US009347035B1

(12) United States Patent (10) Patent No.: US 9,347,035 B1 Carney et al. (45) Date of Patent: May 24, 2016

(54) HAEMATOCOCCUS PLUVIALISCULTURE (56) References Cited COMPOSITIONS U.S. PATENT DOCUMENTS (71) Applicant: HELIAE DEVELOPMENT LLC, 2007,0196.383 A1 8, 2007 Murakami Gilbert, AZ (US) 2011/0312063 A1 12/2011 Malm (72) Inventors: Laura Carney, Chandler, AZ (US); FOREIGN PATENT DOCUMENTS Kristine Sorensen, Chandler, AZ (US) EP 1724337 11, 2006 (73) Assignee: Heliae Development LLC, Gilbert, AZ WOJP 2006138271O9173050 AA1 12/20067/1997 (US) WO 2013O96770 A1 6, 2013 WO 2014O16612 A1 1, 2014 (*) Notice: Subject to any disclaimer, the term of this WO 2014O74769 A2 5, 2014 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 0 days. Chang et al., “Hydrogen Peroxide Production Protects Chlamydomonas Reinhardtii Against Light-Induced Cell Death by (21) Appl. No.: 14/796,732 Preventing Singlet Oxygen Accumulation through Enhanced Carotenoid Synthesis,” Journal of Plant Physiology, 170 (2013) pp. (22) Filed: Jul. 10, 2015 976-986. Schreier et al., “Efficacy of formalin, hydrogen peroxide, and sodium chloride on fungal-infected rainbow trout eggs.” Aquaculture 140 Related U.S. Application Data (1996) pp. 323-331. (63) Continuation of application No. 14/667,917, filed on Pririmary Examiner — Tekchandc ana SaidhSatana Mar. 25, 2015, now Pat No. 91136O7 (74) Attorney, Agent, or Firm — Heliae Development LLC; • 1-a-s s • L vs 8 - 3 u. --- sw • Justin Kniep; Len Smith (51) Int. Cl. (57) ABSTRACT CI2N I/12 (2006.01) Culture compositions comprising Haematococcus pluvialis (52) U.S. Cl. and hydrogenydrogen pperoxide are described herein. The composip CPC ...... CI2N 1/12 (2013.01) tions comprise a concentration of hydrogen peroxide based (58) Field of Classification Search on the stage of the cells in the culturing process to increase the CPC ...... C12P 23/OO likelihood of the cells surviving until the process of accumu USPC 435/67. 257.1 lating carotenoids, Such as astaxanthin, is complete. See application file for complete search history. 13 Claims, No Drawings US 9,347,035 B1 1. 2 HAEMATOCOCCUS PLU/LALLS CULTURE In some embodiments, the calculated concentration of COMPOSITIONS hydrogen peroxide may be in the range of 0.005-0.010 mL/L. In Some embodiments, the calculated concentration of hydro CROSS REFERENCE TO RELATED gen peroxide may be in the range of 0.010-0.015 mL/L. In APPLICATIONS Some embodiments, the calculated concentration of hydrogen peroxide is in the range of 0.015–0.020 mL/L. This application is a continuation of U.S. application Ser. In some embodiments, the growth conditions may com No. 14/667,917, filed Mar. 25, 2015, now U.S. Pat. No. 9,113, prise a photosynthetically active radiation intensity in the 607, entitled Methods for Treating a Culture of Haematococ range of 30-60 molm'd', nitrate concentration in the range cus pluvialis for Contamination Using Hydrogen Peroxide, 10 of 20-50 ppm in the culture medium, and less than 1 ppt of the entire contents of which are hereby incorporated by ref sodium chloride in the culture medium. In some embodi CCC. ments, the reddening conditions may comprise the present of 1-5 ppt sodium chloride in the culture medium. BACKGROUND In some embodiments, the method may further comprise 15 determining a level of chytrids in the culture of Haematococ Haematococcus is a microalga that is capable of producing cus pluvialis cells as a percentage of infected cells out of the astaxanthin, a high value carotenoid with antioxidant proper total cells in a culture. In some embodiments, the culture of ties. The culturing process from beginning to end is relatively Haematococcus pluvialis cells may be contacted with the long compared to other common microalgae, Such as Chlo hydrogen peroxide when the level of chytrids is less than rella or Nannochloropsis, and results in a number of chal 20%. In some embodiments, the culture of Haematococcus lenges to the Survival of Haematococcus cells due to the pluvialis cells is contacted with the hydrogen peroxide when nature of Haematococcus as a slow growing microalga. Over the level of chytrids is at least 5%. the course of the culturing process, the Haematococcus cells In some embodiments, the level of chytrids in the culture must go through a growth and cell division phase to accumu may be maintained below the level of chytrids at the time of late biomass before entering a second stage where growth and 25 contact with hydrogen peroxide while culturing the Haema motility is halted but astaxanthin is accumulated in the cells tococcus pluvialis cells in reddening conditions to produce before harvest. Operating this long multi-stage culturing pro cells in the red cyst stage for the accumulation of carotenoids. cess as an open culture increases exposure of the cells to the In some embodiments, the chytrid level after contacting the dangers of contamination, a Sub-optimal environment, or culture with hydrogen peroxide may be 20-95% less than a other conditions which reduce the survival rate of the cells 30 control culture not receiving treatment with hydrogen peroX and ultimately the quantity and quality of the astaxanthin ide. harvest. In some embodiments, the cells may be contacted with the Developing treatments for increasing the Survival rate of hydrogen peroxide multiple times. In some embodiments, the Haematococcus cultures must take into account the sensitivi cells may be contacted with the hydrogen peroxide every 6-24 ties of the cells at the different stages, impact on biomass 35 hours. In some embodiments, the cells may be contacted with growth, and impact on astaxanthin production, as well as the hydrogen peroxide every 6-12 hours. In some embodi effectiveness of the treatment over the long culturing process. ments, the cells may be contacted with the hydrogen peroxide Treatments developed for faster growing microalgae or every day over the course of 1-14 days. In some embodi microalgae cultured for production of whole biomass, lipids, ments, the cells may be contacted with hydrogen peroxide or proteins. Such as treatment with oxidative agents or com 40 every other day over the course of 3-15 days. mercially available herbicides, fungicides, and pesticides, In some embodiments, the biomass yield of the Haemato have not been shown to be easily translatable to Haematococ coccus pluvialis cells contacted with the hydrogen peroxide cus cultures due to the unique stages of the Haematococcus may be equivalent to or greater than a control culture not culturing process, the sensitivities of Haematococcus cells, receiving treatment with hydrogen peroxide. In some and the desire to use the targeted end product of Haemato 45 embodiments, the biomass yield of the Haematococcus plu coccus cultures in human consumption product industries vialis cells contacted with the hydrogen peroxide may be (e.g., nutritional Supplements, food enhancers, therapeutic 0.01-0.25 g/L greater than a control culture not receiving compositions). Therefore, there is a need in the art to devel treatment with hydrogen peroxide. opment treatment methods for increasing the Survival rate of In some embodiments, the carotenoids yield of the Hae Haematococcus cells before and during theastaxanthin accu 50 matococcus pluvialis cells contacted with the hydrogen per mulation stage, without adversely affecting the cells and oxide may be equivalent to or greater than a control culture value of the end product. not receiving treatment with hydrogen peroxide. In some embodiments, the carotenoid yield of the Haematococcus SUMMARY pluvialis cells contacted with the hydrogen peroxide may be 55 0.10-1.50% greater than a control culture not receiving treat In one non-limiting embodiment of the invention, a method ment with hydrogen peroxide. of culturing Haematococcus pluvialis, may comprise: cultur ing a population of Haematococcus pluvialis cells in growth DETAILED DESCRIPTION OF THE INVENTION conditions in a liquid culture medium to obtain a culture of Haematococcus pluvialis cells in which the cells are prima 60 Overview rily in the green Swimmer stage; contacting the primarily green Swimmer stage culture with hydrogen peroxide to form Haematococcus is a genus of microalgae classified in the a calculated concentration in the range of 0.005-0.020 mL of Eukaryota domain, Viridiplantae kingdom, Chlorophyta phy hydrogen peroxide per L of culture medium (mL/L); and lum, Chlorophyceae class, Chlamydomonadales order, and culturing the Haematococcus pluvialis cells in reddening 65 Haematococcaceae family. The species Haematococcus plu conditions to form cells in the red cyst stage for accumulation vialis is typically grown in phototrophic conditions and is of of carotenoids. particular interest commercially for the production of astax US 9,347,035 B1 3 4 anthin, a high value carotenoid (i.e., organic pigment) with the internal cytoplasm without halo breakage, and is strong antioxidant properties. While Haematococcus pluvia expressed as a % of the total Haematococcus pluvialis cells in lis produces astaxanthin, the level of astaxanthin in the cell is the culture. dependent on the culturing conditions and is not present at a For example, the occurrence of lysis in the green Swimmer constant level in the cell over the life of the cell. stage and a chytrid infection in the non-motile cell stages, Haematococcus pluvialis has been studied academically including green and red cyst stage, have been observed to and produced commercially, and thus conventional culture rapidly kill the majority of Haematococcus cells in a culture. conditions may be found in literature in the public domain. A Chytrids are a basal fungus which operates by attaching to culture of Haematococcus pluvialis cells begins in growth microalgae cells, growing into the microalgae cell, reproduc conditions, where the cells are primarily (i.e., at least 80% of 10 cells) in the green Swimmer stage in which the cells may grow ing in the cell, and Subsequently attacking more microalgae and divide but have low levels of astaxanthin. The term “green cells. Such a loss of a Haematococcus culture after resources Swimmer refers to a state of the Haematococcus pluvialis have been expended to culture the cells for multiple days or cell in which the cell is in a motile state, contains cilia, and has weeks, but before a harvest of the cells with a desirable level a larger proportion of chlorophyll (i.e., green pigment) than 15 of astaxanthin can be obtained, may be devastating for a carotenoids (i.e., red pigment from astaxanthin). Haemato commercial operation. The Vulnerability of the Haematococ coccus pluvialis cells may also exist in a non-motile or cyst cus cells is further amplified in open cultures (e.g., open pond stage when the cell has a larger proportion of chlorophyll (i.e., bioreactors), where conditions are harder to control and con green pigment) than carotenoids (i.e., red pigment from tamination is more easily introduced. astaxanthin), which may be referred to as a “green cyst'. The The length of the culturing process for Haematococcus term “growth conditions' refers to culture conditions that increases the necessity for treatments to the culture be facilitate the growth and cell division of the Haematococcus capable of application multiple times over the course of the pluvialis cells, and minimize the stressors that may cause a culturing process without harming the Haematococcus cells, cell to enter a resting state. Growth conditions for Haemato or application of a high initial concentration that remains coccus pluvialis cells may comprise light in the photosyn 25 effective for a long period but does not harm the Haemato thetically active radiation (PAR) wavelengths, carbon diox coccus cells at the initial application. Treatments where the ide, and a liquid medium comprising primarily water, Haematococcus could not tolerate a one-time application at nitrogen, phosphorus and trace metal nutrients. an initial concentration high enough to maintain effectiveness As Haematococcus pluvialis cells mature, they transition against contamination over time, or where multiple