Goldsmiths’ 2013

Liquid biofuel production using microorganisms

Professor Rod Scott, Department of Biology and Biochemistry, Bath University


The Great Bath — the entire structure above the level of the pillar bases is a later construction. Bath was charged with responsibility for the hot springs in a Royal Charter of 1591 granted by Elizabeth I. This duty has now passed to Bath and North East Somerset Council, who carry out monitoring of pressure, temperature and flow rates. The thermal waters contain sodium, calcium, chloride and sulphate ions in high concentrations. The green colour is not just caused by algae.

Throughout history people have been worried that the Earth’s resources won’t keep up with population growth. One of the first people to put this into print was Thomas Malthus.

The Reverend (Thomas) Robert Malthus FRS (13 February 1766 – 23 December 1834) was a British cleric and scholar, influential in the fields of political economy and demography. Malthus himself used only his middle name Robert.

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Malthus became widely known for his theories about change in population. His An Essay on the Principle of Population observed that sooner or later population will be checked by famine and disease. At the time that he wrote his essay the population of the world was around 1 billion. He worried about the scramble for resources.

He is buried in Bath Abbey and his epitaph is just inside the entrance.

Of course Malthus wasn’t aware of the effect of technology.

How will we meet the challenge of providing the resources for a growing population?

Population – competition for resources


If there are 249 births a minute and 103 deaths a minute then there is a net growth of 146 people per minute. This equates to 77 million people a year.

In developed countries there are not enough young people to look after the old. In Japan they are developing robots to do this.

In less developed countries there are far more babies born per minute. This means lots of mouths to feed.

Resources: Food

By 2050 the population is expected to reach 9.3 billion. Global grain production must double to feed everyone.

Children are an asset in agricultural areas and less well developed economies, particularly ones heavily dependent on unreformed (subsistence) agriculture but they are a liability in the more developed areas of the Earth where the costs of rearing children is very high so parents can’t afford to rear many children, therefore the birth rate goes down. And because infant mortality is very low, it’s sensible to invest a lot of resources in few children. In short, in the developed world children are financial liabilities not assets.

Expensive children are a contraceptive that doesn’t require government intervention (with the exception of the one child policy as practiced in China and forced sterilisation as practiced in India).


In 1798 Thomas Malthus incorrectly predicted that population growth would outrun food supply by the mid-19th century. In 1968, Paul R. Ehrlich reprised this argument in The Population Bomb, predicting famine in the 1970s and 1980s.

Paul Ralph Ehrlich (born May 29, 1932) is an American biologist and educator who is the Bing Professor of Population Studies in the department of Biological Sciences at Stanford University and president of Stanford’s Centre for Conservation Biology.

The predictions of Ehrlich and other neo-Malthusians were vigorously challenged by a number of economists, notably Julian Lincoln Simon, and advances in agriculture, collectively known as the Green Revolution, forestalled any potential global famine in the late 20th century.

However, neo-Malthusians point out that the energy for the Green Revolution was provided by fossil fuels, in the form of natural gas-derived fertilizers, oil-derived pesticides, and hydrocarbon-fuelled irrigation, and that many crops have become so genetically uniform that a crop failure could potentially have global repercussions.

The rising cost of oil and the increased growth of biofuels along with global climate change and the loss of agricultural land may also create resource shortages. There have been food riots in some countries.

Resources: Land

There is plenty of land but some of it has limited use and some of it is in the wrong place. So are we reaching resource limitation e.g. the amount of arable land?



There are actually less than a billion people in Africa. The Chinese are buying up land in sub-Sahara Africa so they can feed themselves.

Resources: Water

70% of global water consumption is used for agriculture and up to 95 % of consumption in some developing countries (IWMI 2006). Irrigated agriculture, which accounts for only 20 % of cultivated land but over 40 % of the global harvest, has significant implications for the future of water availability and food security worldwide. With economic development and higher incomes around the world, per capita food consumption–and subsequently water consumption–will also rise. In particular, as more people in developing countries can afford diversified diets including meat and vegetables, agricultural water use will rise dramatically; it takes ten times more water to raise a kilogram of beef than a kilogram of wheat.

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Resource: Fuel

There is a rising demand for the finite resources of fossil and nuclear fuels. There is also a demand for security of supply. An added problem is that fuel prices are going up, and this is likely to be a long term trend.


Could microalgae make a contribution to our fuel needs?

Rising temperature and atmospheric CO2 levels

To keep rising temperatures below 2oC the most urgent reduction in Co2 emissions needs to be 20-40% by 2020 and 80-90% by 2050. The limit was agreed at the 2009 Copenhagen climate change summit.

