Module 5 - Exploiting microorganisms Flashcards
What two microbes are involved in the production of cheese?
Lactobilli reduces the pH of milk, creating a suitable environment for Rennet (chymosin) to function
Rennet protease cleaves surface glycopeptides from soluble casein molecules in milk
Cleavage of the negatively-charged side chains in kappa-casein forms para-kappa casein which allows for coagulation - the formation of hard cheese
Milk ripening
Surface ripening - the use of ripening at the surface of the food (ie mould on top of the camembert cheese)
Invasive ripening - the use of ripening within the food (ie mould within Roquefort cheese)
Ales
Top fermenters (Saccharomyces cerevisiae) cause yeast to rise to the surface
Ferment (between 20 and 25ºC) in just a few days
Ales tend to have heavier bodies, more alcohol, a darker hue and are cloudier than lagers
Lagers
Involves bottom fermenters (Saccharomyces carlsbergensis) causing yeast to settle at the bottom
Ferment anywhere from one to three months at a much colder temperature than ales (7 to 15ºC)
Lagers have a cleaner taste and appearance and have a lighter body than ales
Lagern means “to store” in German.
Flavour development: what compounds affect the flavour of alcohols?
Esters and fusel alcohols
Esters in flavour development
The most important aroma compounds in beer (ales) which also give a “fruity” character to beer, less desirable in lagers.
Fusel alcohols in flavour development
Group of long-chain alcohols that contribute to beer flavour directly or indirectly as ester precursors
Strong flavours (“alcoholic” or “solvent-like“ aroma) in beer (ales) and lager
Citric acid in beverages
Widely used in the preparation of food and sugar confectionery, soft drinks and beverages, detergent/cleaning industry, and as a stabiliser in medicines
Originally obtained from lemons, produced from a fungus since the 1940s, production worldwide is around 2.4 million tonnes p.a (expected to be close to 3 million tonnes by 2026)
Citric acid production
Citric acid produced from the tricarboxylic acid (TCA) cycle
Normal conditions - little if any citric acid is formed, however, with low Mn and Fe concentrations, citric acid is induced
Sugars purified by precipitation or ion exchange and stainless steel bioreactor used to avoid iron
Non-starch polysaccharides
High content of arabinoglucans increases viscosity in the gut, reducing absorption
A particular problem in the poultry industry
We can reduce viscosity by including ß-glucanase with the feed
Enables other cereals with high glucan content to be included with the feed e.g. barley
Phytate and phytase
Phytate (organic phosphate) cannot be degraded by monogastric stomachs
Phytase is produced by microbes to liberate phosphate from phytic acid, reducing phosphate supplementation by 40%
Food-bourne diseases
Two-thirds of all food poisoning outbreaks involve bacteria (the rest are caused by viruses, parasites, fungi and chemicals)
~ 600 million (1 in 10 people in the world) fall ill after eating contaminated food, >420 000 deaths every year (WHO)
Major disease in developing countries and most cases in children under 5 (40% of foodborne disease burden - for 125,000 deaths per year)
Food-bourne disease outbreak
Caused by many potential risk factors:
- Globalization and mass transport and production of processed foods (ie fast foods)
- Change in population (ageing, malnutrition, etc)
- New strains arising, (ie antibiotic resistance)
- Changes in food production/supply
Listeriosis: the process of infection
Fresh, raw vegetables (especially salads), unpasteurised milk, cheese, ice cream, uncooked fish (including smoked) & meats
Growth: When shredded but not cooked or treated with preservatives, plant saps (sugars etc) released, aiding Listeria growth (which grows at 4°C)
Contaminated water, manure added to soil directly contaminates vegetables, and indirectly contaminate milk
- Rare but has high mortality rates (20-30%)
Listeriosis: the process of infection
Rarely affect healthy adults unless a large dose, very young, elderly, sick and pregnant women are most susceptible
- Causes fever, fatigue, nausea, vomiting, and diarrhoea
- More serious symptoms can result in meningitis (brain infections) and septicemia (bacteria in the bloodstream)
Pregnant women: can result in miscarriage, stillbirth, or meningitis in the newborn
Mycotoxins: what are they, what do they produce, and what was its infection first called?
Toxic fungal metabolites that accumulate in cereal grains, nuts and other foodstuffs, particularly in storage
They produce aflatoxins, highly toxic and carcinogenic which affect man and livestock
First called Turkey X disease in the 1960s in the UK killed >100,000 turkeys due to contaminated groundnuts from Brazil
Aflatoxins: symptoms and testing
Symptoms:
Short-term:
* Abdominal pain and diarrhoea
* Headaches
* Convulsion (seizures)
* Emesis
Long-term:
* Cancer
* Liver/kidney failure
* Brain damage
Testing:
All cereals and nuts must be tested and contain <20ppb if given to humans and must contain <100ppb if given to livestock
Ergot poisoning: what does it do, what microbe causes it, and what are the symptoms of it?