applica to a red cyst stage where cell division halts or slows but 30 tions would accumulate a concentration level toxic to the astaxanthin is accumulated as the cell is stressed by reddening Haematococcus would not achieve the goal of getting the conditions. The term “red cyst” refers to a state of the Hae culture to a successful harvest. Additionally, the sensitivity of matococcus pluvialis cell in which the cell is in a resting state, the Haematococcus cells is dependent on the stage or state of has lost the cilia, and has a larger proportion of carotenoids the cells, with the green Swimmer cells being more sensitive (i.e., red pigment from astaxanthin) than chlorophyll (i.e., 35 than the red cyst cells. For example, lysis is more likely to green pigment). The term “reddening conditions' refers to occur withina culture ofgreen Swimmercells than inaculture culture conditions that stress the Haematococcus pluvialis of red cyst cells, and green Swimmer cells are less tolerant of cells to facilitate the transition to a resting state and accumu salt than red cyst cells. A general treatment may only be lation of carotenoids (e.g., astaxanthin) in the cells. Redden effective for one stage of a Haematococcus culture or may be ing conditions for Haematococcus pluvialis may comprise 40 harmful to cells in a certain state, therefore a successful nitrogen or other nutrient deprivation, addition of bi-carbon treatment over the life of a Haematococcus culture must take ate, addition of bleach, and increased levels of salinity, light into account the state of the cells in order to maximize effec intensity, and/or temperature as compared to growth condi tiveness and minimize or eliminate adverse effects on the tions. cells. Due to the size of Haematococcus cells, the culture is 45 Tests of available treatments, including chemical biocides, actively mixed by means known in the art such as, but not blends of natural organic herbs, bleach, Sodium hydroxide, limited to, paddlewheels, gas sparging, and mechanical stir and biological agents were found to have varying levels of rers, in order to prevent the cells from settling to the bottom of effectiveness against chytrids in examples 13-17. However, the bioreactor and to circulate the cells for exposure to avail the public domain knowledge for these available treatments able light and nutrients. Haematococcus pluvialis may be 50 does not address how these treatments will affect Haemato cultured in a number of systems known in the art that meet the coccus cells in the green Swimmer and red cyst stages, and shear sensitivity requirements for green Swimmer cells Such therefore do not provide immediately available solutions to as, but not limited to, column bioreactors with gas sparger the described challenges faced in culturing Haematococcus. mixing, raceway pond bioreactors with paddlewheel mixing, Known methods of adaptation or genetic modification may and bag bioreactors with gas sparger mixing. 55 be used to alter the Haematococcus cells for increased resis The process of culturing a small Volume Haematococcus tance to lysis or contamination, however the process may be culture through the green Swimmer stage to a large Volume in long and expensive. Additionally, genetic modification may the red cyst stage may take weeks due to the slow rate at which limit product markets available for using the Haematococcus Haematococcus grows, divides, and accumulates caro derived astaxanthin. tenoids. During this time period the Haematococcus cells are 60 The inventors have developed the described methods spe Vulnerable to weakening of the physical integrity of the cells cific to the green Swimmer and cyst stages, including red cysts (e.g., lysis) and to attacks by contamination (e.g., bacteria, and green cysts, for use in the contexts of prevention of lysis fungi, predator organisms, other microalgae) which reduce or fungal infection of a Haematococcus culture and treating a the chances of Haematococcus Survival in both the green culture of Haematococcus cells with existing levels of lysis or swimmer and red cyst stages. The term “lysis” refers to Hae 65 fungal infection. Some embodiments of the methods may be matococcus pluvialis cells losing the integrity of the cell used multiple times to treat the same culture of Haematococ membrane and breaking open the outside of the halo or lysing cus, including treating the same culture multiple times in a US 9,347,035 B1 5 6 single day, while minimizing or eliminating any negative viding lethal stress conditions for programmed death of a effect on the biomass yield and carotenoid yield of the cells. genetically modified microalgae using hydrogen peroxide (at The inventors surprisingly found that treatment of a culture least 0.1769 mL/L). The wide ranges of hydrogen peroxide of Haematococcus pluvialis with hydrogen peroxide was suc for different purposes disclosed in the prior art do not address cessful in preventing and treating lysis in a culture of Hae effective concentrations that treat or prevent lysis and fungal matococcus cells without negatively effecting biomass accu infections while avoiding adverse effects on Haematococcus mulation and productivity of the cells, even when in green Swimmer and red cyst cell stages. Due to this defi administered multiple times. The inventors also Surprisingly ciency in the publically available information, such determi found that treatments of a culture of Haematococcus pluvialis nations for the proper concentrations of hydrogen peroxide with hydrogen peroxide, salt, or hydrogen peroxide in com 10 and methods of application for Haematococcus cells in vari bination with Salt were successful in preventing and treating ous stages of the culturing process were determined through a chytrid infection in a culture of Haematococcus cells with extensive experimentation of two different Haematococcus out negatively affecting biomass accumulation, productivity, pluvialis strains by the inventors. Additionally, the extensive and carotenoid accumulation, even when administered mul experimentation by the inventors resulted in the methods of tiple times. Hydrogen peroxide was also found to be advan 15 effectively promoting Survival of Haematococcus cells tageous due to the ability to dissipate quickly in the Haema through prevention and treatment of lysis and fungal infec tococcus culture (e.g., degrades to undetectable levels within tions while also not adversely affecting biomass accumula 2 hours of application), which allows multiple applications to tion, productivity, and carotenoid accumulation of the cells, be applied without the danger of buildup of residual concen which is an additional step of commercial importance not trations or detection in the final harvested product. Salt was addressed by the prior art. treatments were found to be advantageous in that the concen tration of salt in the culture may be a result of an single dosing Method Embodiments at a desired concentration or multiple doses building up to the desired concentration, but remain effective over time without In one non-limiting embodiment, a method of preventing determinant to the Haematococcus cells or harvested product. 25 and/or treating a chytrid infection in a culture of Haemato Hydrogen peroxide may be purchased commercially at coccus may comprise: culturing a population of Haematococ different stock concentrations, therefore a calculated concen cus cells in growth conditions in a liquid culture medium to tration was used to describe the inventive methods. The term obtaina culture of Haematococcus cells in which the cells are "calculated concentration' is a concentration value for a con primarily (i.e., at least 80%) in the green Swimmer stage; tamination treatment Solution calculated by multiplying the 30 contacting the primarily green Swimmer cell stage culture Volume of the treatment solution per culture Volume (e.g., mL with an effective amount of hydrogen peroxide; and culturing of hydrogen peroxide/L of microalgae culture medium) by the Haematococcus cells in reddening conditions to form the percent Stock concentration of the contamination treat cells in the red cyst stage for the accumulation of carotenoids, ment solution. The calculated concentration expressed is in Such as astaxanthin. units of volume/volume (e.g., mL/L) for a theoretical 100% 35 In some embodiments, the Haematococcus cells may be stock concentration of treatment solution applied to a contacted with hydrogen peroxide to form a calculated con microalgae culture. For example, a 1 L. microalgae culture centration in the range of 0.005-0.025 mL/L. In some treated with 10 mL of a hydrogen peroxide solution of a stock embodiments, the Haematococcus cells may be contacted concentration of 50% would have a calculated concentra with hydrogen peroxide to form a calculated concentration in tion=10 mL/Lx0.5=5 mL/L contamination treatment solu 40 the range of 0.005-0.020 mL/L. In some embodiments, the tion. Haematococcus cells may be contacted with hydrogen per The described methods of applying an effective concentra oxide to form a calculated concentration in the range of tion of hydrogen peroxide, salt, or combination of hydrogen 0.005-0.010 mL/L. In some embodiments, the Haematococ peroxide and salt to a culture of Haematococcus may prevent cus cells may be contacted with hydrogen peroxide to form a the occurrence of lysis or a chytrid infection, prevent an 45 calculated concentration in the range of 0.010-0.015 mL/L. In increase in the lysis or chytrid infection level (i.e., maintain Some embodiments, the Haematococcus cells may be con the level), slow the increase of the lysis or chytrid infection tacted with hydrogen peroxide to form a calculated concen level, or decrease the lysis or chytrid infection level in order to tration in the range of 0.015–0.020 mL/L. In some embodi increase the Survival rate of the Haematococcus cells and ments, the Haematococcus cells may be contacted with decrease any negative effect on the accumulation of astaxan 50 hydrogen peroxide to form a calculated concentration in the thin. The level of tolerance a culture of Haematococcus has range of 0.020-0.025 mL/L. for lysis or chytrids may vary depending on the Strain, culture The hydrogen peroxide may be in liquid form. In some conditions, and bioreactor system. Some cultures may be able embodiments, the hydrogen peroxide may be introduced into to survive a lysis or chytrid infection level above 50%, while the culture at a location of active mixing Such as, but not other cultures may only survive at lower levels such as below 55 limited to at the point of paddlewheel or mechanical stifling, 30%, 20%, 10%, or 5%. and a point of high turbulence caused by gas sparging. In While the prior art has generally disclosed the presence of Some embodiments, the hydrogen peroxide may be added all hydrogen peroxide in a microalgae or cyanobacteria culture at a single point. In some embodiments, the hydrogen peroX for a variety of functions, the teachings of the prior art relate ide may be evenly applied over a surface area. to the use of hydrogen peroxide in contexts not directly trans 60 In some embodiments, the cells may be contacted with latable to culturing Haematococcus for astaxanthin product, hydrogen peroxide multiple times. In some embodiments, the Such as: Sterilizing bioreactors with vapor hydrogen peroxide cells may be contacted with hydrogen peroxide multiple and culturing cyanobacteria genetically modified for resis times while the cells are in the green Swimmer stage. In some tance to the residual hydrogen peroxide (0.0024-1.1790 embodiments, the cells may be contacted with hydrogen per mL/L) from the bioreactor sterilization step: killing bacteria 65 oxide multiple times while the cells are in the red cyst stage. in a microalgae culture with intermittent doses of hydrogen In some embodiments, the cells may be contacted with hydro peroxide (0.00001-0.2 mL/L, 0.0590-0.7074 mL/L); and pro gen peroxide multiple times while the cell stages span the US 9,347,035 B1 7 8 green Swimmer and red cyst stage. In some embodiments, the radiation (PAR) intensity in the range of 30-60 molmd'. In cells may be contacted multiple times while the culture is in Some embodiments, the growth conditions for a culture of growth conditions. In some embodiments, the cells may be Haematococcus may comprise a nitrate concentration in the contacted multiple times while the culture is in reddening range of 20-50 ppm. In some embodiments, the growth con conditions. In some embodiments, the cells may be contacted ditions for a culture of Haematococcus may comprise a con multiple times while the culture conditions span growth and centration of less than 1 ppt salt, such as sodium chloride, in reddening conditions. the culture medium. In some embodiments, the cells may be contacted with In some embodiments, the reddening conditions for a cul hydrogen peroxide 2-4 times per day. In some embodiments, ture of Haematococcus may comprise the presence of salt in the cells may be contacted with hydrogen peroxide every 6-24 10 the culture medium. In some embodiments, the concentration hours. In some embodiments, the cells may be contacted with of sodium chloride in the reddening conditions may comprise hydrogen peroxide every 6-8 hours. In some embodiments, 1-5 ppt. In some embodiments, the concentration of sodium the cells may be contacted with hydrogen peroxide every 6-12 chloride in the reddening conditions may comprise 1-2 ppt. In hours. In some embodiments, the cells may be contacted with Some embodiments, the concentration of sodium chloride in hydrogen peroxide every 12-18 hours. In some embodiments, 15 the reddening conditions may comprise 1-3 ppt. The salt may the cells may be contacted with hydrogen peroxide every comprise but is not limited to sodium chloride. 