HOB Culture Medium Ingredients

Bugs
1

Hi @“NarcisP”#9

I've been trying to source the ingredients from your [2023 paper](https://doi.org/10.1016/j.jece.2023.111550) so we can start an HOB culture. I'd like the culture medium to be entirely edible by humans, and as easy for people to procure as possible so my purchasing priority is:

A. Food ingredients from mainstream supermarkets
B. Food ingredients from specialist stores
C. Food grade chemicals from mainstream online retailers
D. Food grade chemicals from specialist chemical retailers
E. Food grade chemicals from chemical manufacturers/wholesalers (who don't appear to normally sell to the public)

You mentioned at the Journal Club that you changed from ammonium chloride to ammonium sulphate to reduce chlorine formation - I was wondering if we could go a step further to ammonium bicarbonate (like [Entirely Ingredients](https://amzn.eu/d/hBfaVPj) - C) which seems easier to procure, and would reduce the likelihood of sulphur-reducing bacteria, or is the sulphate necessary?

Also, might you be able to list the ingredients of the micronutrients solution?

Here's what I've found so far:

  • 1. 0.47 g/L (NH4)2SO4 - ASPURE - E (They don’t have pricing, but offer samples, however they don’t even take an address in the sample application process, so I think I may buy Entirely Ingredients Ammonium Bicarbonate - C instead)

  • 1. 1.05 g/L NaHCO3 - [Dr Oetker](https://www.tesco.com/groceries/en-GB/products/282996938) - A
  • 1. 2.3 g/L KH2PO4 - [APC Pure](https://apcpure.com/product/potassium-dihydrogen-phosphate-99-102-00-anhydrous-acs-fg/) - D
  • 1. 2.9 g/L Na2HPO4 - [APC Pure](https://apcpure.com/product/di-sodium-hydrogen-phosphate-anhydrous-98-5-acs-food-grade/?attribute_pack-size=100gm) - D
  • 1. 0.5 g/L MgSO4⋅7 H2O - [Health Leads Epsom Salts](https://amzn.eu/d/49Etx5x) - C
  • 1. 0.01 g/L CaCl2 - [Intralabs](https://amzn.eu/d/21TBBSU) - C
  • 1. 5 mL/L micronutrients solution based on the [formulation by Rabaey and colleagues](http://refhub.elsevier.com/S2213-3437(23)02289-3/sbref36) - is behind a paywall
    • 2

    Hi @Martin. Sure, you can use ammonium bicarbonate instead of ammonium sulphate without any problem.

    3

    @“NarcisP”#p104 Brilliant, they’ll hopefully reach me in time for next Wednesday. Do you know how I can get my hands on the micronutrients though?

    4

    Crazy Idea:

    Instead of sourcing many food grade chemicals to make up a micronutrient solution, how about we try human multi-vitamin and mineral supplements…?

    I've been looking at the options, wanting to find one that is:

  • 1. Internationally available
  • 2. Comprehensive in vitamin and mineral content
  • 3. Affordable
  • 4. Fairly easy to get into solution

    • So far I'm thinking [Centrum Advance Women 50+](https://www.centrum.co.uk/products/multivitamins/centrum-women-50-plus/) which claims to have:

  • - Vitamin A (RE) (50% as beta-carotene) - 800 µg
  • - Vitamin E (α-TE) - 16 mg
  • - Vitamin C - 80 mg
  • - Vitamin K - 30 µg
  • - Thiamine - 1.5 mg
  • - Riboflavin - 1.8 mg
  • - Vitamin B6 - 2.1 mg
  • - Vitamin B12 - 7.5 µg
  • - Vitamin D - 15 µg
  • - Biotin - 75 µg
  • - Folic acid - 300 µg
  • - Niacin (NE) - 20 mg
  • - Pantothenic acid - 9 mg
  • - Calcium - 340 mg
  • - Phosphorus - 105 mg
  • - Magnesium - 107 mg
  • - Iron - 4.2 mg
  • - Iodine - 100 µg
  • - Copper - 500 µg
  • - Manganese - 2.4 mg
  • - Chromium - 40 µg
  • - Molybdenum - 50 µg
  • - Selenium - 30 µg
  • - Zinc - 5 mg

    • The 50+ range seem to have higher concentrations of most things than their regular Advance other than iron, and the Women variety has more of most things than the mens (including Iron). I also looked at [Sanatgen A-Z Complete](https://www.sanatogen.co.uk/products/sanatogen-az-complete-multivitamin) which contains concentrations of each of the above bar Potassium (possibly not an issue since we have KH2PO4 in the main solution. I also looked at [H&B Expert](https://www.hollandandbarrett.com/shop/product/h-b-expert-multivitamin-tablets-60049854), [Lamberts Multi-Guard](https://www.lambertshealthcare.co.uk/multiple-formulas/multis-for-adults/multiguard/) and [Performance Lab Nutrigeneses](https://uk.performancelab.com/products/nutrigenesis-multi-men) the last two of which contained more ingredients than the Centrum options but was concerned that these may not be easily available outside the UK. The Performance Lab were attractive as they come in capsules, so we wouldn't need to grind up the pills, but some of their ingredients (like fibre) may cause issues and they are significantly more expensive.

    Anyway what do you think? Is there anything that our Hydrogen Oxidising Bacteria may be missing, or indeed anything that may inhibit their growth, and do you recon one tablet crushed into 1L would be the right concentration? I have reached out to Prof Rabaey and will try to compare the above with his micronutrients solution when I get my hands on a copy.