Sporadic throughout history, occasional epidemics
Caused by claviceps purpurea
Causes severe joint pain, hallucinations, seizures, (Gangrene of the fingers, toes, nose and ears in severe cases)
Genetic manipulation: the definition
Involves the direct manipulation of genes
Genetically modified organism
GMO - an organism whose genetic material has been altered using genetic engineering techniques
Agrobacterium: what do they do and what use do they have in plants?
Live in the soil naturally and enter plants through wounds near the base of shoots
By using tumour-inducing (Ti) plasmids, they can use their T-DNA in it to biosynthesis plant growth hormones and octopine (an opine) - an amino acid that can only be utilised by the bacteria
Agrobacterium: the infection process
Wounded plants release acetosyringone and VirA and VirG detect this which begins the movement towards the wound
Once near the wound, a pillar made of VirB and held to the bacterium by VirD4 is made between itself and the plant which allows transport from the bacteria, including VirF, VirE2, and T-DNA supported and moved by VirD2
Plants: how do they detect an infection?
Flagellin (the protein compartment of flagella) is highly conserved and remains the same wherever it is found so essentially all plant cells have a receptor (leucine-rich repeat (LRR)) for flagellin to detect it
If the receptors are triggered, an MKK4/5 cascade is triggered where, eventually, VIP1 is phosphorylated and moves to the nucleus for pathogenesis-related protein (PRPs) transcription.
How does agrobacterium not get immediately destroyed by the plants’ defence system?
By using VirF to degrade VIP1 transcription factors, preventing the manufacturing of PRPs
The bacterium then coats its T-DNA with VirE2 which can bind to and use VIP1 to enter the nucleus and have its T-DNA integrated into the plant’s genome (the mechanism behind this not fully understood)
Why do agrobacteria infect plants?
Their T-DNA causes excessive auxin and cytokinin production which makes tumours which produce opines which can be used as a food source for these bacteria
What can we do to exploit agrobacteria?
Remove both the hormone and opine biosynthesis genes with two genes of interest (likely including an antibiotic-resistant gene)
What are the issues and solutions of agrobacteria exploitation?
Typical Ti plasmid is around 200kb large - way too large for practical use
This is solved by creating a T-DNA plasmid and a helper plasmid. Helper plasmid contains everything excluding the T-DNA and the T-DNA plasmid can have its genes manipulated with its much smaller size
Plant transformation I - co-cultivation
Grow cells taken from leaves with the bacteria
Plant transformation II - selection
The process is inefficient and manipulated cells have to be discovered among countless not manipulated cells, for this reason, signals are often used
Common markers used in genetic engineering
Neomycin phosphotransferase (nptII); confers kanamycin resistance
A bar (bialaphos resistance) gene encodes a phosphinothricin acetyl transferase (PAT); confers resistance to phosphinothricin (antibiotic) or gluphosinate (herbicide)
Plant transformation III - regenration
After manipulated cells are detected, the plant will regenerate naturally due to plant cells keeping their totipotency even after differentiation
After growth hormones used and a shoot is grown, whole plant growth is relatively easy
What is the normal ‘lab rat’ plant?
Arabidopsis - a type of weed
What organisms can be manipulated by agrobacteria?
Many types of Plants, Fungi, and even Chordata (Hominidae)
Botulinum toxin
Bacillus thuringienesis - produces spores with large crystal-like structures made of proteins
These proteins can exhibit toxic activity which is highly specific, only affect insects and may affect different types of insects differently
The potential use of the Bt toxin: insecticide spray
If used as a spray, the active toxin is released and gives some protection from insects for a few days, however, there is not complete stability and the crystals are released and the toxins are broken down eventually
The potential use of the Bt toxin: organic pesticide
If used as an organic pesticide after having the BT gene inserted into other bacteria, then the effect persists for longer as the crystals remain even after death
The potential use of the Bt toxin: gene insertion
Place the Bt gene in front of the 35S promoter causing a more efficient transmission of the toxin
The problem now is to prevent resistance from arising
Advantages of inserting the Bt toxin
- More effective pest control
- Lower rates of disease
- Higher yields
Weed control
Plants are autotrophic, synthesise all vitamins essential amino acids de novo and this causes the pathways to be potential targets for herbicides (another pathway is photosynthesis)
Herbicides must be selective – kill the weeds but do not harm the crop, this is helped by how relatively easy it is to distinguish very different species
Advantages of the use of glyphosate as a herbicide
- Broad-based, i.e. kills everything
- Resistance is rare; although becoming more of a problem
- Relatively environmentally friendly
- Does not persist - broken down rapidly in contact with water
- Very specific inhibitor of EPSP synthase
- Not toxic to humans
Animals (e.g. humans) don’t make aromatic amino acids, don’t have a shikimate pathway.