18-24 hours. In some embodiments, a level of chytrids or chytrid infec In some embodiments, the cells may be contacted with tion in a culture of Haematococcus may be determined and hydrogen peroxide every day over the course of 1-14 days. In expressed as a percentage of infected cells out of the total some embodiments, the cells may be contacted with hydro cells in a culture. In some embodiments, the culture of Hae gen peroxide every day over the course of 1-2 days. In some matococcus cells may be contacted with hydrogen peroxide embodiments, the cells may be contacted with hydrogen per when the level of chytrids is less than 60%. In some embodi oxide every day over the course of 1-3 days. In some embodi ments, the culture of Haematococcus cells may be contacted ments, the cells may be contacted with hydrogen peroxide with hydrogen peroxide when the level of chytrids is less than every day over the course of 1-5 days. In some embodiments, 25 50%. In some embodiments, the culture of Haematococcus the cells may be contacted with hydrogen peroxide every day cells may be contacted with hydrogen peroxide when the over the course of 5-7 days. In some embodiments, the cells level of chytrids is less than 40%. In some embodiments, the may be contacted with hydrogen peroxide every day over the culture of Haematococcus cells may be contacted with hydro course of 7-10 days. In some embodiments, the cells may be gen peroxide when the level of chytrids is less than 30%. In contacted with hydrogen peroxide every day over the course 30 Some embodiments, the culture of Haematococcus cells may of 10-12 days. In some embodiments, the cells may be con be contacted with hydrogen peroxide when the level of tacted with hydrogen peroxide every day over the course of chytrids is less than 20%. In some embodiments, the culture 12-14 days. of Haematococcus cells may be contacted with hydrogen In some embodiments, the cells may be contacted with peroxide when the level of chytrids is less than 10%. In some hydrogen peroxide every 24-72 hours. In some embodiments, 35 embodiments, the culture of Haematococcus cells may be the cells may be contacted with hydrogen peroxide every contacted with hydrogen peroxide when the level of chytrids 36-60 hours. In some embodiments, the cells may be con is less than 5%. In some embodiments, the culture of Haema tacted with hydrogen peroxide every 42-54 hours. In some tococcus cells may be contacted with hydrogen peroxide embodiments, the cells may be contacted with hydrogen per when the level of chytrids is less than 3%. In some embodi oxide every other day over the course of 3-15 days. In some 40 ments, the culture of Haematococcus cells may be contacted embodiments, the cells may be contacted with hydrogen per with hydrogen peroxide when the level of chytrids is less than oxide every other day over the course of 3-5 days. In some 2%. In some embodiments, the culture of Haematococcus embodiments, the cells may be contacted with hydrogen per cells may be contacted with hydrogen peroxide when the oxide every other day over the course of 5-7 days. In some level of chytrids is less than 1%. In some embodiments, the embodiments, the cells may be contacted with hydrogen per 45 culture of Haematococcus cells may be contacted with hydro oxide every other day over the course of 7-9 days. In some gen peroxide when the level of chytrids is 0%. embodiments, the cells may be contacted with hydrogen per In some embodiments, the culture of Haematococcus cells oxide every other day over the course of 9-11 days. In some may be contacted with hydrogen peroxide when the level of embodiments, the cells may be contacted with hydrogen per chytrids is at least 1%. In some embodiments, the culture of oxide every other day over the course of 11-13 days. In some 50 Haematococcus cells may be contacted with hydrogen per embodiments, the cells may be contacted with hydrogen per oxide when the level of chytrids is at least 2%. In some oxide every other day over the course of 13-15 days. embodiments, the culture of Haematococcus cells may be In some embodiments, the cells may contacted with hydro contacted with hydrogen peroxide when the level of chytrids gen peroxide in a combination of days over an extended time is at least 5%. In some embodiments, the culture of Haema period. Such as every day for a period of days and then every 55 tococcus cells may be contacted with hydrogen peroxide other day for a period of days, or vis versa. For example, the when the level of chytrids is at least 10%. In some embodi cells may be contacted with hydrogen peroxide every day for ments, the culture of Haematococcus cells may be contacted a period of 1-5 days and then every other day for a period of with hydrogen peroxide when the level of chytrids is at least 3-15 days. In other embodiments, the cells may contacted 20%. In some embodiments, the culture of Haematococcus with hydrogen peroxide with more than one day in between 60 cells may be contacted with hydrogen peroxide when the applications, such as but not limited to, 2 days, 3 days, 4 days, level of chytrids is at least 30%. In some embodiments, the 5 days, 6 days, 7 days, or more between applications. In some culture of Haematococcus cells may be contacted with hydro embodiments, an application of hydrogen peroxide at a gen peroxide when the level of chytrids is at least 40%. In scheduled time (e.g., after 6 hours, after 24 hours) may be Some embodiments, the culture of Haematococcus cells may skipped and application may be resumed at a later time. 65 be contacted with hydrogen peroxide when the level of In some embodiments, the growth conditions for a culture chytrids is at least 50%. In some embodiments, the level of of Haematococcus may comprise a photosynthetically active chytrids in a culture of Haematococcus cells may be main US 9,347,035 B1 9 10 tained below the level of chytrids at the time of contact with treatment with hydrogen peroxide. In some embodiments, the hydrogen peroxide while culturing the Haematococcus cells biomass yield of the Haematococcus cells contacted with in reddening conditions to form cells in the red cyst stage for hydrogen peroxide is 0.10-0.15 g/L greater than a control the accumulation of carotenoids. culture not receiving treatment with hydrogen peroxide. In In some embodiments, the chytrid level after contacting the Some embodiments, the biomass yield of the Haematococcus culture with hydrogen peroxide may be less than a control cells contacted with hydrogen peroxide is 0.15-0.20 g/L culture not receiving treatment with hydrogen peroxide. In greater than a control culture not receiving treatment with some embodiments, the chytrid level after contacting the hydrogen peroxide. In some embodiments, the biomass yield culture with hydrogen peroxide may be reduced by 20–95% of the Haematococcus cells contacted with hydrogen peroX compared to a control culture not receiving treatment with 10 ide is 0.20-0.25 g/L greater than a control culture not receiv hydrogen peroxide. In some embodiments, the chytrid level ing treatment with hydrogen peroxide. after contacting the culture with hydrogen peroxide may be In some embodiments, the biomass yield of the Haemato reduced by 20-30% compared to a control culture not receiv coccus cells contacted with hydrogen peroxide in combina ing treatment with hydrogen peroxide. In some embodiments, tion with Salt is equivalent to or greater than a control culture the chytrid level after contacting the culture with hydrogen 15 not receiving treatment. In some embodiments, the biomass peroxide may be reduced by 30-40% compared to a control yield of the Haematococcus cells contacted with hydrogen culture not receiving treatment with hydrogen peroxide. In peroxide in combination with salt is 0.01-0.30 g/L greater some embodiments, the chytrid level after contacting the than a control culture not receiving treatment. In some culture with hydrogen peroxide may be reduced by 40-50% embodiments, the biomass yield of the Haematococcus cells compared to a control culture not receiving treatment with contacted with hydrogen peroxide in combination with salt is hydrogen peroxide. In some embodiments, the chytrid level 0.01-0.10 g/L greater than a control culture not receiving after contacting the culture with hydrogen peroxide may be treatment. In some embodiments, the biomass yield of the reduced by 50-60% compared to a control culture not receiv Haematococcus cells contacted with hydrogen peroxide in ing treatment with hydrogen peroxide. In some embodiments, combination with salt is 0.10-0.20 g/L greater than a control the chytrid level after contacting the culture with hydrogen 25 culture not receiving treatment. In some embodiments, the peroxide may be reduced by 60-70% compared to a control biomass yield of the Haematococcus cells contacted with culture not receiving treatment with hydrogen peroxide. In hydrogen peroxide in combination with salt is 0.20-0.30 g/L some embodiments, the chytrid level after contacting the greater than a control culture not receiving treatment. culture with hydrogen peroxide may be reduced by 70-80% In some embodiments, the carotenoid yield of the Haema compared to a control culture not receiving treatment with 30 tococcus cells contacted with hydrogen peroxide is equiva hydrogen peroxide. In some embodiments, the chytrid level lent to or greater thana control culture not receiving treatment after contacting the culture with hydrogen peroxide may be with hydrogen peroxide. In some embodiments, the caro reduced by 80-90% compared to a control culture not receiv tenoid yield of the Haematococcus cells contacted with ing treatment with hydrogen peroxide. In some embodiments, hydrogen peroxide is 0.10-1.50% greater than a control cul the chytrid level after contacting the culture with hydrogen 35 ture not receiving treatment with hydrogen peroxide. In some peroxide may be reduced by 90-95% compared to a control embodiments, the carotenoid yield of the Haematococcus culture not receiving treatment with hydrogen peroxide. cells contacted with hydrogen peroxide is 0.10-0.25% greater In some embodiments, the chytrid level after contacting the than a control culture not receiving treatment with hydrogen culture with hydrogen peroxide in combination with salt may peroxide. In some embodiments, the carotenoid yield of the be less than a control culture not receiving