reservoir, more resistant than E. coli to chlorine and other environment antagonist

Cryptosporidium parvum

severe dehydration, sexual and asexual stages, oocysts, low infectious dose, highly resistant to chlorine

adenovirus

dsDNA, resp disease, conjunctivitis, concentrated in sewage

enteroviruses (poliovirus, coxsackievirus, echovirus, enterovirus)

ssRNA, species specific, replicate in intestine, shed in large numbers, infections subclinical, feacal/ oral route

Hepatitis viruses

ssrRNA, inflammation of liver

Viral gastroenteritis

reovirus, rotavirus, calcivirus, astrovirus, coronavirus, norwalk virsuses, vomiting and diarrhea

Diarrhoea

directly related to water and sanitation, leading cause of childhood death, person dies every 30sec from water related illness

Common waterborne diseases

cholera - acute bacterial infection 

schistosomiasis - parasitic flatworm 

guinea worm disease - contaminated water 

trachoma - bacterial eye infection

malaria - mosquitoes 

Life Cycle - Guinea Worm Disease

1. Pain relief behaviour: infected person soaks blister (with exposed worm) in water; worm bursts and releases larvae 

2. intermediate host: water fleas ingest larvae, which mature into infective third-stage larvae within ~ 2 weeks 

3. transmission: another person drinks contaminated water 

4. larval release: water fleas are digested in stomach

5. development: larvae penetrate intestinal wall and mature into adult worms in body cavity, mate 

6. migration: fertilised female worm (up to 3 feet) move to skin surface, susually in lower limbs, causing a painful blister a year later

7. continuation

measuring water quality

QLD - Class A+ to D Class, based on E. coli levels, related to level of treatment/ quality of water 

reference pathogens 

those that present a worst-case combination of high occurrence, high concentration in water to be recycled, high pathogenicity, low removal in treatment, long survival in environment

Outline Activates Sludge Process

1. Screening: Removes large objects from wastewater

2. primary settling: heavy solids sink, oils float and are removed 

3. aeration tank: air is added so bacteria can break down waste

4. settling tank: bacteria and solids settle to the bottom as sludge 

5. sludge recycling: some sludge is reused

6. disinfection: clean water is treated (e.g., with chlorine or UV) before release

FLOCS

form in aeration tank, heavy enough to settle in settling tank, seperating clean water from sludge complex heterogeneous structures, shape and structure determine how successfully the solids phase separates from the liquid pahse in secondary clarifiers

Role of heterotrophs in activated sludge

require organic carbon as carbon source, feed on bacteria and control bacterial numbers and reduce turbidity

Role of metazoa in activated sludge

acts as predators contributing to the removal of bacterial cells

Role of bacteria in activated sludge

removal of C, N, P, toxins, complex organic compounds and pathogens, FLOC formation

Bacteria responsible for reducing carbon

heterotrophs (require organic carbon as C source), nitrifiers (autotrophs that use ammonia as energy source), denitrifiers (heterotrophs that require organic carbon)

Bacteria responsible for reducing phosphorus

Polyphosphate-accumulating organisms, heterotrophs, work in both aerobic and anaerobic zones

role of viruses in Activated Sludge

many human viral particles (like HIV) unlikely to survive process, more robust like rotavirus, reovirus and adenovirus probably survive, bacteriophages probably act to reduce population densities

Role of fungi in activated sludge

role as predators on nematodes, come from air or soil

factors affecting survival of microorganisms in activated sludge

growth rate, mean cell resident time, dilute rate, advantage being in floc, growth factors, tolerance to abiotic factors and toxins, ability to contribute to floc formation

inorganic forms of N include 

NH4/ NH3 - toxic to aquatic organisms

NH4 and NO3- contribute to ozone depletion and the greenhouse effect and acid rain formation

NO2- can cause ‘blue baby’ disease and form carcinogens called nitrosamines

Nitrogen redox cycle

1. nitrogen fixation (bacteria change nitrogen gas from air —> ammonia in soil) 

2. ammonification (organic nitrogen —> ammonia) 

3. nitrification (ammonia —> nitrite —> nitrate) - supply energy for bacterial growth

4. denitrification (in oxygen low places, nitrate —> nitrogen gas)

bacteria that carry out ammonia oxidation

nitroso bacteria (aerobic and chemolithoautotrophs)

bacteria that oxidise nitrite to nitrate

nitro bacteria (aerobic and chemolithoautotrophs)

Autotrophic Ammonia Oxidising Bacteria (AOB) or nitroso bacteria 

gram negative, aerobic chemolithoautotrophs

1. members of betaproteobacteria 

2. members of gammaproteobacteria 

Biochemistry of AOB

two step process

1. ammonia + oxygen + H —> Hydroxylamine + water 

Enzyme = ammonia monooxygenase (No ATP)

