TLDR:

1. Identified needle moving variables for Algae growth and optimal inputs

   1. Medium — Wastewater

   2. Choosing and Indoor (instead of an outdoor) operation

   3. Photobioreactor using Vertical Tubular photobioreactor

   4. Light — white LED lights with a light intensity of less than 20 mol m−2 day−1, 12 h:12 h light and dark period.

   5. Temperature — 25 to 30°C

   6. Mixing, Aeration, and Flowrate — Peristaltic pump at speed of 100RPM for a mixing time below 17.5s, and an aeration rate of 0.05vm

   7. pH — most papers suggesting between 7.0 and 8.0

2. Identified key performance indicators: Biomass, lipid, protein, and optical density (growth rate).

Medium — Decided to Use Wastewater

Normally Chlorella Vulgaris requires freshwater to grow. Based on some of the research papers below wastewater of pretty much any type seems to be a functional medium. The benefit of having wastewater as a medium is companies and governments pay for wastewater to be cleaned. Instead, they can give us the culture medium for free and pay money for it to be cleaned by algae.

The algal pond could remove pollutants, especially N and P, more efficiently compared to the conventional activated sewage process (Zhou et al., 2014). C. Vulgaris, achieved a nitrogen reduction percentage of 100% in wastewater, under high light supply conditions (Gonçalves et al., 2014).

For cleaning wastewater. The study showed 80% TN (total nitrogen) removal efficiency. This coincides with a study reported by Iasimone et al. (2018). Affected by C. Vulgaris, 94% TP (total phosphorus) removal efficiency was recorded at the end of the cultivation period.

Chemical oxygen demand (COD) is the amount of oxygen needed to oxidize (take away electrons from) the organic matter present in the water — seems like taking away the electrons is how you clean the water. COD removal was recorded at the exponential growth phase of C. Vulgaris with 72.24% removal efficiency.

Previous studies have shown that Chlorella can grow and produce biomass in wastewater such as urban wastewater (Tercero et al., 2014), domestic wastewater (Aziz and Ng, 1992), textile wastewater (Chu et al., 2009), piggery wastewater (Ji et al., 2012), etc. This study showed that cultivating Chlorella with membrane-treated distillery wastewater MTDW was also a feasible strategy. Biogas slurry (another form of waste) can also be used. Seems like any type of waste works out well with Chlorella.

Biomass productivity of Chlorella in different wastewaters ranged from 0.029 g L−1 d−1 to 0.64 g L−1 d−1. In contrast, 0.04 g L−1 d−1 biomass productivity of Chlorella cultured in MTDW is not high, indicating that the use of MTDW to cultivate Chlorella to produce biomass still has much room for improvement.

Conclusions: Wastewater seems to be an effective method of cultivating Chlorella. Chlorella seems to be effective at cleaning wastewater — according to the metrics above. You can get paid by companies/governments to clean wastewater + you don’t need to pay anything for the medium which is good.

The question I need to answer is: Is it better than water treatment plants in terms of cost, time to clean, and effectiveness (how clean it is?)

Outdoor vs Indoor Operation.

The reason you want indoor instead of outdoor is you produce higher yields, reduce your chance of contamination (which could kill your algae), avoid variable temperatures (too much or too little heat could decrease growth rate), that the light frequency is on the spectrum of 400 to 700nm (having 1 specific color of light can optimize growth. The clear downside is indoor operations cost more.

Note: This seems to be a consensus among papers I have read.

The way I think about cost is that it is very similar to vertical farming and traditional agriculture. Vertical farming has better yield, less land used, less water used, no need for pesticides because the environment is controlled, etc. The main downside of vertical farming was it cost more. Even if it isn’t cheaper right now they are fairly new (the oldest came in 2012). Tech always decreases cost over time.

Type of Photobioreactor (PBR)

Why you would want to use PBR?

My main idea: Traditional agriculture vs vertical farming. On a yield per space, you are doing much better. If you get costs down then indoor wins. Running on the assumption the cost of photobioreactors can be decreased similar to how vertical farming companies figured it out.

Types of PBRs — Review Paper

• Tubular, Plastic Bag, Column airlift, and flat-panel airlift reactors are recommended for large-scale operations.

• Lower photosynthetic efficiencies were obtained with the horizontal tubular photobioreactor and the raceway pond.

• High photosynthetic efficiencies were obtained in Vertical Tubular and flat panels.

Light

Photon Flux Densities (light intensity) — The photosynthetic photon flux density is the amount of photosynthetically active photons (400–700nm) hitting a surface per unit area per unit time. Measured in μmol m−2 s−1 or some similar measurement.

Effect of photon flux density on photosynthetic efficiency — Photosynthetic efficiency is the efficiency at which solar light energy is captured as stored chemical energy in biomass and it allows the estimation of the productivity for other locations if the photon flux density is known.