treatment. In some 40 Haematococcus cells contacted with hydrogen peroxide is embodiments, the chytrid level after contacting the culture 0.25-0.50% greater than a control culture not receiving treat with hydrogen peroxide in combination with salt may be ment with hydrogen peroxide. In some embodiments, the reduced by 10-95% compared to a control culture not receiv carotenoid yield of the Haematococcus cells contacted with ing treatment. In some embodiments, the chytrid level after hydrogen peroxide is 0.50-0.75% greater than a control cul contacting the culture with hydrogen peroxide in combina 45 ture not receiving treatment with hydrogen peroxide. In some tion with salt may be reduced by 10-30% compared to a embodiments, the carotenoid yield of the Haematococcus control culture not receiving treatment. In some embodi cells contacted with hydrogen peroxide is 0.75-1.00% greater ments, the chytrid level after contacting the culture with than a control culture not receiving treatment with hydrogen hydrogen peroxide in combination with Salt may be reduced peroxide. In some embodiments, the carotenoid yield of the by 30-60% compared to a control culture not receiving treat 50 Haematococcus cells contacted with hydrogen peroxide is ment. In some embodiments, the chytridlevel after contacting 1.00-1.25% greater than a control culture not receiving treat the culture with hydrogen peroxide in combination with salt ment with hydrogen peroxide. In some embodiments, the may be reduced by 60-95% compared to a control culture not carotenoid yield of the Haematococcus cells contacted with receiving treatment. hydrogen peroxide is 1.25-1.50% greater than a control cul In some embodiments, the biomass yield of the Haemato 55 ture not receiving treatment with hydrogen peroxide. coccus cells contacted with hydrogen peroxide is equivalent In some embodiments, the method may further comprise to or greater than a control culture not receiving treatment transferring the culture of Haematococcus cells to a new with hydrogen peroxide. In some embodiments, the biomass culturing vessel after contacting the culture with the hydro yield of the Haematococcus cells contacted with hydrogen gen peroxide. In some embodiments, the culture may be peroxide is 0.01-0.25 g/L greater than a control culture not 60 contacted with hydrogen peroxide when the culture is at an receiving treatment with hydrogen peroxide. In some optimal temperature. In some embodiments, the culture may embodiments, the biomass yield of the Haematococcus cells be contacted with hydrogen peroxide when the culture is at an contacted with hydrogen peroxide is 0.01-0.05 g/L greater optimal culture density. than a control culture not receiving treatment with hydrogen In another non-limiting embodiment, a method of prevent peroxide. In some embodiments, the biomass yield of the 65 ing and/or treating a chytrid infection in a culture of Haema Haematococcus cells contacted with hydrogen peroxide is tococcus may comprise: culturing a population of Haemato 0.05-0.10 g/L greater than a control culture not receiving coccus cells in reddening conditions in a liquid culture US 9,347,035 B1 11 12 medium comprising 1-5 ppt of salt to obtain a culture of ments, the culture of Haematococcus cells may be contacted Haematococcus cells in which the cells are primarily (i.e., at with salt when a level of cells infected by chytrids is less than least 80%) in the red cyst stage for the accumulation of 2%. In some embodiments, the culture of Haematococcus carotenoids, such as astaxanthin; and contacting the primarily cells may be contacted with salt when a level of cells infected red cyst cell stage culture with an effective amount of hydro 5 by chytrids is less than 1%. In some embodiments, the culture gen peroxide. In some embodiments, the Haematococcus of Haematococcus cells may be contacted with salt when a cells in which the cells may be primarily (i.e., at least 80%) in level of cells infected by chytrids is 0%. In some embodi the green cyst stage in reddening conditions, a combination of ments, the level of chytrids in the culture may be maintained green and red cysts in reddening conditions, or a non-motile below the level of chytrids at the time of contact with the salt state in reddening conditions when contacted with an effec 10 while culturing the Haematococcus cells in reddening condi tive amount of hydrogen peroxide. tions to form cells in the red cyst stage for the accumulation of In another non-limiting embodiment, a method of treating carotenoids. a chytrid infection in a culture of Haematococcus may com In some embodiments, the culture of Haematococcus cells prise: culturing a population of Haematococcus cells in red may be contacted with salt when a level of cells infected by dening conditions in a liquid culture medium to obtain a 15 chytrids is at least 1%. In some embodiments, the culture of culture of Haematococcus cells in which the cells are prima Haematococcus cells may be contacted with salt when a level rily (i.e., at least 80%) in the red cyst stage for the accumu of cells infected by chytrids is at least 2%. In some embodi lation of carotenoids, such as astaxanthin; detecting a pres ments, the culture of Haematococcus cells may be contacted ence of chytrids in the culture; and contacting the culture with salt when a level of cells infected by chytrids is at least comprising chytrids and primarily red cyst cells with an effec 5%. In some embodiments, the culture of Haematococcus tive amount of salt. In some embodiments, the Haematococ cells may be contacted with salt when a level of cells infected cus cells in which the cells may be primarily (i.e., at least by chytrids is at least 10%. In some embodiments, the culture 80%) in the green cyst stage in reddening conditions, a com of Haematococcus cells may be contacted with salt when a bination of green and red cysts in reddening conditions, or a level of cells infected by chytrids is at least 20%. In some non-motile state in reddening conditions when contacted with 25 embodiments, the culture of Haematococcus cells may be an effective amount of salt. contacted with salt when a level of cells infected by chytrids In some embodiments, the salt contacting the chytrids and is at least 30%. In some embodiments, the culture of Haema red cyst cells may be sodium chloride. In some embodiments, tococcus cells may be contacted with salt when a level of cells the effective amount of sodium chloride is at least 1.5 times infected by chytrids is at least 40%. In some embodiments, the amount of Sodium chloride found in typical reddening 30 the culture of Haematococcus cells may be contacted with conditions, and may be up to 10 times. In some embodiments, salt when a level of cells infected by chytrids is at least 50%. the concentration of sodium chloride contacting the chytrids In another non-limiting embodiment, a method of prevent and red cyst cells may be 1-20 ppt. In some embodiments, the ing a chytrid infection in a culture of Haematococcus may concentration of sodium chloride contacting the chytrids and comprise: culturing a population of Haematococcus cells in a red cyst cells may be 1-2 ppt. In some embodiments, the 35 liquid culture medium; determining a number of Haemato concentration of sodium chloride contacting the chytrids and coccus cells infected with chytrids in the culture; contacting red cyst cells may be 1-3 ppt. In some embodiments, the the culture with an effective amount of hydrogen peroxide concentration of sodium chloride contacting the chytrids and when the percentage of Haematococcus cells infected with red cyst cells may be 1-5 ppt. In some embodiments, the chytrids is less than a threshold level of the total cells; con concentration of sodium chloride contacting the chytrids and 40 tinuing to culture the Haematococcus cells; and Verifying that red cyst cells may be 5-10 ppt. In some embodiments, the a percentage of Haematococcus cells infected with chytrids is concentration of sodium chloride contacting the chytrids and less than a threshold level of the total cells after contact with red cyst cells may be 10-15 ppt. In some embodiments, the the hydrogen peroxide. concentration of sodium chloride contacting the chytrids and In some embodiments, the threshold level of cells infected red cyst cells may be 15-20 ppt. 45 with chytrids may be 1% of the total cells. In some embodi In some embodiments, the culture of Haematococcus cells ments, the threshold level of cells infected with chytrids may may be contacted with salt when a level of cells infected by be 2% of the total cells. In some embodiments, the threshold chytrids is less than 60%. In some embodiments, the culture level of cells infected with chytrids may be 3% of the total of Haematococcus cells may be contacted with salt when a cells. In some embodiments, the threshold level of cells level of cells infected by chytrids is less than 50%. In some 50 infected with chytrids may be 4% of the total cells. In some embodiments, the culture of Haematococcus cells may be embodiments, the threshold level of cells infected with contacted with salt when a level of cells infected by chytrids chytrids may be 5% of the total cells. In some embodiments, is less than 40%. In some embodiments, the culture of Hae the threshold level of cells infected with chytrids may be 10% matococcus cells may be contacted with Salt when a level of of the total cells. In some embodiments, the threshold level of cells infected by chytrids is less than 30%. In some embodi 55 cells infected with chytrids may be 15% of the total cells. In ments, the culture of Haematococcus cells may be contacted some embodiments, the threshold level of cells infected with with salt when a level of cells infected by chytrids is less than chytrids may be 20% of the total cells. In some embodiments, 20%. In some embodiments, the culture of Haematococcus the threshold level of cells infected with chytrids may be 25% cells may be contacted with salt when a level of cells infected of the total cells. In some embodiments, the threshold level of by chytrids is less than 10%. In some embodiments, the 60 cells infected with chytrids may be 30% of the total cells. culture of Haematococcus cells may be contacted with salt In another non-limiting embodiment, a method of prevent when a level of cells infected by chytrids is less than 5%. In ing and/or treating lysis in a culture of Haematococcus may Some embodiments, the culture of Haematococcus cells may comprise: culturing a population of Haematococcus cells in a be contacted with salt when a level of cells infected by liquid culture medium in growth conditions to obtaina culture chytrids is less than 4%. In some embodiments, the culture of 65 of Haematococcus cells in which the cells are primarily (i.e., Haematococcus cells may be contacted with salt when a level at least 80%) in the green Swimmer stage; contacting the of cells infected by chytrids is less than 3%. In some embodi primarily green Swimmer cell stage culture with an effective US 9,347,035 B1 13 14 amount of hydrogen peroxide prior to the formation of cell may be reduced by 10-15x10 CFU/mL after contact with the cysts; and continuing to culture the Haematococcus cells in hydrogen peroxide. In some embodiments, the live bacteria growth conditions. count may be reduced by 15-20x10 CFU/mL after contact In some embodiments, the method may further comprise with the hydrogen peroxide. In some embodiments, the live determining a level of lysis in the culture of Haematococcus bacteria count may be reduced by 20-25x10 CFU/mL after cells as a percentage of the total cells in the culture. In some contact with the hydrogen peroxide. In some embodiments, embodiments, the culture of Haematococcus cells may be the live bacteria count may be maintained below 107 CFU/mL contacted with hydrogen peroxide when the level of lysis is following contact with the hydrogen peroxide. less than 30%. In some embodiments, the culture of Haema In another non-limiting embodiment, a method of prevent tococcus cells may be contacted with hydrogen peroxide 10 ing lysis in a culture of