2. Hydroxylamine + oxygen —> nitrite + water + ATP 

enzyme = hydroxylamine oxido-reductase 

Nitro or Nitrite Oxidising Bacteria (NOB)

gram negative chemoautotrophic bacteria using CO2 as carbon source, require lots of ATP 

Taxonomy of NOB

nitrospira, 2 species = N. marina, N. moscoviensis

Biochemistry of Nitro Bacteria

2NO2 + O2 —> 2NO3

enzyme = nitrite oxido reductase in cell membrane

using an anoxic tank to turn nitrate —> nitrogen gas and aeration tank to turn ammonia—> nitrate with nitrate recycled between the two tanks to complete the cycle

Nitroso bacteria in activated sludge

only rarely detected in activated sludge plants, better adapted for low substrate concentration and so out-competed in activated sludge

nitro bacteria in activated sludge systems

in clusters, and physically located next to clusters of AOB, syntrophy, AOB supply their energy source Nitrite, NOB remove toxic nitrite from AOB cells

dissimilatory nitrate reduction

reduction of NO3 or NO2 to gaseous products of N2, NO or N2O by chemoorganoheterotrophc microbes called anaerobic respiration

why does denitrificaiton require an anoxic zone

because N2O is 300X worse than CO2, in the anoxic zone there is no O2 but plenty of NO3, an organic carbon (often added) and energy source

denitrification steps

Nitrate —> nitrite —> nitric oxide —> nitrous oxide —> dinitrogen

harmful intermediates like N2O can accumulate in activated sludge systems

The Anammox process

Ammonia + nitrite —> dinitrogen + H2O (via hydrazine)
surrounded by membrane with unique lipids called ladderanes
protect rest of cell from hydrazine

microbiology of anammox

nitrite first oxidised to NO by nitrite reductase

hydrazine hydrolase —> hydrazine (from NO + ammonium) —> N2 (by hydroxylamine oxidoreducatase

using anammox for treating wastewater

suitable for N removal with high ammonium content and low C:N ratios

requires no C addition or aeration

needs nitrite as an energy source 

P reduction from wastewater with lime or alum

low inital cost, dose flexibility, ease of control, low energy use, low maintanence, improved clarifyer performance, reliable, high chemical cost, environmental damage, reduced bio-available P

P reduction from wastewater with enhanced biological phosphorus removal EBPR

lower long term costs, only biosolids produced, environmentally more acceptable, higher reduction of P achievable, recyclable, high initial costs, relatively higher energy costs, larger foot-print, less reliable, potentially higher maintenance costs

EBPR

uses special bacteria to remove phosphorus from water - without chemicals

1. anaerobic zone: PAOs take in organic matter and release phosphate into the water 

2. aerobic zone: the same PAOs use oxygen to grow and take up more phosphate than they released before, storing it inside their cells

3. sludge removal: when sludge is removes from the system, the phosphorus leave with it 

EBPR biomass from anaerobic zones

stains for poly B-hydroxybutyrate (PHB) but not polyphosphate (POLY-P)

stored polyphosphate is utilised by PAO to provide energy (ATP) which cells use to assimilate the Readily Biodegradable COD (RBCOD) for synthesis of PHB

EBPR biomass for aerobic zones

stains for polyphosphate (POLYP) but not for PHB

utilise PHB as a carbon and energy source, provide cells with selective advantage in absence of other carbon sources
PHB provide energy for P assimilation and synthesis of intracellular POLYP, when sludge is wasted P removed from system inside biomass = BIOSOLIDS

cells from EBPR plants on artificial media

most bacteria that grow are Acinetobactor spp, belong to gram negative gammaproteobacteria 
acinetobactor will synthesise polyP aerobically but not PHB anaerobically

Culture independent methods

use 16srRNA, design FISH proves against dominant clones, apply staining techniques to see if any possible PAO

most are from Betaproteobacteria

acinetobacter spp detected in very small numbers by FISH in EBPR biomass samples

FISH data

confirm that phodocyclus like bacteria are a POA called Candidatus "‘accumulibacter phosphatis’ 
FISH/ MAR reveals it has correct phenotype assimilates acetate anaerobically to synthesis PHB and phosphate aerobically

MAR

microautoradiography

FISH

fluorescent in situ hybridisation

Why EBPR plants often fail

PAO are out competed by other non-PAO bacteria for substrates in anaerobic zone

bacteria = glycogen accumulating organisms (GAOs) - assimilate acetate and sysnthesise PHB anaerobically, then use PHB aerobically to syntheise glycogen with no polyP accumulation

GAOs

bacteria found in wastewater systems - they compete with PAOs

cannot stain for glycogen inside bacterial cell = cannot idenitfy GAO from microscopic exam

How GAOs work?

1. anaerobic - GAOs take up volatile fatty acids from wastewater, energy from breaking down stored glycogen, do not release or take up phosphate

2. aerobic - use oxygen to rebuild glycogen stores using energy from oxidisng the stored carbon, focus is on carbon and energy storage not phosphorus removal