Finding herbicide-resistant genes
Salmonella synthesise aromatic amino acids
Easy to find mutants - AroA mutants will grow on glyphosate due to an altered EPSP synthase gene
Using herbicide-resistant genes
The whole plant must be resistant to the herbicide
Place mutant Aro A gene in front of a strong promoter (e.g, CAMV 35S (expressed everywhere)) makes the plant immune to the herbicide -herbicide still inhibits the plant gene, but the mutant bacterial gene still works and, since every cell has the gene expressed, resistance is given to the herbicide
CP4 gene
Obtained from agrobacterium, retain low km for the substrate (high affinity) and keep resistance
Disadvantages of using transgenic soya beans
- Farmers pay premiums to supplier
- No increase in yield
- May still have some toxicity
- Reliance on one herbicide
- Spread of glyphosate-resistance
- Concentrated supply in a small number of large companies
Advantages of using transgenic soya beans
- More flexible, pre- or post-emergence
- Only need to be sprayed when required
- Decreased tillage, less erosion
- Increase in glyphosate application offset by a decrease in application of other herbicides
- ~10% decrease in total herbicide applications
- On average other herbicides are at least 3x as toxic and persist in the environment twice as long
- Cost
What are the issues with untreated sewage production?
- Organic compounds are readily oxidised by aerobic bacteria which results in decreased dissolved oxygen, killing aquatic species
- Solids cause silting of rivers and estuaries
- Toxic compounds e.g. ammonia and heavy metals - death to aquatic life/build-up in the food chain
- Pathogenic bacteria and viruses - risk to human health
What are the solutions to untreated sewage production?
Treatment using microbes:
- Reduce organic compounds, resulting in less decreased dissolved oxygen
- Resuspension of solids
- Transformation of toxic compounds e.g ammonia (NH₄) converted into nitrate (NO₃)
These treatments only apply to domestic waste and specialised treatment is needed for different types of waste such as industrial/agricultural
Water and sewage treatment: what happened in 2011?
In 2011, 1x10⁹ tonnes of water was purified in the UK pa. (equivalent to 100 tonnes per person, £250 - £300 per head)
Costs have risen due to legislation to increase environmental standards
Severn Trent water
Treats domestic waste of over eight million people through the use of 38,500 kilometres of sewers
Sewage is a thin liquid containing < 1% solids (food materials, human waste, some detergents, fats, oils and greases, sand, clay, paper fibres etc. )
Severn Trent - 1,048 sewage works with 100 people as the smallest serving and the largest serving in Birmingham with over one million people
How is waste measured?
Using biochemical oxygen demand (BOD) - the amount of oxygen consumed by bacteria in a set time
By law, the BOD must not exceed 20 mgL⁻¹
Domestic waste purification: the primary process
- Primary screening - solids removed using a mesh or filter
- Primary settling tank - settling of fine particles
Primary settling tank leads to either:
* Primary sludge -> anaerobic digestion -> sludge cake
* Secondary treatment
Domestic waste purification: the secondary process
- Secondary treatment - either percolating filter bed process or activated sludge response
- Secondary settling tank - settling of fine particles
Secondary settling tank leads to either:
* Secondary sludge -> anaerobic digestion -> sludge cake
* Tertiary treatment
* Discharged into the water course
Domestic waste purification: the tertiary process
- Tertiary treatment - filtration (sand) or passing over grass plots
Discharged into the water course
Percolating filter tank
Large tanks filled with rocks. As the liquid is sprayed on the rocks, the liquid is filtered before moving into a settling tank where sludge is gathered and removed and the fluid is moved onward in the treatment
- Long-term (30-50 years) - the balance of increasing biofilm due to growth and removal by sloughing and grazing by invertebrates
- Microbes form biofilm on the surface (top 0.5m) of the clinker (oxidising organic compounds)
- Microbes below this oxidize ammonia (NH₄) to nitrate (NO₃) - nitrification
- Regulation of rate of waste spray critical - too high, biofilm gets too thick, causing blockages
Activated sludge processes
Same as PFT but at a faster rate (up to 10x): compressed air pumped into air diffusers
- Differs from percolating filtration as microbes are not surface attached
- Microbes grow as flocs and oxidise organic compounds and NH₄
- Waste spend 4 -8h in the tank
- Running costs are higher and more sensitive to the composition of incoming water compared to percolating filtration
Anaerobic digesters
Used to treat sludge from the aerobic treatment process and reduce organic matter by up to 50%
The process involves incubation in a sealed stirred tank reactor at 35°C which causes anaerobic conditions due to rapid drop in O₂ by aerobes
CH₄, CO₂, and NH₄ produced can be used as a fuel (mainly CO₂ and CH₄ used as biofuel) which can be used to heat the anaerobic digester
Anaerobic digestion issues
Methanogens doubling time is 8-10h compared to 1-4h for acetogens and 30 min for fermentative bacteria
Can cause an increase in H₂ which leads to acetogens forming longer chain organic compounds (e.g. butyric acid).
Are not used by methanogens, the process is therefore continuously monitored (????)
Sludge removal
Sludge has a low odour and is pathogen free
Sludge is dried down to form “Sludge cake” and may be supplied free to farmers as fertiliser (not from cities due to high metal content) or may be incinerated or dumped at sea
At larger plants, linked to the national grid
The efficiency of sewage treatment
BOD reduced by >95%