Haematococcus may comprise: cul when the level of lysis is less than 25%. In some embodi turing a population of Haematococcus cells in a liquid culture ments, the culture of Haematococcus cells may be contacted medium in growth conditions to obtain a culture of Haema with hydrogen peroxide when the level of lysis is less than tococcus cells in which the cells are primarily (i.e., at least 20%. In some embodiments, the culture of Haematococcus 80%) in the green swimmer stage; determining a level of cell cells may be contacted with hydrogen peroxide when the 15 lysis for the Haematococcus cells; contacting the primarily level of lysis is less than 15%. In some embodiments, the green Swimmer cell stage culture with an effective amount of culture of Haematococcus cells may be contacted with hydro hydrogen peroxide prior to the formation of cell cysts when gen peroxide when the level of lysis is less than 10%. In some the lysis level of the Haematococcus cells is less than a embodiments, the culture of Haematococcus cells may be threshold level; continuing to culture the Haematococcus contacted with hydrogen peroxide when the level of lysis is cells in growth conditions; and verifying that the level of lysis less than 5%. In some embodiments, the culture of Haema of Haematococcus cells is less than the threshold level after tococcus cells may be contacted with hydrogen peroxide contact with the hydrogen peroxide. when the level of lysis is less than 4%. In some embodiments, In some embodiments, the threshold lysis level may be 1% the culture of Haematococcus cells may be contacted with of the total cells. In some embodiments, the threshold lysis hydrogen peroxide when the level of lysis is less than 3%. In 25 level may be 2% of the total cells. In some embodiments, the Some embodiments, the culture of Haematococcus cells may threshold lysis level may be 3% of the total cells. In some be contacted with hydrogen peroxide when the level of lysis embodiments, the threshold lysis level may be 4% of the total is less than 2%. In some embodiments, the culture of Haema cells. In some embodiments, the threshold lysis level may be tococcus cells may be contacted with hydrogen peroxide 5% of the total cells. In some embodiments, the threshold when the level of lysis is less than 1%. In some embodiments, 30 lysis level may be 10% of the total cells. In some embodi the culture of Haematococcus cells may be contacted with ments, the threshold lysis level may be 15% of the total cells. hydrogen peroxide when the level of lysis is 0%. In some embodiments, the threshold lysis level may be 20% In some embodiments, the level of lysis in the culture may of the total cells. In some embodiments, the threshold lysis be maintained at or below the level of lysis at the time of level may be 25% of the total cells. In some embodiments, the contact with hydrogen peroxide while continuing to culture 35 threshold lysis level may be 30% of the total cells. the Haematococcus cells in growth conditions. In some In some embodiments, the described methods may be embodiments, the lysis level of the culture after contact with applied to an open culture of Haematococcus. In some hydrogen peroxide may be 1-80% less than a lysis level in a embodiments, the described methods may be applied to an control culture not receiving treatment with hydrogen peroX outdoor culture of Haematococcus. In some embodiments, ide. In some embodiments, the lysis level of the culture after 40 the described methods may be applied to a closed culture of contact with hydrogen peroxide may be 1-3% less thanalysis Haematococcus. In some embodiments, the described meth level in a control culture not receiving treatment with hydro ods may be applied to an indoor culture of Haematococcus. gen peroxide. In some embodiments, the lysis level of the The use of hydrogen peroxide and/or salt in the described culture after contact with hydrogen peroxide may be 3-6% methods does not function as a further stress to the Haema less than a lysis level in a control culture not receiving treat 45 tococcus cells for the accumulation of astaxanthin, but rather ment with hydrogen peroxide. In some embodiments, the provides the function of prevention and treatment of lysis and lysis level of the culture after contact with hydrogen peroxide chytrid infections to increase the survival of the Haematococ may be 6-10% less than a lysis level in a control culture not cus cells. The application of the described methods to a cul receiving treatment with hydrogen peroxide. In some ture of Haematococcus in a batch process also does not pro embodiments, the lysis level of the culture after contact with 50 vide the necessary time period to adapt the Haematococcus hydrogen peroxide may be 10-20% less than alysis level in a cells for increased resistance to lysis or fungal infections, control culture not receiving treatment with hydrogen peroX which would require many applications over multiple gen ide. In some embodiments, the lysis level of the culture after erations coupled with selection of the positively performing contact with hydrogen peroxide may be 20-40% less than a cells. lysis level in a control culture not receiving treatment with 55 The level of lysis, level of chytrid infection, and stage of the hydrogen peroxide. In some embodiments, the lysis level of Haematococcus cells may be assessed by means known in the the culture after contact with hydrogen peroxide may be art such as, but not limited to, visual observation under a 40-60% less than a lysis level in a control culture not receiv microscope, or automated monitoring with cameras and ing treatment with hydrogen peroxide. In some embodiments, visual recognition Software, spectrometers, or fluorimeters. the lysis level of the culture after contact with hydrogen 60 The monitoring and detection of the Haematococcus culture, peroxide may be 60-80% less than a lysis level in a control whether manual or automated, may be used to determine culture not receiving treatment with hydrogen peroxide. when the hydrogen peroxide and/or salt of the described In some embodiments, the method may comprise deter methods is administered to a culture by manual means, auto mining a live bacteria count in the culture of Haematococcus mated means, and combinations thereof. Automated moni cells. In some embodiments, the live bacteria count may be 65 toring and detection data may also be recorded or utilized by reduced by 10-25x10 CFU/mL after contact with the hydro a programmable logic controller to control the application of gen peroxide. In some embodiments, the live bacteria count hydrogen peroxide and/or salt to a culture of cells. Visual US 9,347,035 B1 15 16 observation under a microscope of the Haematococcus cul Swimmer cells of a first strain of Haematococcus pluvialis tures using the described methods intests also showed that the (Strain 1) were tested in well plates by adding a single dose methods aided in breaking up filamentous fungus and lower (0.06, 0.10, 0.13, 0.16 mL/L) of hydrogen peroxide 25% ing the background bacteria population of the culture, which stock concentration to 2 mL cultures of cells. The calculated may contribute to lysis. 5 concentrations of the hydrogen peroxide doses tested were In some embodiments, a microalgae culture composition 0.0150, 0.0250, 0.0325, and 0.0400 mL/L. The amount of cell may comprise: a population of Haematococcus cells in a lysis was quantified using visual observation under a micro liquid culture medium; and a calculated concentration of hydrogen peroxide in the range of 0.005-0.025 mL of hydro Scope 18 hours after administration of hydrogen peroxide. gen peroxide per L of culture medium (mL/L), wherein the The results are presented in Table 1, with standard error hydrogen peroxide has been added to the culture medium in 10 denoted as “SE. the previous 120 minutes. In further embodiments, the culture may comprise a concentration of 1-20 ppt of sodium chloride. TABLE 1 In some embodiments, the Haematococcus cells of the cul H2O2 dosage ture composition may be primarily (i.e., at least 80%) in the calculated green Swimmer stage. In some embodiments, the Haemato 15 concentration % Cell Lysis at coccus cells of the culture composition may be primarily (i.e., (mL/L) 18 hours (+1SE) at least 80%) in the red cyst stage. 0.00 (control) O.8% O.3% O.O150 1.6% 1% EXAMPLES O.O2SO 1.9% O.2% O.O325 2% 0.07% Embodiments of the invention are exemplified and addi O.O4(OO 5.2% O.48% tional embodiments are disclosed in further detail in the fol lowing Examples, which are not in any way intended to limit As shown in Table 1, significant lysis (i.e., more than 5%) the scope of any aspects of the invention described herein. of the cells occurred at hydrogen peroxide dosage levels Within these Examples two different strains of Haematococ 25 cus pluvialis were tested and are identified as "Strain 1” and above 0.0325 mL/L calculated concentration. “Strain 2, respectively. Green Swimmer and red cyst cells of a second strain of Haematococcus pluvialis (Strain2) were tested in well plates Example 1 by adding a dose (0.03, 0.04, 0.05, 0.06, 0.07 mL/L) of hydrogen peroxide 35% stock concentration to 2 mL cultures Experiments were conducted to determine the degradation 30 of cells of both green swimmer cells and red cyst cells three rate of hydrogen peroxide in a culture of Haematococcus times per day (i.e., morning, noon and evening). The calcu pluvialis. An Amplex Red Hydrogen Peroxide Kit (CAT. No. lated concentrations of the hydrogen peroxide doses tested A22188), commercially available from Life Technologies were 0.0105, 0.0140, 0.0175, 0.0210, and 0.0245 mL/L. The (Grand Island, N.Y.), was used to assay hydrogen peroxide amount of cell lysis was quantified using visual observation concentrations according to the detection of a fluorescent 35 under a microscope after 24, 48, and 72 hours. The results are product formed in the oxidation of a reagent in the presence of presented in Tables 2-3, with standard error denoted as “SE''. hydrogen peroxide. Samples of Haematococcus pluvialis (Strain 1) were taken from cultures in a carboy bioreactor (axenic conditions) and an open raceway pond bioreactor TABLE 2 disposed in a greenhouse with paddlewheel mixing (non 40 Green Swimmer Cells % Cell lysis at time periods axenic conditions). Samples from both cultures were dosed H2O2 dosage calculated h) after dosing (t1SE with 0.06 mL/L of hydrogen peroxide 25% stock concentra tion (effective concentration of 0.015 mL/L), and the hydro concentration (mL/L) 24 48 72 gen peroxide concentration was monitored every 30 minutes 0.00 (control) 3.3% 1.7% S.0% 1.7% S.O% O.0% O.O1 OS 3.3% 1.7% S.0% 1.7% 3.3% O.O% for 180 minutes using the Amplex Red Hydrogen Peroxide 45 Kit. Samples from both cultures showed an exponential decay O.O140 4.2% 0.8% 5.8% 2.5% 9.2% 4.2% in the concentration of hydrogen peroxide, resulting in a O.O175 2.5% O.8% 9.2% 2.5%. 13.3% 3.3% concentration below a level that is expected to be effective O.O210 4.2% 2.5% 6.7% 1.7%. 20.0% 3.3% against uniflagellates (e.g., fungi Zoospores) namely greater O.0245 15.0% O.0%. 22.5% O.8%. 27.5% 2.5% than 0.03 mL/L of 25% stock (calculated concentration greater than 0.0075 mL/L) after 30 minutes, and an concen 50 tration of below detectable limits in 120 minutes. These TABLE 3 results show that treating a culture with an calculated concen Red Cyst Cells tration of hydrogen peroxide of 0.015 mL/L will maintain a H2O2 dosage concentration above the levels expected to be effective calculated against contamination (e.g., uniflagellates) in the short term 55 concentration % Cell lysis at time periods (h) after dosing (t1SE (i.e. <30 minutes) and will thereafter dissipate to levels that are not harmful to any of the desired microorganisms in the (mL/L) 24 48 72 96 culture, including the Haematococcus cells. The results also 0.00 (control) 1.7% 1.0% O.0% O.0% O.8% O.8% 2.5% 0.8% demonstrate that repeated doses of hydrogen peroxide over O.O140 1.7% 1.0% O.8% O.8% 3.3% O.O% 3.3% 1.7% time do not create the risk of building up a residual concen 60 O.O175 1.1% 0.6%. 