Growing Botryococcus

A vital element of the economic production of Algae Biofuels is growing the algae efficiently. Botryococcus is often described as relatively difficult to grow; however, this should be viewed as a relative comparison to other algae rather than an absolute statement. It is certainly not as difficult as many tissue culture systems and the like, but it is easy to neglect its needs because it is such a slow grower (and then it equally slow to recover). There is also very limited well-documented efforts where one has gone beyond simple 'maintenance' of cultures. We hope to provide within these documents a description that will convey experience to allow someone to rapidly get to 'happy cultures' and try to minimize the 'art' as opposed to rationale considerations that are based on expereince.

We have grown Botryococcus on very high light (~1000 uE/m2/s), low light, very high density, 5% CO2 and even air. All are possible, though remember that adaptation for a slow growing organism requires many months.

General Plant Growth Standards

Plant Growth Measurements	Metric	Units
Dry Weight Measurements	grams Dry Weight per Liter	gDW/L
Optical Density	Absorbance Unit	AU

Foreign Bacteria/Contaminants

There are very few reports of axenic, or non-contaminated, B. braunii race B cultures in the literature. There are only two labs in the world that are known to hold axenic cultures of B. braunii, Curtislab at Penn State University and a lab in Japan (Citation needed).

One method to create axenic samples of B. braunii race B is to use "a serial n-hexane pretreatment followed by a 15-second wash with sodium hypochlorite and distribution of 200 microliters of aliquots into sterile eppitubes." Making an axenic culture of B. braunii will facilitate large-scale growth of the algae for economical biofuel production. Axenic cultures are also significantly easier to sequence than contaminated ones.

B. braunii Growth

Volumetric productivity of 22.5 mg oil/L/photo-hour is the highest found in systems using B. braunii. This productivity is "more than an order of magnitude higher than previous reports..."

B. braunii has achieved dry Weight densities greater than 30gDW/L, with "daily replacement of 5-15% of the respective cultures."

Botryococcus braunii

Race B

B. braunii race B has been grown using a "feed-forward culturing strategy", or "predicting the nutrient requirements of a culture based on a nitrogen-based balance."

As a precaution before growing B. braunii, some labgroups keep "back-up, stock cultures... due to the difficulty of maintaining high-density B. braunii race B cultures.

Some conditions to consider growing B. braunii are the following:

Light Limitations	Nutrient Limitations	pH Levels
Gas Transfer Rates	Conductivity ( Ions)

Light Limitations

Algal cultures tend to become light-limited at high cell densities. Therefore, in order to maintain those higher cell densities, light intensity must be increased sequentially to facilitate an increase in cell density. This increase is done slowly, to allow the plants to adapt to higher light levels without photoinhibition caused by rapidly changing light intensity.

Nutrient Limitations

B. braunii can be grown in many media, but at Curtislab the WFAM medium is used. WFAM is short for Wayne's Freshwater Algae Medium.

pH Levels

Stoichiometric control of the pH level allows for optimization of algal growth. Through the use of Nitrate (NO3) and Ammonium (NH4), one can control the nitrogen uptake of the algae while also managing its pH levels and minimizing the acidifying effects of liquid CO2.

Previous studies have shown that algal cultures preferentially utilize ammonium when both ammonium and nitrate are available in the presence of excess CO2. These studies reported that when a sufficient amount of ammonium was present in the medium, algal cultures assimilated ammonium until the pH of the culture dropped to inhibitory levels which led to algal death although a switch to nitrate assimilation could have maintained culture viability.

Gas Transfer Rates

CO2-limitation is a problem to consider when growing B. braunii. Having an insufficient quantity of CO2 in an algal growth system can cripple and eventually kill algae.

The Gas Transfer Rate of CO2 in B. braunii for a 50mL sample is 0.0816mol/Liter-hr for B. braunii. Determined experimentally, this information is utterly crucial to having efficient growth rates for the algae.

Conductivity (Ions)

The conductivity of a bioreactor can be used to determine "an accumulation of ions" that is sometimes responsible for "rapid culture crash and washout". Work with B. braunii in bioreactors has found that "gradual but significant increase[s] in electrical conductivity... suggests the accumulation of ions in the media, and most likely the accumulation of counter-ions of the various salts that are very difficult to balance due to the constraints on the major nutrients such as nitrogen and phosphorous." The conductivity of a bioreactor is simply another tool that can be used to diagnose issues in bioreactor systems.