Research led by Professor Martin Warren at the Quadram Institute and the University of Kent has led to several scientific and technological developments for sustainable vitamin B12 production. The traditional process for synthesising B12 uses bacteria and needs cyanide and cobalt, a heavy metal that is damaging to the environment. A novel strain of Escherichia coli (E. coli) was developed that required significantly less cobalt, leaving no surplus.
Further work produced a metalation calculator that enables producers to calculate the exact amount of cobalt needed. Used together, these developments could dramatically improve the sustainability of B12 production, with a much lower risk of environmental damage. This is also not just limited to cobalt, being potentially applicable to similar processes that use other damaging heavy metals.
With increasing levels of B12 deficiency due to changing diets and an ageing population, this improved production on an international scale will also address a growing need for the vitamin.
The importance of vitamin B12
Essential nutrients for life include vitamins and minerals, along with proteins, fats and carbohydrates. There are 13 essential vitamins needed for the proper functioning of our metabolism. Most organisms cannot produce enough (or any) of these nutrients, so they must be sourced from our diets.
Vitamin B12, also known as cobalamin, is an essential micronutrient. It is important in red blood cell production, nerve function, and DNA production. A lack of B12 can cause a wide range of symptoms, including cognitive changes, tiredness, shortness of breath, muscle weakness and anaemia.
If left untreated, deficiency can cause permanent damage. This also applies to newborns, with adequate amounts needed for foetal neural tube development.
Where do we get B12 from?
Despite being essential for life, humans and other animals cannot produce B12 for themselves. It is only produced by certain species of bacteria that live in the gut. However, in humans, these bacterial species produce a form of B12 that is non-functional in human cells. In contrast, bacteria that live in the intestines of ruminants, such as cows, produce the correct form of B12 that humans can use.
Humans can acquire their B12 from beef and dairy products, including milk, yoghurt, and cheese, as well as other meats, eggs, fish and shellfish.
For vegans, fortified foods, including breakfast cereals and nutritional yeast, are an alternative source. These fortified foods or supplements are required by individuals on vegan and vegetarian diets who face challenges sourcing B12 due to how it’s not made nor required in plants.
This is also a particular issue for individuals with the autoimmune disease pernicious anaemia, which prevents the absorption of B12 in the gut due to a depletion in the main B12-binding protein involved in B12 absorption. Certain medications, inadequate stomach acid, intestinal surgeries and digestive disorders can also prevent proper B12 uptake. This creates a wide and varied group of affected individuals in need of commercially produced B12.
As we move towards more sustainable diets, undernutrition or ‘hidden hunger’ will become a wider issue. We must be able to produce safe and sustainable B12 quickly and easily.
Vitamin deficiencies in the UK
For many vitamin deficiencies, prevalence is not well-defined or studied. It’s estimated that 6% of both UK and US adults below 60 are B12 deficient, which jumps to 20% for those older than 60 and 11% for vegans.
In comparison, one in six UK adults and nearly 20% of children have low vitamin D levels, with the UK government suggesting supplements between October and March. Vitamin C deficiency, which leads to scurvy, is much rarer for UK adults, sitting around 5% for men and 3% for women, but these figures increase if sampling low-income groups or those older than 65.
Malnutrition and scurvy are both on the rise in the UK, with healthcare admissions for both malnutrition and scurvy more than doubling between 2008 and 2018. Ricketts was also on the rise before dropping back to 2008 levels in 2016.
What counts as a deficiency?
One of the reasons B12 deficiency may be a ‘hidden hunger’ is that there is a lack of national and international clarity and cohesion regarding normal cobalamin serum levels.
Current UK guidelines for serum cobalamin levels suggest anything below 200 nanograms per litre would be abnormal, and those with less than 100 nanograms per litre are definitively deficient. Many NHS hospitals also regard 150 to 180 nanograms per litre to be ‘early deficiency’.
There is ongoing conversation and research within the scientific and medical communities on reference ranges for serum cobalamin. It’s suggested that many countries’ reference ranges are too low, creating a large ‘grey area’ when diagnosing B12 deficiency. This could lead to individuals of all backgrounds experiencing symptoms but not receiving the treatment needed due to these barriers to diagnosis.
It’s generally agreed that the trend is towards more people becoming B12 deficient. If more people are diagnosed there will be a pressing need to improve the production of this essential vitamin sustainably.
B12 commercial production
The challenges to commercial, environmentally responsible, and economically viable B12 production are multifactorial. Structurally, B12 is the most complex of all the vitamins. The complete biosynthesis pathway requires 30 different genes in the bacterial genome to be activated in a specific order. The total chemical synthesis of vitamin B12 has also been achieved but involves over 70 different steps, making it non-viable on an industrial scale. For that, we rely on the bacteria.
Commercially, vitamin B12 is produced through bacterial fermentation. B12-producing bacteria are grown in large vats of over 100,000 litres but, even at this scale, produce relatively low yields. Once produced, the B12 must be extracted and purified. It can take two weeks to go from fermentation to the pure product.
On top of the production costs, B12 production leads to potentially harmful surplus or waste products that need responsible and costly handling to prevent environmental damage. B12 production requires cobalt and cyanide. Cobalt is a carcinogen, so any excess after B12 production needs to be safely disposed of, preventing negative effects on the surrounding environment. Cyanide is also a hazardous compound, needing the same careful treatment and disposal. B12 production factories abide by strict environmental rules and restrictions to guarantee this.
This extensive process makes B12 the most expensive nutrient on the market, raising costs for healthcare or industrial needs. Rising costs and lack of steady sources has also seen the price of B12 vary from £3,000 to £20,000 per kilogram.