3.3% 1.7% S.8% 5.8% S.8% 2.5% tration that would be harmful to the Haematococcus or detect O.O210 3.9% 2.0% 0.8% O.8%. 1.7% O.O%. 18.3% 1.7% able in the harvested end product. O.0245 3.9% 0.6% S.0% 1.7% 9.2% 0.8% S1.7% S.0% Example 2 As shown in Table 2, the green Swimmer cells maintained 65 a cell lysis level below 10% when dosed with hydrogen per Experiments were conducted to evaluate the level of hydro oxide at a 0.0105 mL/L calculated concentration for at least gen peroxide tolerance of Haematococcus pluvialis. Green 72 hours. At a calculated concentration of 0.0210 mL/L the US 9,347,035 B1 17 18 green swimmer cells maintained lysis levels below 10% for #2320 starting before the second hydrogen peroxide treat 48 hours. These results show that the cells treated with lower ment and during the period of the lysis event. concentration doses experienced low levels of lysis (i.e., A Haematococcus pluvialis culture containing green below 10%) for all time periods, but the cultures receiving swimmer cells in an open raceway pond bioreactor #2330 higher concentration doses only maintained low levels of 5 disposed in a greenhouse, using paddlewheel mixing, and lysis (i.e., below 10%) for 48 hours or less, thus indicating operating in conditions: reactor volume of 55,000 L; Daily that the concentration of hydrogen peroxide in the method is PAR of 55 mol mid'; pH of 7.5; and paddlewheel speed of critical and simply adding more does not equate to better 40%; was treated once with 0.03 mL/L hydrogen peroxide results regarding lysis in green Swimmer cells. 25% stock concentration (calculated concentration of 0.0075 As shown in Table 3, the red cyst cells maintained a cell 10 mL/L) two days before transfer and twice after transfer to lysis level below 10% at dosages of hydrogen peroxide below open raceway bioreactor #2430, which was disposed in a 0.0175 mL/L calculated concentration for at least 96 hours, greenhouse, using paddlewheel mixing, and operating in con but dosages at or above 0.0175 mL/L calculated concentra ditions: reactor volume of 140,000 L; Daily PAR of 55 mol tion experienced cell lysis above 10% after 48-96 hours. mid'; pH of 7.3; and paddlewheel speed of 70%. High These results also show that simply increasing the dosages of 15 amounts of cell lysis were visually observed before the first hydrogen peroxide does not produce better results with treatment and remained high for 4 days. The effect of the regards to lysis in red cyst cells, however the results were not hydrogen peroxide treatment to prevent or decrease lysis exactly the same for the concentrations when applied to green appeared to be limited due to the fact that treatment began swimmer and red cyst cells. Therefore, the results of Table 2 after the lysis had occurred. Chytrid sporangia were not and Table 3 together demonstrate the state of the cell, con detected in the culture. Rheinheirmera, Flectobacillus, centration of hydrogen peroxide, and the length of time the Runella, and Flavobacterium, began increasing in relative to treatment is applied are factors that should be considered other bacteria in the bacterial community analysis of the when hydrogen peroxide is used to treat a culture of Haema culture during the start of lysis, however only Runella became tococcus pluvialis to maintain cell lysis at acceptable levels or dominant towards the end of the lysis period. prevent lysis. 25 Another Haematococcus pluvialis culture containing green swimmer cells in open raceway pond bioreactor #2330 Example 3 operating in conditions: reactor volume of 60,000 L; Daily PAR of 57 mol mid'; pH of 7.5; and paddlewheel speed of A series of Haematococcus pluvialis (Strain 1) cultures 40%; was treated twice with 0.03 mL/L hydrogen peroxide were compared using bacterial community sequencing (SSU 30 25% stock concentration (calculated concentration of 0.0075 rRNA 16s) to analyze the bacterial community of the cultures mL/L) before transfer to another bioreactor. The culture of during a lysis event, a chytrid infection, and during treatment bioreactor #2330 was transferred to open raceway pond with hydrogen peroxide to determine if the bacteria commu bioreactor #2420 disposed in a greenhouse, using paddle nity shifts or produces a detectable pattern. wheel mixing, and operating in conditions: reactor Volume of Haematococcus pluvialis cultures containing green Swim 35 140,000 L; Daily PAR of 58 mol mid'; pH of 7.3; and mer cells in open raceway pond bioreactors identification paddlewheel speed of 70%); but the culture was not treated numbers (#) 2210 and 2220 disposed in a greenhouse, using after the transfer. This culture was observed to continue cell paddlewheel mixing, and operating in conditions: reactor division after transfer to bioreactor #2420. Observations volume of 16,000 L; Daily photosynthetically active radia showed that motility decreased by about 30% after the first tion (PAR) of 57 mol mid'; pH of 7.5; and paddlewheel 40 treatment and 60% lysis occurred after the second treatment. speed of 40%; were inoculated on the same day and cultured Chytrid sporangia were observed to be present at the end of in growth conditions. The culture in bioreactor #2210 was not the culture period, 7 days after hydrogen peroxide treatment treated with hydrogen peroxide before or after transfer to ended. Bacteria including Pseudomonas, Flectobacillus, and bioreactor #2310. The culture in the bioreactor #2220 was Cytophaga were present in the bacterial community in both treated every 24 hours with 0.03 mL/L of hydrogen peroxide 45 the bioreactors #2330 and #2420. The percentage of caro 25% stock concentration (calculated concentration of 0.0075 tenoids in the culture was approximately 3.5%, quantified by mL/L) before and after the culture was transferred to open UV method. raceway pond bioreactor #2320 operating in conditions: reac tor volume of 50,000 L; Daily PAR of 54 mol mid'; pH of Example 4 7.5; and paddlewheel speed of 40%; which was also disposed 50 in a greenhouse and used paddlewheel mixing. The results An experiment was conducted to determine if treating a showed that the treated culture in bioreactor #2220 retained culture of Haematococcus pluvialis with hydrogen peroxide high motility (95%) one day longer than the untreated culture affects the live bacteria count. Cultures of Haematococcus in bioreactor #2210, and chytrids were detected three days pluvialis (Strain 1) containing green Swimmer cells in open later in the treated culture in bioreactor #2320 than they did in 55 raceway ponds (250 L Volume) with paddlewheel mixing and the untreated culture of bioreactor #2310. disposed in awarehouse were split into open pond bioreactors Nitrogen fixing bacteria including Rhizobium, Enticicia, #2210 and 2230 disposed inside a greenhouse, using paddle and Sinorhizobium, were identified as dominant species in the wheel mixing, and operating in conditions: reactor Volume of bacterial community analysis of the cultures and were present 16,000 L; Daily PAR of 50 mol mid'; pH of 7.5; and in the beginning and middle of the culture period for each 60 paddlewheel speed of 40%. The culture in bioreactor #2210 culture. However, the relative amount of nitrogen fixing bac was treated with 0.33 mL/L hydrogen peroxide 3% stock teria decreased from 20-40% to less than 10% of the dominant concentration (calculated concentration of 0.0099 mL/L) bacterial community, after the hydrogen peroxide treatments every 6 hours for the duration of the culture's green swimmer in bioreactor #2320. High amounts of cell lysis were visually stage (90 hours). The culture in bioreactor #2230 was not observed in the treated culture (bioreactor #2320), but not 65 treated to serve as a control for comparison. Samples were until five days after the treatment ended. Runella was domi taken to assess motility, lysis, and the live bacteria count in the nant in the bacterial community of the culture in bioreactor cultures at 11, 34, 52, and 66 hours. The live bacteria count US 9,347,035 B1 19 20 was obtained by plate count using Petrifilm available from TABLE 6 3M (St. Paul, Minn.), and the results are shown in Table 4. % Lysis in samples from TABLE 4 Bioreactor #2430 24 hours after transfer (+1SD) Live Bacteria Count CFU mL. BacT Untreated H2O2 Untreated H2O2 Time (h) Control Treatment Time (h) Control treatinent 10 O O.O.O.O% O.O.O.O% 11 3.7 x 107 4.8 x 10 24 85.83.5% 7.5 - 1.2% 34 1.0 x 10 5.5 x 10 52 1.5 x 10 1.8 x 10 48 75.8. 20.0% O.O.O.O% 66 2.9 x 10 4.5 x 10 96 13.3 4.7% 4.11.2% 90 8.1 x 10 No data 15 Motility was maintained above 60% for the entire experi TABLE 7 ment but was slightly higher in the untreated control culture. Lysis greater than 15% did not occur in either culture. As % Lysis in samples from Bioreactor #2430 shown in Table 4, the culture that received the hydrogen 48 hours after transfer peroxide treatment had an approximately 1 log reduction in the bacteria count. This result may be useful in analyzing the Untreated H2O2 potential for lysis in the culture, as findings from previous Time (h) Control Treatment experiments showed a correlation between bacteria concen O 3.32.9% 3.32.9% trations above 107 cells/mL and the occurrence of lysis in 24 63.32.4% 25.0 9.4% cultures of Haematococcus pluvialis. Therefore, reducing the 25 96 97.5 - 1.2% 16.7 4.7% live bacteria count of a Haematococcus pluvialis culture with a hydrogen peroxide treatment can be used to prevent condi As shown in Table 5, lysis remained at low levels in both tions that are favorable for lysis and reduce the risk of losing the control and hydrogen peroxide treatment cultures. The Haematococcus pluvialis cells to lysis. 30 results in Tables 6 and 7 show lysis occurred at high levels (greater than 80%) in the control cultures and was held to Example 5 lower levels with the hydrogen peroxide treatments (less than A series of experiments were conducted to determine if 30%), demonstrating a treatment with hydrogen peroxide treating a culture of Haematococcus pluvialis with hydrogen may successfully reduce lysis by 60-80%. peroxide prevents lysis. Samples of a culture of Haematococ 35 The findings from the flask test samples were then applied cus pluvialis (Strain 1) containing green Swimmer cells were to commercial scale cultures of Haematococcus pluvialis taken from open raceway pond bioreactor #2330 disposed in (Strain 1) containing green Swimmer cells that were trans a greenhouse, using paddlewheel mixing, and operating in ferred from open raceway pond bioreactor #2310 disposed in conditions: reactor volume of 60,000 L; Daily PAR of 57 mol 40 a greenhouse, using paddlewheel mixing, and operating in mid'; pH of 7.5; and paddlewheel speed of 40%; before the conditions: reactor volume of 55,000 L. Daily PAR of 42 mol culture was transferred to open raceway pond bioreactor mid'; pH of 7.5; and paddlewheel speed of 40%; to open #2430 disposed in a greenhouse, using paddlewheel mixing, raceway pond bioreactor #2420 disposed in a greenhouse, and operating in conditions: reactor volume of 140,000 L.; using paddlewheel mixing, operating in conditions: reactor Daily PAR of 58 mol mid'; pH of 7.3; and paddlewheel 45 volume of 140,000 L; Daily PAR of 40 molm’d'; pH of 7.3; speed of 70%); as well as 24 and 48 hours after transfer to and paddlewheel speed of 70%. Similarly, the culture in bioreactor #2430. The samples were divided into flasks with some flasks receiving treatment with 0.028 mL/L hydrogen bioreactor #2320, operating in conditions: reactor volume of peroxide 25% stock concentration (calculated concentration 60,000 L; Daily PAR of 42 mol mid'; pH of 7.5; and of 0.007 mL/L) three times per day, and some flasks receiving paddlewheel speed of 40%; was transferred by being split into no treatment to serve as controls for comparison. Lysis was 50 pond bioreactors #2410 and 2430 operating in conditions: quantified daily through visual observation of samples under reactor volume of 150,000 L; Daily PAR of 40 mol mid': a microscope. The results are shown in Tables 5-7, with pH of 7.3; and paddlewheel speed of 70%. The cultures in standard deviation denoted as “SD'. pond