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We cannot continue to burn oil, even if there are infinite quantities available. Despite that we are still looking for more gas to guzzle.

Shale gas is natural gas that is found trapped within shale formations.

Hydraulic fracturing is the fracturing of rock by a pressurised liquid. The technique is very common in wells for shale gas, tight gas, tight oil, and coal seam gas.

Global, per day, oil consumption

80-90 million barrels of oil are used worldwide per day. 80% of this is used for liquid fuels. This equates to 14,310,000,000 litres per day and is enough to fill 5,724 Olympic swimming pools. The pool is 50 metres long, 25 metres wide, minimum of 2 metres deep giving a liquid volume of 2,500,000L.


Units above are gallons and 1 barrel of oil = 159 litres

This has generated a thirst for renewable fuels

Electricity = 33% of global energy market

Liquid fuels = 66% and liquid fuels are needed in the long term for heavy machinery, haulage, shipping and aviation.

We need a renewable energy source that generates liquid fuel.

How is oil made and how long does it take?

Petroleum is a fossil fuel derived from ancient fossilized organic materials, such as zooplankton and algae.


The process occurs naturally over 10-30 million years when buried to a depth sufficient to achieve high temp & pressure (in the Mississippi River delta, it may take 10 million years). In some respect it is wrong to call it a non-renewable fuel because it is being made all the time. We are simply using it up faster that it can be made.

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The process can occur in the lab in hours or days at high temperature and pressure in a process called pyrolysis.

Pyrolysis is a thermochemical decomposition of organic material at elevated temperatures in the absence of oxygen. It involves the simultaneous change of chemical composition and physical phase, and is irreversible.

If we were to use 30 barrels/day for 200 million years this gives a total 2.2 trillion barrels. This means consumption is 2-3 million times faster than formation.

The cost of fuel


From the January issue of Car and Driver


Renewable energy sources include photovoltaics, solar thermal, geothermal and wind and wave but these can’t be used for vehicles and machinery.


Ethanol biodiesel, methane butanol and liquid hydrogen are needed.

Liquid renewables represents 33% but we need it to be at least 66%. An increase of 35% by 2025 is needed for all the new cars. In China alone there were 17m new cars in 2010.

We need a renewable source of liquid fuel.

Global climate change and fossil fuel depletion is driving the need for solutions to the ever increasing energy demand.

One such solution is biofuels – which in theory provide sustainable energy usage via a closed carbon cycle.

Biofuel is a fuel derived from ‘fresh’ biological material, unlike fossil fuels which utilise dead organic matter, millions of years old. Ideally it should use the whole plant.

‘First-generation biofuel’ feedstocks include sugary or starchy materials fermented into bioethanol, or oil from seeds that can be used in biodiesel.

How bioethanol is made



Second generation biofuel use special-energy non-food crops (e.g. Miscanthus) and waste biomass containing lignin & cellulose. This does not divert food away from the food chain and is relatively abundant.

Third generation biofuel is the production of biodiesel from algal oils.


The process is carbon neutral? Energy to grow the crops only causes a slight gain in C emissions.

It can be economical especially 2nd generation biofuels.

It helps tackle poverty issues (Thailand 2nd generation biofuel installations have aided strengthening of the agricultural sector).


Rapid cycling reduces soil fertility

It competes with agricultural land and freshwater – upsetting the balance between Energy, Food and Water Security (availability and socio-political and economic implications) e.g. 1st generation biofuels divert food from the human food chain, leading to shortages and price increases.

– All of these factors can put developing countries at greatest risk.

Biofuels – solar to chemical energy

Use photosynthesis to capture solar energy in combustible molecules such as ethanol and other hydrocarbons.

Solar energy could give us 5,700 times the global energy demand. Captured at 100% efficiency this would only require 0.017% of Earth’s surface. However Photosynthetically active radiation (PAR) is only 40% of this and typical actual solar energy to biomass conversion efficiency is a paltry 0.1-2.0%. We would actually need 1.13-2.10% of the Earth’s surface where 29-54% is arable land.


Sugar cane conversion efficiency is better at 8% but it requires 3.6-6.4% of arable land.


Algae’s current biomass conversion efficiency is less than sugar cane at 1-4% but is requires 3% of land mass where 1.5-2.7% can be NON-arable land.