The inherent inefficiencies of production, the environmental risks and the increasing need to combat B12 undernutrition demand a novel approach.
A safer and better way of production
Professor Martin Warren, who leads the Quadram Institute’s synthetic biology and biosynthetic pathways group, has been working on a solution to the multivarious B12 problem.
Professor Warren’s current area of research interest is vitamin B12, how B12 affects the gastrointestinal microbiome, and how this is related to B12 deficiency. Via an international collaboration, Professor Warren’s team engineered an E. coli strain that can produce B12 on a similar scale to current commercial strains.
Crucially, the team was able to modulate the strain’s uptake of cobalt and metal transportation mechanisms, reducing the amount of cobalt that was added to the growth medium by an order of magnitude. All the cobalt that is added is absorbed during the process, so there is no surplus cobalt to act as an environmental risk, making the strain more environmentally friendly and affordable than current strains. Many other bacterially mediated production processes that require heavy metals could benefit from this.
This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) LINK grant, which supports academic-industry collaborative research. It was also supported by BBSRC’s Elements of Bioremediation, Biomanufacturing and Bioenergy (E3B): Metals in Biology network. This is one of six BBSRC Networks in Industrial Biotechnology and Bioenergy (NIBB), a BBSRC-Engineering and Physical Sciences Research Council-funded effort to build capacity and capability in the UK for sustainable and bio-based manufacturing.
Professor Warren says:
The LINK grant was definitely the right way to go. I am always supportive of anything we can do to get industry involved more. If we’re going to exploit science, we have to work with industry. The funding and support from BBSRC have always been incredibly positive. I think the link with the whole network, the NIBB, is brilliant.
I’m very excited to see what’s going to happen with the Diet and Health Open Innovation Research Club (OIRC) because it allows you to interact with others interested in food, nutrients and health. It gets people talking.
Calculating for sustainability
One such interaction between Professor Warren, Professor Nigel Robinson, and Dr Tessa Young brought further success. The collaboration worked on a metalation calculator that allows researchers to optimise metal uptake in cells. For B12 production, the calculator would allow manufacturers to pinpoint how much cobalt would be needed. If used with the new E. coli strain, the calculator would further support more sustainable B12 production.
This calculator is not just limited to cobalt in B12 production and could be applied to many other production processes that use metal.
Professor Warren says:
About 50% of enzymes have a type of metal in them. Quite a few of those are heavy metals like copper, iron, and nickel. So, there are many other bacterially produced enzymes and commodities that also require the addition of metals, which need to abide by EU environmental rules.
BBSRC funded several grants that contributed to the development of the calculator, including several responsive mode grants and support from the Metals in Biology BBSRC NIBB.
Purification with proteins
Outside of BBSRC-funded research, Martin’s team have also been working on improving the lengthy purification process for vitamin B12.
One popular and traditional method for protein purification is column affinity chromatography. This is the scientific equivalent of pushing your solution, which is full of the protein of molecules you want to be purified, through a sticky tube or column. The method uses a protein with high affinity to your desired product, which is stuck onto the column. It plucks out the product, keeping it in the column, while everything else gets flushed out.
Some bacterial species naturally produce proteins that have a high binding affinity for B12. By taking these proteins and using them in affinity chromatography, researchers could easily purify B12. Some of the binding proteins Martin’s team has been experimenting with could cut the purification time from a week to 20 minutes. This could significantly improve production rates.
Purified B12 also allows for a more controlled addition of cyanide, which can reduce contamination risk.
This research, however, is still ongoing. While these proteins successfully bind and purify B12, they need to be able to release both the column and the B12 to be reused, which has yet to be achieved.
The future for B12 in the UK
With a dedicated partner and the developments made by Martin’s team, the UK could produce sustainable and environmentally friendly B12. This could prove powerful in the fight against B12 deficiency and ‘hidden hunger, in turn reducing NHS costs.
And there are other initiatives underway, beyond improving the commercial B12 production process, for working towards this goal.
Martin emphasises the need for educational programs around B12 nutrition and hidden hunger. He says:
I think it’s up to us as scientists and the science funders to try and hammer home these messages as much as possible. Otherwise, it’s going to be a generation, possibly even more, of children who are growing up eating bad food. Through education, we can try to get people to think a little bit more savvily about what they’re eating.
Martin’s research on B12 is also continuing, including through several ongoing BBSRC grants. This includes a collaboration with Professor Anthony Dodd at the John Innes Centre, which is another BBSRC strategically funded institute. This project builds on previous research that fortified pea shoots with B12. The collaboration aims to do so on a commercial scale, which could enable us to get our daily B12 needs from mixed salad bags.
Martin says:
We can now get the recommended daily allowance of B12 into a single pea shoot using an aeroponic approach. BBSRC, through the Follow-on Fund, has given the money needed to take that further.
Martin also initiated cluB-12, an international discussion group that is working to raise awareness around B12 deficiency, targeting policymakers and the public to prevent health implications. Members of the group include patients, patient group representatives, scientists, GPs, and medics. CluB-12 will continue to work on a global clinical determination of B12 levels and the availability of treatments for deficiency.
Find out more
The Quadram Institute’s impact case study: an environmentally friendly vitamin B12 production method that makes manufacture more affordable.
The Quadram Institute’s news post: addressing the vitamin B12 insufficiency pandemic.
The Quadram Institute’s blog post: the global group understanding vitamin B12’s role in our health.
Durham University, E3B: Metals in Biology.
BBSRC’s Diet and Health OIRC: diet and health innovation boosted by new funding partnership.
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