The photosynthetic efficiency of plants is directly linked to their ability to convert atmospheric carbon dioxide energy in the form of sugar in a process referred to as carbon fixation.

Status Quo of light — Sun and fluorescent light. The problem with the sun is you are reliant on good weather to grow algae, which is not optimal for algae growth. You could have rainy days, super hot (will why in temperature section) negatively affecting growth or not having consistent algae output due to input being variable. When you compare fluorescent light to LED lights turns out LEDs have a smaller size, are lighter weight, last longer, are more efficient in terms of longer operating life, and the amount of light given off is much higher compared to their power consumption

What are the best-LED lights?

Chlorella Vulgaris (the type of algae that seems to have the highest capture potential. Has two types of chlorophyll: chlorophyll-a and chlorophyll-b (best for Biodiesel growth). Chlorophyll-b primarily absorbs blue light which has wavelengths from 400 nm to 450 nm, and chlorophyll-a absorbs red light which has wavelengths from 650 nm to 700 nm. Using blue and red LED light created 60% more biodiesel compared to broad-spectrum LEDs (white).

White LED Light seems to be the best performing and another this paper, white light seems to create the highest biomass.

What is the best light intensity?

Maximal photosynthetic efficiencies were obtained for the three closed photobioreactors below 20 mol m−2 day−1. This was in a pilot-scale operation.

Light and Dark Periods — In Chlorella Vulgaris, higher biomass production were achieved under 12 h:12 h light and dark period

When grown under more than 17h of light, the plants of greenhouse fruiting vegetable crops usually become damaged, showing signs of leaf chlorosis (reduce chlorella), reduced photosynthesis, and ultimately, a reduction in yield (Hao et al., 2018). It has been hypothesized that this photo-injury is controlled by circadian rhythms through an integrated temperature and light signaling pathway in the plant.

Temperature

There are different strains of Chlorella Vulgaris — curious how much they move the needle.

Paper 1 suggests 25–28°C

Paper 2 suggests 25 to 30°C

Paper 3 suggests 25 to 30°C

Paper 4 suggests 25°C.

The more stable culture temperatures in the closed photobioreactors could have contributed to the higher photosynthetic efficiencies.

Mixing and Aeration and Flowrate

Based on this review paper — a peristaltic pump would be what is used for a larger scale. A Peristaltic Pump puts pressure inside your system (photobioreactor) to move the algae around without causing damage to the cells. The reason you need a pump is to make sure all of the algae gets exposed to the light source. Without the pump, only part of the algae grows.

In this paper, 100RPM seems to be performing better than 120RPM. It seems that 350 is the max that has been identified before you start damaging the algae cells. Probably wanna keep at a lower RPM to save power. Lower mixed time of bubbling in PBR is correlated with higher biomass production. Lipid and biomass have an inverse relationship. When biomass increases, lipids decrease.

Three identical bubbling PBRs with the flow rates of 2.7 L/min, 1.3 L/min, and 0.2 L/min were used to investigate the effects of flowrate on biomass production and lipid yield. The bubbling PBR with the highest flow rate (2.7L/min) produced the highest biomass yield (16 × 106 cells/mL), whereas the PBR with the flow rate of 0.2 L/min was not successful to produce biomass.

It may be due to the higher flow rate providing better mixing effects in the bubbling PBR, which not only prevents microalgae settling but also absorbs more light energy for photosynthesis to have higher biomass productivity.

Another way that Aeration (or flowrate is measured)

1.0 volume per minute (vvm) — VVM stands for volume of air (in culture) per unit volume of growth medium per minute, it is calculated by dividing measured airflow rate (units: L/m, using a rotameter) with the volume (L) of growth medium (including cultured cells). In this paper, an aeration rate of 0.05 vim produced the maximum biomass productivity (268.1 mg/L/d) and protein productivity (155.4 mg/L/d).

Applied Dilution

It is how much culture you are adding. For example, using a volume of 300 ml a dilution rate of 0.1 means that 30 ml of media is added to the culture per increment of time (per hour or per day)

The highest dilution rates in the flat panel photobioreactor (0.4 day−1) and open raceway pond (0.24 day−1) were only applied for a short period, 6 and 11 days, respectively, as these resulted in a strong decrease in biomass concentration.

So high dilution is not the way

The highest photosynthetic efficiencies were obtained with the vertical photobioreactors with intermediate dilution rates; 0.2 day−1 for VT and 0.3 day−1 for Flat panel.

Size of Glass

The system with the shortest optical path (0.02 m), the flat panel photobioreactor, resulted in the overall highest average areal (biomass) productivity. The overall lowest average areal productivity was obtained with the open raceway pond, because of the long optical path (0.2 m).

We need to have smaller distances for the glass to minimize the optical path.