Another way of explaining the above: 100% sunlight → non-bioavailable photons waste is 47%, leaving 53% (in the 400–700 nm range) → 30% of photons are lost due to incomplete absorption, leaving 37% (absorbed photon energy) → 24% is lost due to wavelength-mismatch degradation to 700 nm energy, leaving 28.2% (sunlight energy collected by chlorophyll) → 32% efficient conversion of ATP and NADPH to d-glucose, leaving 9% (collected as sugar) → 35–40% of sugar is recycled/consumed by the leaf in dark and photo-respiration, leaving 5.4% net leaf efficiency.

Not all wavelengths of light can support photosynthesis. The photosynthetic action spectrum depends on the type of accessory pigments present. For example, in green plants, the action spectrum resembles the absorption spectrum for chlorophylls and carotenoids with peaks for violet-blue and red light. In red algae, the action spectrum overlaps with the absorption spectrum of phycobilins for red blue-green light, which allows these algae to grow in deeper waters that filter out the longer wavelengths used by green plants. The non-absorbed part of the light spectrum is what gives photosynthetic organisms their color (e.g., green plants, red algae, purple bacteria) and is the least effective for photosynthesis in the respective organisms.

There is a conflict for resources: Food vs Fuel

30% of the USA corn harvest is used to produce bioethanol. For North Americans this isn’t a problem as they have plenty of food available, but 30% of corn in a developing country may mean that the people will go hungry.

Because of this conflict it matters how we proceed with new technologies, such as algae for biofuel production.

Microalgae offer efficient land-use

Microalgae may avoid the food vs fuel problem. It is highly productive, and does not need arable land.

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Chisti 2007


In organic chemistry, transesterification is the process of exchanging the organic group R″ of an ester with the organic group R′ of an alcohol. These reactions are often catalysed by the addition of an acid or base catalyst. A mixture of methanol and sulphuric acid can be used for this process.

Economics – algal biofuel

Algae fuel or Algal biofuel is an alternative to fossil fuel that uses algae as its source of natural deposits.

At the moment algal biofuels are not currently economic, even at $90/barrel. To improve matters capital costs of production facilities (open pond/PBR) need to be reduced and productivity needs to be increased (biomass yield g/m2/d).

A photobioreactor (sometimes abbreviated PBR) is a bioreactor that incorporates some type of light source to provide photonic energy input into the reactor.


Large scale production of algae

There is a problem with light penetration and stirring costs money.


Open Ponds have low CapEx, are low yielding with the possibility of contamination.


Photobioreactors have high CapEx, are high yielding with reduced contamination.

Bioprospecting at Bath


So what sort of algae is found at the roman baths?

Roman algae – thermo-tolerance


The water in the Roman baths (Aquae Sulis) has a temperature range of between 39- and 46°C. The water flow rate is 1,170,000 l/day.

The Roman temple was constructed in 60-70 AD but not enclosed for more than 100 years.

The aims of the project

There are many obstacles to the realisation of microalgal biodiesel outlined below. Knowledge is very fragmented and outdoor culture, which is cheapest, is fraught with problems (variable conditions/contamination).

Prof. Scott’s project focuses on cost;

Maintaining the temperature, which is necessary if you want the algae exposed to intense sunlight to give the highest productivity. This may require a source of heat. You may also need to produce a source of carbon dioxide for photosynthesis;

Extracting the oil as the cell walls are tough;

Making sure there is enough algae to replace what has been taken;

Finding ways to integrate the growth and separation processes (e.g. programmed apoptosis or selective culturing for thinner/weaker cell walls).

Cooling is also expensive. Sunlight will give you the product but also heat. HOT flue gas needed for a CO2 feed? à Roman baths

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Most microalgae show optimal growth in a narrow temperature range (20) with temperatures of 35oC or more being lethal or at least causing reduction in productivity.

In the interest in finding algae with high temperature tolerance they analysed water samples from the roman baths for potential candidate algae.

The picture on the left shows the Kings Bath (hottest source 46oC) whilst the picture on the right shows collection from the cooler (39oC) ‘Great Bath’. To their surprise they found a variety of microalgae some of which are shown below.



Algae typically have a similar density to water hence centrifugation is often employed as a means of harvesting which is energy intensive.

Microstraining is cheapest way to harvest algae (no additives required but only applies to filamentous strains)

Isolated and identified 7 species

DNA bar-coding: U16S (cyanobacteria) or U18S (eukaryotic algae)

Coelastrella saipanensis, Chlorophyta, Klebsormidium sp., Chlorophyta, Hantzschia sp.., Bacillariophyta, Chroococcidiopsis thermalis, Cyanophyta, Microcoleus chthonoplastes, Cyanophyta, Mastigocladus laminosus, Cyanophyta

Oscillatoria sancta and Cyanophyta


Improving productivity: mutation breeding for reduced chlorophyll antennae size


Light reactions produce chemical energy and reducing power for the dark reactions to make sugar molecules

Photosystems contain more chlorophyll than required to service the dark reactions. This captures excess photons, the energy from which is dissipated as heat and fluorescence.