bioreactors #2310 and 2320 had previously received treatments of 0.02 mL/L hydrogen peroxide 35% stock con 55 TABLE 5 centration (calculated concentration of 0.007 mL/L) every six hours, which continued after the cultures were transferred % Lysis in Samples from until the fourth day of treatment. Lysis was quantified upon Bioreactor #2330 (1SD harvest of the culture through visual observation of samples Untreated H2O2 60 under a microscope. Carotenoid content of the cells was mea Time (h) Control Treatment sured by UV method upon harvest, and the average growth O 8.3 - 7.6% 8.3 - 7.6% rate of the culture was calculated based on periodic dry 24 6.7 2.4% 10.0 - 4.7% weight samples. The results were compared to data from 48 4.23.5% 12.S 3.5% 72 O.O.O.O% O.O.O.O% previous culture runs in the same bioreactors or bioreactors 120 17 O.O% O.8 1.2% 65 with similar operating conditions during the same month (September 2014) at the same location. The results are shown in Tables 8-10. US 9,347,035 B1 22 TABLE 8 170,000 L; Daily PAR of 53 mol mid'; pH of 7.3; and paddlewheel speed of 70%. Bioreactor #2420 had just been H2O2 Maximum 9.6 inoculated with cells in the green Swimmer stage into redden Bioreactor Treatment Lysis of Culture ing conditions that comprised of culture medium comprising 2410 No 25% a 1 ppt concentration of sodium chloride (NaCl). The flask 242O No 25% 2430 No 90% experiment consisted of duplicate untreated controls and 2440 No 95% duplicate treatments. The treated flask cultures were dosed 2410 Yes 70% with 0.03 mL/L hydrogen peroxide of 25% stock concentra 242O Yes 20% tion (calculated concentration of 0.0075 mL/L) 2-3 times per 2430 Yes 35% 10 day. Samples were taken from the flask cultures daily to monitor the percentage of chytrids in the total cells of the culture (% chytrid infection) through visual observation TABLE 9 using a microscope and measurement of cell dry weighting/L H2O2 Maximum 96 15 for a duration of about 9 days (217 hours). The results are Bioreactor Treatment Carotenoid (UV) shown in Tables 11-12. 2410 No 2.84 242O No 0.79 TABLE 11 2430 No 1.88 2440 No 2.38 Average Chytrid Infection (% 2410 Yes 3.22 242O Yes 3.58 Time (h) Control HO, Treatment 2430 Yes 1.21 O O.O. O.0% O.O. O.0% Ended early 24 O.O. O.0% O.O. O.0% 48 O.8 1.2% O.O. O.0% 25 72 O.O. O.0% O.O. O.0% 144 38.354.2% O.O. O.0% TABLE 10 168 40.0 - 56.6% O.O. O.0% 197 43.3 44.8% O.O. O.0% H2O2 Average Growth 217 SO.OS8.9% O.O. O.0% Bioreactor Treatment Rate (g/m/day) 2410 No 2.1 30 242O No 2.3 2430 No 3.2 TABLE 12 2440 No 3.6 Average Cell Dry Weight (gfL 2410 Yes 4.7 242O Yes 4.3 2430 Yes 2.3 35 Time (h) Control H2O, Treatment Ended early O O.1 O.O O.1 O.O 24 O.10.1 O.1 O.O 48 O3 + O.O O2 O.O As shown in Table 8, lysis remained in the range of 20-35% 72 O.S. O.1 O3 + 0.1 in two of the commercial scale cultures treated with hydrogen 144 No data No data peroxide, which was an improvement over the historical data 40 168 O.8 O2 O.7 O.O 197 O.7 O.O O.6 O.O that showed untreated cultures may reach lysis levels over 217 O.9 O.O O.8 O.O 80%. As shown in Tables 9 and 10, the cultures treated with hydrogen peroxide produced the higher levels of carotenoids and had a higher average growth rate than the untreated cul Results showed that the 96 chytrid infection in the untreated tures, indicating that the hydrogen peroxide treatment may be 45 controls varied widely (15-90%) starting on day six (144 h), directly improving growth and carotenoid accumulation or with the average steadily increasing over the remaining days may be indirectly improving growth and carotenoid accumu to over 40% (as shown in Table 11). In the treated cultures, '% lation by creating an environment with Suppressed lysis. chytrid infection was 0% the entire nine day period. As shown Viewing the results from the flask tests and the commercial 50 in Table 12, the dry cell weight increased in both the control scale cultures together, treating a culture of Haematococcus and treated cultures. pluvialis green Swimmer cells with hydrogen peroxide at a Culture samples for a second experiment were taken three calculated concentration of at least 0.007 mL/L every 6 hours days after inoculation when the salt concentration was at 1 ppt can reduce the level of cell lysis by at least 60% without from open raceway bioreactor #2440 disposed within agreen negatively affecting the culture growth rate (i.e., biomass 55 house, mixed with paddlewheels, and operating in condi tions: reactor volume of 175,000 L; Daily PAR of 40 mol accumulation) and accumulation of carotenoids. mid'; pH of 7.3; paddlewheel speed of 70%. Samples were Example 6 placed in flasks for culturing, consisting of duplicate untreated controls and duplicate treatments. The treated flask A series of experiments were conducted to evaluate the 60 cultures were dosed with 0.03 mL/L hydrogen peroxide of effectiveness of hydrogen peroxide alone and in combination 25% stock concentration (calculated concentration of 0.0075 with concentrations of salt as a treatment for controlling mL/L) 2-3 times per day. The flask cultures contained Hae chytrid infections in Haematococcus pluvialis cultures. Cul matococcus pluvialis (Strain 1) cells in both the green Swim ture samples of Haematococcus pluvialis (Strain 1) cultures mer stage and early red cyst stage. Samples were taken from were taken and placed into flasks from open raceway biore 65 the flask cultures daily to quantify 96 chytrid infection and cell actor #2420 disposed within a greenhouse, mixed with dry weight for a duration of about 6 days (144 hours). The paddlewheels, and operating in conditions: reactor Volume of results are shown in Tables 13-14. US 9,347,035 B1 23 24 TABLE 13 TABLE 16-continued Average Chytrid Infection (% Average Cell Dry Weight (gfL Time (h) Control H2O, Treatment 5 Time (h) Control H2O, Treatment O O.O.O.O% O.O.O.O% 120 2.31 O.32 2.26 OOO 24 O.O.O.O% 1.72.9% 144 No data No data 96 59.4 7.7% 250 2.9% 168 2.20 O.37 22 OOO 120 68.3 - 4.4% S1.7 S.O% 192 2.73 O.O7 2.68 O.91 144 67.24.2% 43.9 4.2% 264 2.19 O.325 2.O2 O.65 10 As shown in Table 15, the 96 chytrid infection varied TABLE 1.4 widely in the first 8 days (192 h), but was reduced in the Average Cell Dry Weight (g/L treatments as compared to the control by day 11 (264 h). As 15 shown in Table 16, the dry cell weight increased in both the Time (h) Control H2O, Treatment control and treated cultures but was highly variable by the end O O.16 O.OO O.16 OOO of the experiment. 24 O32- 0.05 O.31 O.O2 Culture samples of Haematococcus pluvialis (Strain 1) for 96 O.S3 O.OS O55, O.O4 a fourth experiment were collected from open raceway biore 120 O56 - 0.16 O49, O.O9 2O actor #2310 disposed within a greenhouse, mixed with a 144 No data No data paddlewheel, and operating in conditions: reactor Volume of 48,000 L; Daily PAR of 50 molm d'; pH of 7.5; and As shown in Table 13, the '% chytrid infection reached 67% paddlewheel speed of 40%. The culture samples were inocu in the controls and was reduced in the treated cultures. As lated in the green Swimmer stage in growth conditions i.e., in shown in Table 14, the dry cell weight increased in both the 25 less than 1 ppt of sodium chloride (NaCl). The flask cultures control and treated cultures. were then diluted 3:1 with nitrate free HMB media and Culture samples of Haematococcus pluvialis (Strain 1) for brought to a 1 ppt concentration of NaCl, consisting of dupli a third experiment were collected from open raceway biore cate untreated controls and duplicate treatments. The treated actor #2210 disposed within a greenhouse, mixed with a flask cultures were dosed with 0.03 mL/L hydrogen peroxide paddlewheel, and operating in conditions: reactor volume of 30 of 25% stock concentration (calculated concentration of 18,000 L. Daily PAR of 48 mol mid'; pH of 7.5; paddle 0.0075 mL/L) 3 times per day. Samples were taken from the wheel speed of 40%. The collected samples were inoculated flask cultures daily to % chytrid infection and cell dry weight in the green Swimmer stage in growth conditions i.e., in the for a duration of about 9 days (216 hours). The results are absence of sodium chloride (NaCl). The samples were dis shown in Tables 17-18. tributed in flasks, consisting of duplicate untreated controls 35 and duplicate treatments. The treated flask cultures were TABLE 17 dosed with 0.03 mL/L hydrogen peroxide of 25% stock con centration (calculated concentration of 0.0075 mL/L) two Average Chytrid Infection (% times per day. Samples were taken from the flask cultures daily to quantify '% chytrid infection and cell dry weight for a 40 Time (h) Control H2O, Treatment duration of about 11 days (264 hours). The results are shown O O.O.O.O% O.O. O.0% in Tables 15-16. 24 O.O.O.O% O.O. O.0% 48 O.O.O.O% O.O. O.0% 120 16.13.5% 1.7 1.7% TABLE 1.5 144 60.6 - 7.5% 18.3 - 18.5% 45 168 62.8 12.5% 17.2 - 14.4% Average Chytrid Infection (% 192 67.25.4% 32.2 28.1% 216 90.6 5.4% 44.4 46.8% Time (h) Control H2O, Treatment O O.O. O.O% O.O. O.O% 24 O.O. O.O% O.O. O.O% 50 48 O.O. O.O% O.O. O.O% TABLE 1.8 96 O.O. O.O% O.O. O.O% Average Cell Dry Weight (gfL 120 O.O. O.O% O.O. O.O% 144 6.79.4% 4.25.9% 168 36.737.1% 18.3 25.9% Time (h) Control H2O, Treatment 192 49.2 - 29.5% 26.6 37.1% 55 O OOOOOO OOOOOO 264 81.7 2.4% S1.7 7.1% 24 O45 O.O3 O46 O.O7 48 O.96 - 0.15 O.94 O.18 120 1.71 - 0.2O 1.60 O.OS 144 1.29 O.O9 1.56 O.35 TABLE 16 168 1.55 0.41 2.01 - O.O2 60 192 1.21 O.24 2.34 - 0.39 Average Cell Dry Weight (g/L 216 1.77 0.15 2.96 O.26 Time (h) Control H2O, Treatment O O.26 OOO O.26 OOO As shown in Table 17, a 50-90% reduction in % chytrid 24 O.93 + O.04 O.76 O.O6 infection in the treatments as compared to the control was 48 No data No data 65 observed starting 5 days after inoculation (144 h). The vari 96 2.16 O.O8 1.90 O.OO ability in the results were attributed to the fact that one treat ment flask became infected while the other did not. As shown US 9,347,035 B1 25 26 in Table 18, the dry cell weight increased in both the control either 1 or 3 ppt salt (NaCl). The flask cultures containing 1 and treated cultures but was higher in treated flasks. ppt salt were dosed three times per day (morning, noon and Culture samples of Haematococcus pluvialis (Strain 1) evening) with 0.02 or 0.04 mL/L of hydrogen peroxide 35% were collected for a fifth experiment from open raceway stock concentration (calculated concentrations of 0.007 or bioreactor #2310 operating in conditions: reactor volume of 5 0.014 mL/L). The flask cultures containing 3 ppt salt were 66,000 L; Daily PAR of 48 mol mid'; pH of 7.5; and dosed three times per day (morning, noon and evening) with paddlewheel speed of 40%. The culture samples were distrib 0.02 mL/L of hydrogen peroxide 35% stock concentration uted into flasks for culturing, consisting of duplicate (calculated concentration of 0.007 mL/L). The percent of the untreated controls and duplicate treatments. The flask cul cells infected with chytrids was quantified via visual obser tures were then diluted 1:3 into reddening media comprising 10 vation under a microscope at 72 and 96 hours after the cul 1 ppt NaCl or 2 ppt NaCl (33.3 mL and 66.6 mL culture tures were inoculated in the flask. The results are shown in media). The treated flask cultures were dosed with 0.03 mL/L hydrogen peroxide of 25% stock concentration (calculated Table 21. concentration of 0.0075 mL/L) 3 times per day. Samples were TABLE 21 taken from the flask cultures daily to analyze 96 chytrid infec 15 tion and cell dry weight for a duration of about 8 days (192 % Chytrids Infection hours). The results are shown in Tables 19-20. 1 ppt salt 1 ppt salt & 3 ppt salt Time Untreated & O.OO7 0.001.4 mL.L & O.OO7 TABLE 19 (h) Control mL/L H2O2 H2O2 mL/L H2O2 Average Chytrid Infection (% 72 71.7 O.O% 57.55.9% 36.7 4.7% 72.51.2% 96 96.7 4.7% NODATA 97.5 - 1.2% NODATA Time 1 ppt NaCl 1 ppt NaCl and 2 ppt NaCl 2 ppt NaCl and (h) Control H2O, Treatment Control H2O, Treatment As shown in Table 21, the hydrogen peroxide treatments O O.O.O.0% O.O.O.O% O.O.O.O% O.O. O.0% 25 24 O.O.O.0% O.O.O.O% O.O.O.O% O.O. O.0% for the cultures containing 1 ppt salt showed a reduction in the 12O 27.5 - 20.0% O.O.O.O% 86.7 2.4% O.O. O.0% chytrids infection 96 compared to the untreated control and 144 76.79.4% O.O.O.O% 94.25.9% O.O. O.0% 168 93.30 - 7.1% O.O.O.O%. 