    5

    I was discussing this with Bard (who you may be interested to know can do comparison tables, which Bing CoPilot can’t yet do) and saw that the Centrum Advance tablets contain fish oil.

    So, while the Centrum are suitable for human consumption, they aren't suitable for vegans. According to Bard Sanatogen A-Z Complete also contains bovine gelatin and most of the options I was looking at contain cholecalciferol (vitamin D3) from lanolin. Bard found these [Vegan Vitality](https://vegan-vitality.co.uk) and while I bizarrely can't directly see the contents either on their website or their [amazon.co.uk listing](https://amzn.eu/d/ankwzAB) Bard managed to give the following:

    #### Vitamins

  • - Vitamin A (as Beta-Carotene): 800 μg RE (100% NRV)
  • - Vitamin D3 (as Cholecalciferol from Lichen): 400 IU (100% NRV)
  • - Vitamin E (as mixed tocopherols): 400 IU (267% NRV)
  • - Vitamin K2 (as Menaquinone-7): 75 μg (50% NRV)
  • - Vitamin C (as Ascorbic Acid): 200 mg (250% NRV)
  • - Thiamine (Vitamin B1): 1.4 mg (100% NRV)
  • - Riboflavin (Vitamin B2): 1.6 mg (114% NRV)
  • - Niacin (Vitamin B3): 18 mg (112% NRV)
  • - Pantothenic Acid (Vitamin B5): 6 mg (120% NRV)
  • - Pyridoxine (Vitamin B6): 2 mg (143% NRV)
  • - Biotin (Vitamin B7): 50 μg (100% NRV)
  • - Folic Acid (Vitamin B9): 400 μg (200% NRV)
  • - Vitamin B12 (as Methylcobalamin): 50 μg (300% NRV)

    • #### Minerals

  • - Calcium (as Calcium Carbonate and Citrate): 220 mg (28% NRV)

  • - Magnesium (as Magnesium Citrate): 400 mg (100% NRV)
  • - Iron (as Ferrous Bisglycinate): 18 mg (129% NRV)
  • - Zinc (as Zinc Bisglycinate): 25 mg (250% NRV)
  • - Selenium (as L-Selenomethionine): 55 μg (100% NRV)
  • - Iodine (as Potassium Iodide): 150 μg (100% NRV)
  • - Chromium (as Chromium Picolinate): 40 μg (100% NRV)
  • - Molybdenum (as Molybdenum Chelate): 75 μg (100% NRV)
  • - Manganese (as Manganese Citrate): 2 mg (100% NRV)
  • - Copper (as Copper Bisglycinate): 1 mg (111% NRV)
  • - Potassium (as Potassium Citrate): 100 mg (2.5% NRV)
  • - Boron (as Boron Citrate): 1 mg (167% NRV)

    • #### Other Ingredients

  • - Bulking Agent (Microcrystalline Cellulose),

  • - Anti-Caking Agents (Silica Dioxide, Magnesium Stearate, Stearic Acid),
  • - Glazing Agent (Hydroxypropyl methylcellulose), Coating (Hypromellose),
  • - Colour (Titanium Dioxide).

    • While they are made in the UK, and can apparently be shipped most places, we may want to find a more global brand. I need to head to dinner now, so I'll buy some on [Amazon](https://amzn.eu/d/ankwzAB), if it transpires that the bugs can't eat them, at least I can...

    7

    OK, unless anyone disagrees, I’m about to make up the following:

    1. 0.56 g/L NH4HCO3 1 - Entirely Ingredients
    2. 1.05 g/L NaHCO3 - Dr Oetker
    3. 2.3 g/L KH2PO4 - APC Pure
    4. 2.9 g/L Na2HPO4 - APC Pure
    5. 0.5 g/L MgSO4⋅7 H2O - Health Leads Epsom Salts
    6. 0.01 g/L CaCl2 - Intralabs
    7. 1 pill/L Vegan Multivitamins & Minerals with High B12, D3 & K2 - Vegan Vitality 2
    8. Make up to 1L with raw water 3

    The next big question is then whether I should start the culture with 100% culture media rather than adding it gradually to a sample of pure raw water that we electrolyse. I'm thinking the former otherwise the ionic strength may be too low for successful electrolysis.

    ---

    1 to give the same mass of NH4 as 0.47 g/L (NH4)2SO4 - please check my maths
    2 see https://forum.amybo.org/d/25-trace-element-solution/3 for further discussion of trace minerals
    3 rather than risk washing out the bugs should the chemostat flow be too high, I'm thinking of making up the initial culture media with the raw water that we're enriching.

    8

    @“Martin”#p111 I did a quick calculation and your maths for the concentration of NH4 looks correct. Good luck!

    9

    Thanks @SandrineH

    10

    I just noticed that there is CaCl2 in both the culture medium and the trace mineral solution?

    11

    Well spotted. I’d want to avoid Chlorides in these mesonutrients at least (see @NarcisP’ reply here) although I’ve just checked that they used CaCl2 according to their 2023 paper[1] I have a feeling that avoiding chlorides altogether may increase yield (unless of course the bugs actually need chloride in which case we’re stuck with a balance of meeting that need and bleaching them.

    [1] N. Pous, M. D. Balaguer, P. Chiluiza-Ramos, L. Rovira-Alsina, L. Bañeras, and S. Puig, ‘Electricity-driven microbial protein production: Effect of current density on biomass growth and nitrogen assimilation’, Journal of Environmental Chemical Engineering, vol. 11, no. 6, p. 111550, Dec. 2023, doi: 10.1016/j.jece.2023.111550.

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