Reducing the size of the chlorophyll antenna enables more photons to penetrate into the algal culture.

The desire is to increase efficiency with less chlorophyll.

Open pond culture

pH 3-4

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Flocculation (100x concentration)

Flocculation, in the field of chemistry, is a process wherein colloids come out of suspension in the form of floc or flake; either spontaneously or due to the addition of a clarifying agent. The action differs from precipitation in that, prior to flocculation, colloids are merely suspended in a liquid and not actually dissolved in a solution. In the flocculated system, there is no formation of a cake, since all the flocs are in the suspension.

If the pH is right then the algae binds together and simply falls out of the water.


Centrifugation: Wet paste

Waste water is useful as algae love waste ammonia.


Wastewater treatment and reduced biomass production costs


The N and P problem of wastewater

Wastewater ‘polishing’ is costly (energy, chemicals, plant), generates greenhouse gases, is technically challenging, particularly for P (ferric chloride) chemical precipitation usually with salts of iron (e.g. ferric chloride).

Each person excretes between 200 and 1000 grams of phosphorus annually.

It seems sensible to use it on algae.

Too much nitrogen and phosphorus in the water kills fish.


Eutrophication or more precisely hypertrophication, is the ecosystem response to the addition of artificial or natural substances, such as nitrates and phosphates, through fertilizers or sewage, to an aquatic system. One example is the “bloom” or great increase of phytoplankton in a water body as a response to increased levels of nutrients. Negative environmental effects include hypoxia, the depletion of oxygen in the water, which induces reductions in specific fish and other animal populations. Other species (such as Nomura’s jellyfish in Japanese waters) may experience an increase in population that negatively affects other species.


The above graph shows that far too much nitrogen and phosphorus is ending up in the water.



Why a PBR? >>> a closed system means you can control conditions, have monocultures, higher growth rates and a higher productivity…

Pilot Plant

Welsh water

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PBR growing of Chlorella emersonii to produce biodiesel for engine testing.

Chlorella is a genus of single-cell green algae, belonging to the phylum Chlorophyta. It is spherical in shape, about 2 to 10 μm in diameter, and is without flagella. Chlorella contains the green photosynthetic pigments chlorophyll-a and -b in its chloroplast. Through photosynthesis, it multiplies rapidly, requiring only carbon dioxide, water, sunlight, and a small amount of minerals to reproduce.

With some algae you have to “starve” it to get it to produce oil.

Heterotrophic oil production from abundant carbon sources

Miscanthus is a genus of about 15 species of perennial grasses native to Subtropical and tropical regions of Africa and southern Asia, with one species (M. sinensis) extending north into temperate eastern Asia.

The rapid growth, low mineral content, and high biomass yield of Miscanthus make it a favourite choice as a biofuel. Miscanthus can be used as input for ethanol production, often outperforming corn and other alternatives in terms of biomass and gallons of ethanol produced.

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EM Rubin Nature 454, 841-845 (2008) doi:10.1038/nature07190

Alternatives to microalgae: oleaginous yeast

Metschnikowia pulcherrima

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This a type of yeast that can produce fats using many different carbon sources such as C5, C6 and longer chain sugars such as oligomers, dimers and trimers. It grows on the surface of grapes. Most types of yeast can only use C6, not C5.

It has a high biomass productivity : >10g/L

It is an extremophile: acidophile (pH 2-4)

It can survive in none-sterile culture in open ponds

It doesn’t cost much to produce.

It is a thermophile;

It did produce a small amount of ethanol.

An extremophile (from Latin extremus meaning “extreme” and Greek philiā (φιλία) meaning “love”) is an organism that thrives in physically or geochemically extreme conditions that are detrimental to most life on Earth.

Acidophiles or Acidophilic organisms are those that thrive under highly acidic conditions (usually at pH 2.0 or below).

A thermophile is an organism — a type of extremophile — that thrives at relatively high temperatures, between 45 and 122 °C. Many thermophiles are archaea. Thermophilic eubacteria are suggested to have been among the earliest bacteria.

Open pond culture at pH 2-4


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Produce stuff that doesn’t have much of a use and feed it to the yeast which then excretes the useful stuff.

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