Vertical Tubular vs Horizontal Tubular photobioreactors

In vertical photobioreactors, microalgal cells dissipate less of the absorbed light energy as a result of lower photon flux densities because of light dilution on the reactor surface in comparison to the horizontal systems.

Less light energy is lost in vertical vs horizontal.

Vertical photobioreactors have higher yields than horizontal photobioreactors because of the hydrodynamics, the amount of gas in dispersion, and their gas-liquid mass transfer characteristics (pH, pCO2, pO2). The vertical reactor’s upright orientation allows gas to be introduced at the bottom of the reactor and travel to the top.

The short optical path of the flat panel photobioreactor results in the small dark zone in the culture; respiration takes place in a small part of the culture. The long optical path in the open raceway pond results in a large dark zone in the culture. [11–13].

Limiting the chance for respiration or night time limits the loss of lipids and biomass. The energy that could have been used for growth is used to create o2 which is not the use of CO2 we want.

Higher photon flux densities will penetrate deeper into the culture and will decrease the size of the dark zone present in the culture.

Variations in areal productivity were larger for all photon flux densities and dilution rates in the flat panel photobioreactor and the open raceway pond than in the tubular systems. The large variations in the flat panel photobioreactor are a result of the plug flow regime moving the culture through each panel. The culture is not mixed well overall panels, while in the other systems the entire culture volume is mixed resulting in less variation in areal productivity.

We would probably want to use a tubular system (vertical) to decrease variation, even though flat panel bioreactors have a higher upside with a higher variation.

In the open raceway pond, the large variation in areal productivity is the result of low culture temperatures and automated level control. The low culture temperatures resulted in suboptimal conditions during a large part of the day, for many days throughout the experimental period. The automated level control in the open raceway pond resulted in negative areal productivity; for days with heavy rainfall, dilution rates were higher than intended because of the automated level control. Raceway ponds are not that great.

PH

Most microalgal species grow best at a pH range between 7.0 and 8.4 [64], [65],

Under all light intensities the ph increased. Cell Density was highest was the highest at pH 10. The cell density with pH control at 10.0 was 1.32 times higher than that that of initial pH at 10.0pH

[Optimal rate](https://pubmed.ncbi.nlm.nih.gov/21090111/#:~:text=Using HCl and NaOH regulated,of Chlorella vulgaris was 7.0.) for a waste type culture — 7.0 + if you want to optimize for lipid generation.

Most microalgae species have a favorite pH range of 8.2–8.7, although they can also be cultivated in the pH range between 7 and 9 [81].

Optimal growth occurred when the pH of the medium was adjusted to values of 7.5 and 8.0.

What I would need to test: How pH creates the difference in specifically wastewater. Honestly inclined for 7 to 7.5. Since it is within the general range of plants that I’m aware of of. Probably need to have this as a controlled temperature

Effect of Nutrients — 3 Essential Ones for Biomass Production

Carbon, nitrogen, phosphours. microalgae assimilate sufficient nitrogen and phosphorus from medium for their metabolic activities. Seems to require similar nutrients to most plants.

Nitrogen, is the main component of algal proteins and enzymes catalyst, and are responsible for microalgae growth, photosynthesis and metabolism (Kong et al., 2021).

Phosphorus is another key element for microalgae growth and other cellular activities (energy transfer and biosynthesis of nucleic acids) (Kong et al., 2021).

Main Outcomes (KPIs)

Biomass Production

Biomass production is the most significant indicator to observe when research into CO2 bio-sequestration by microalgae cultivation — high sinking capacity = increased biomass.

Lipid Production

It has been widely accepted that the production of these lipids serves as energy storage to microalgae cells. Through the process of trans esterification, the Triacylglycerol (TAG) found in adipose tissue serves as the major energy storage form in higher eukaryotes (algae being one of them). TAGs can be easily converted into fatty acid methyl esters which are an important and versatile form of biodiesel and the cornerstone for its production.

Protein Production

The species Chlorella vulgaris — 51–58% protein of dry wegth

In fact, there can be as much as six times the amount of the amino acid arginine than that found in whey protein, and almost the same amount of glutamine. Algae protein is on par if not better than whey proteins

It also doesn’t require land used for agriculture since you would operate this indoors where you control all of the variables.

Algae exhibits rapid biomass production containing high oil contents, at least 15 to 20 times higher than land based oleaginous crops.

Optical Density — How Growth Rate is Measured

In general, we grow to our full adult size via an increase in the number — not the size — of our cells. This is the same in cells in plants, the more cells the more cells the more plant growth.

Below is an image of an LB medium. One on the left is a fresh LB medium and the one on the right is the same medium after shaking it overnight in a 37 degree incubator.

The one on the left is clear while the one of the right is cloudily. As bacteria/cells grow it makes the medium more cloudy. The cloudiness is called optical density. The higher the optical density the more cells that are created — more growth. Ideas from this video.