10O.O.O.O% O.O. O.0% treatment containing 3 ppt salt after 72 hours, with the 0.014 192 97.5 - 1.2% O.O.O.O% 97.5 - 1.2% O.O. O.0% mL/L hydrogen peroxide treatment resulting in a larger reduction than the 0.007 mL/L hydrogen peroxide treatment. 30 After 96 hours the 1 ppt salt and 0.014 mL/L hydrogen per oxide treatment did not appear to continue as an effective TABLE 20 treatment for the reduction of chytrids, demonstrating that the Average Cell Dry Weight (g/L combination of Salt and hydrogen peroxide may be effective 35 for reducing chytrids in a culture of Haematococcus with an Time 1 ppt NaCl 1 ppt NaCl and 2 ppt NaCl 2 ppt NaCl and existing infection for a limited period of time after which (h) Control H2O, Treatment Control H2O, Treatment additional action may need to be taken in the form of harvest O O.O3 + O.OO O.O3 + O.OO O.O3 + O.OO O.O3 + O.OO ing the Haematococcus cells or utilizing a different treatment 24 1.16 (0.06 1.01 - 0.04 1.12 O.O3 1.OOOO6 120 1.33 - 0.18 O.89 OO1 1.12 O.23 O.86 - 0.06 method. 144 O.95 O.O1 O.72 OO6 O.58. O.14 O66 0.08 40 168 O64 - 0.06 O.78 O.O3 O.6O O.O3 O.78 O.O3 Example 8 192 O.91 O.O7 1.01 - 0.04 O.66 O.O3 1.OOO.O3 An experiment was conducted to evaluate the effectiveness As shown in Table 19, a chytrid infection appeared within of the combination of salt and hydrogen peroxide as a treat 5 days (120 h) and increased to greater than 90% chytrid 45 ment method for reducing chytrids in an infected culture of infection by day 7 (168h) in the control cultures. In the treated Haematococcus pluvialis. Three 3,000 L open raceway pond cultures, '% chytrid infection was 0% the entire eight day bioreactor it's MP1, MP2, & MP3 with paddlewheel mixing period. As shown in Table 20, the dry cell weight was com disposed outdoors were inoculated with cultures of green parable between control and treated cultures. Across all Swimmer Haematococcus pluvialis (Strain. 2) cells from out experiments, treatment with hydrogen peroxide was show to 50 door reactor #2310 at a dilution ratio of 1 part culture samples be effective in reducing chytrid infections when used with and to 3 parts reddening media containing 1 ppt salt (NaCl). After without salt. the cultures failed to become infected on their own, chytrid Example 7 infection was promoted by adding 5 L of infected culture 55 from another outdoor reactor (#2420) after 6 days. MP1 and An experiment was conducted to evaluate the effectiveness MP3 received treatments of 0.03 mL/L of hydrogen peroxide of the combination of Salt and hydrogen peroxide as a treat 35% stock concentration (calculated concentration of 0.0105 ment method of reducing chytrids in an infected culture of mL/L) every 6 hours. MP2 was not treated with hydrogen Haematococcus pluvialis. Culture samples of Haematococ peroxide to serve as an untreated control for comparison cus pluvialis (Strain 2) were collected from 3,000 L open 60 purposes. The percent of cells infected with chytrids was raceway pond bioreactor #MP1 with paddlewheel mixing quantified using visual observation under a microscope 24 disposed outdoors. At the time the samples was taken, the hours after the chytrids were introduced, and determined to be culture comprised green swimmer cells and chytrids (100% 20% in MP1, 10% in MP2, and less than 5% in MP3. After of culture infected). One mL of infected culture was inocu hydrogen peroxide treatments began, the percent of the cul lated into replicate flasks (100 mL. Volume) containing axenic 65 tures infected with chytrids, percent of carotenoids by UV green Swimmers from a carboy bioreactor culture diluted into method, and cell dry weight (g/L) were quantified daily. The reddening media 1 part culture to 3 parts media comprising results are show in Tables 22-24. US 9,347,035 B1 27 28 TABLE 22 As shown in Table 22, the hydrogen peroxide and salt treatment was effective for maintaining the chytrids infection % Chytrids Infection level below 5% in the MP3 culture which was below 5% when Untreated 0.0105 mLL 0.01.05 mLL the treatments started. The hydrogen peroxide and salt treat Time (days Control (MP2, HO, (MP1, HO, (MP3, ment was also effective in slowing the chytrid infection of the after initial infection initial infection initial infection MP1 culture which started with a moderate level (20%) as inoculation) 10%) 20%) less than 5%) compared to the untreated control (MP2). Visual observation NODATA NODATA NODATA of the red cyst cells under a microscope also showed that the NODATA NODATA NODATA Haematococcus cells were susceptible to infection when the 8.3 - 7.6% 23.35.8% 1.72.9% 10 1.O.O.5.0% 2O.O.5.0% O.O.O.0% cells were transitioning from the green Swimmer stage to the 16.7 - 12.6% 3O.O. 10.0% 1.72.9% red cyst stage before the cyst had fully formed. 450 18.0% 26.72.9% O.O.O.0% 11 76.75.8% 61.724.7% O.O.O.0% As shown in Tables 23 and 24, the continuous treatments 12 90.0 - 10.0% 633 - 16.1% O.O.O.0% with hydrogen peroxide in the presence of salt did not inhibit 13 95.O.S.0% 73.37.6% 3.32.9% 15 the accumulation of carotenoids and biomass in the Haema 14 98.3 2.9% 76.7 17.6% O.O.O.0% tococcus cells. Together these results demonstrate that con tinuously treating a culture of Haematococcus cells infected by chytrids with hydrogen peroxide in the presence of low salt TABLE 23 (1 ppt) is effective in preventing an increase in the infection if treatment is started when the infection is at a low level (below % Carotenoids (UV 5%), or at a minimum the treatment is effective in slowing Untreated 0.0105 mLL 0.01.05 mLL down the increase in the infection that is already at a moderate Time (days Control (MP2, H2O, (MP1, H2O, (MP3, level (e.g., 20%) at the time treatment is initiated without after initial infection initial infection initial infection negatively affecting the carotenoid (e.g., astaxanthin) and inoculation) 10%) 20%) less than 5%) 25 biomass accumulation by Haematococcus. NODATA NODATA NODATA NODATA NODATA NODATA NODATA NODATA NODATA Example 9 3.72 3.71 3.77 3.72 4.03 4.19 30 4.20 4.44 4.SS An experiment was conducted to evaluate if higher con 11 3.24 4.33 3.74 centrations greater than 0.03 mL/L 35% stock (calculated 12 3.27 3.95 4.47 13 NODATA NODATA NODATA concentrations greater than 0.0105 mL/L) of hydrogen per 14 NODATA NODATA NODATA oxide could be used as a treatment method for reducing chytrids in an infected culture of Haematococcus pluvialis. 35 Culture samples of Haematococcus pluvialis (Strain 2) were collected on day 5 of the culture from open raceway bioreac TABLE 24 tor #2420 operating in conditions: reactor volume of 170,000 Cell Dry Weight fL L; Daily PAR not measured; pH of 7.3; and paddlewheel 40 Untreated 0.0105 mLL 0.01.05 mLL speed of 70%). Culture samples were distributed into flasks Time Control (MP2, H2O, (MP1, H2O, (MP3, for culturing, consisting of duplicate untreated controls and (days after initial infection initial infection initial infection duplicate treatments. Treatments compared different fre inoculation) 10%) 20%) less than 5%) quency doses (one to three times daily) of higher concentra O.156 O.216 O.190 tions (0.04 and 0.05 mL/L) of hydrogen peroxide 35% stock O.219 O.283 O.2O1 45 concentration (calculated concentrations of 0.0140 and O.244 O.321 O.311 O.314 O.371 O412 0.0175 mL/L) with the standard treatment to date (calculated O.317 O.349 O.353 concentration of 0.0105 mL/L three times daily). Samples O.346 O.370 O.343 were taken from the flask cultures every few days in order to 11 O.347 O.353 O.349 quantify '% lysis,% chytrid infection, and cell dry weight for 12 0.375 0.357 0.375 50 13 O.366 O.364 O.390 a duration of about 11 days. Due to lysis rates exceeding 10% 14 O.360 0.357 O.427 in all treatments by day 5, treatment was discontinued and recovery from lysis was monitored. Results are shown in Table 25-27. TABLE 25 % Lysis

0.01.05 mL,L, O.O140 mLL 0.0140 mLL, O.O140 mLL 0.0175 mLL Time H2O2 H2O2 H2O2 H2O2 H2O2 (days after Untreated 3 times per 1 time per 2 times per 3 times per 1 time per inoculation) Control day day day day day 4.2 + 1.2 4.2 + 1.2 4.2 + 1.2 4.2 + 1.2 4.2 + 1.2 4.2 + 1.2 O.8 1.2 O.8 1.2 5.024 5.0 - 4.7 2.53.5 2.5 1.2 4.25.9 3.32.4 4.2 + 1.2 3.32.4 9.2 1.2 4.2 + 1.2 1.7 OO 10.8 - 1.2 1O.O 2.4 1O.O 2.4 425 - 15.3 9.2 1.2 O.O.O.O 3.32.4 7.5 - 1.2 9.75.9 SS.O.30.6 6.7 2.4 US 9,347,035 B1 29 30 TABLE 25-continued

% Lysis

0.0105 mLL, O.O140 mLL, O.O140 mLL 0.0140 mLL 0.0175 mLL Time H2O2 H2O2 H2O2 H2O2 H2O2 (days after Untreated 3 times per 1 time per 2 times per 3 times per 1 time per inoculation) Control day day day day day

9 No Data 6.7 2.4 10.8 - 1.2 S.O.O.O 60.0 - 28.3 6.7 - 4.7 11 No Data 4.23.5 7.5 - 1.2 O.O.O.O 56.733.0 4.23.5

TABLE 26 % Chytrid infection 0.0105 mLL, O.O140 mLL, O.O140 mLL 0.0140 mLL 0.0175 mLL Time H2O2 H2O2 H2O2 H2O2 H2O2 (days after Untreated 3 times per 1 time per 2 times per 3 times per 1 time per inoculation) Control day day day day day O O.O. O.O O.O.O.O O.O.O.O O.O.O.O O.O. O.O O.O.O.O 3 28.3 - 23.6 O.8 1.2 O.O.O.O 2.5 1.2 3.3 4.7 O.8 1.2 4 417 - 18.8 2.5 3.5 O.O.O.O 2.53.5 O.O. O.O 17 O.O 5 53.3 O.O 17 O.O 1.724 5.0 - 4.7 O.O. O.O 17 O.O 6 88.3 2.4 2.5 1.2 2.5 - 1.2 5.835 O.O. O.O O.O.O.O 9 97.53.5 7.5 3.5 O.O.O.O 43.34.7 O.O. O.O O.O.O.O 11 No Data 100.O.O.O 35.8. 29.S. 97.53S 11.7 - 16.5 34.227.1

TABLE 27 Culture Dry Weight (g/L 0.0105 mLL, O.O140 mLL, O.O140 mLL 0.0140 mLL 0.0175 mLL Time (days H2O2 H2O2 H2O2 H2O2 H2O2 after Untreated 3 times per 1 time per 2 times per 3 times per 1 time per inoculation) Control day day day day day O O.29 O.O2 0.29 O.O2 O29 O.O2 O.29 O.O2 0.29 O.O2 O.29 O.O2 3 O42 (0.07 O42 O.O7 O42 (0.05 O.44 O.O O4O O.O1 O.47 O.O3 4 No Data No Data No Data No Data No Data No Data 5 No Data No Data No Data No Data No Data No Data 6 No Data O.45 O.O2 O.44 O.O O41 O. 11 O.30 OO6 0.35 - O.O2 9 O59 0.29 O.74 O.O2 O.82 0.04 O.67 + O.O1 0.54. O.11 O.76 O.O1 11 O57 0.16 O.69 O.O9 O.54 - 0.32 O.64 O.O8 0.81 - O.1 O.79 0.13

45 As shown in Table 25, percent lysis reached 10% in all of hydrogen peroxide (calculated concentration 0.0105 cultures treated with hydrogen peroxide by day 5, except for 0.0175 mL/L) could be used as a treatment method for reduc 0.014 mL/L dosed three times per day which were at 40% ing chytrids in an infected culture of Haematococcus pluvia lysis. Treatment was discontinued at this time and the lysis 50 lis. Culture samples of Haematococcus pluvialis (Strain 2) rate remained stable through days 6 to 11. As shown in Table were collected on the second day of the culture from open 26, chytrid infection increased to 100% in the untreated con raceway bioreactor #2430 operating in conditions: reactor trols, but remained less than 10% in all treated flasks until 4-6 volume of 170,000 L; Daily PAR not measured; pH of 7.3; days after treatment ended. Final infection rates were lowest 55 and paddlewheel speed of 70. The culture samples were dis in flasks previously dosed with 0.0140 and 0.0175 mL/L once tributed into flasks for culturing, consisting of duplicate per day. As shown in Table 27, culture dry weights were untreated controls and duplicate treatments. Treatments com highest in the cultures treated once daily on day 9. While the pared different frequency doses (once daily and once daily results showed that the treatments did not demonstrate a 60 followed by every other day) of 0.03, 0.04 and 0.05 mL/L negative effect on lysis, the treatments were effective against 35% stock hydrogen peroxide (calculated concentrations of chytrid infection without negatively affecting dry weight. 0.0105, 0.0140, and 0.0175 mL/L). Samples were taken from Example 10 the flask cultures daily to quantify '% lysis and % chytrid 65 infection and cell dry weight was quantified every other day A second flask experiment was conducted to evaluate if one for a duration of 11 days. The results are shown in Tables daily treatment of 0.03-0.05 mL/L 35% stock concentration 28-30. US 9,347,035 B1 31 32 TABLE 28 20 Lysis

0.01.05 mLL 0.0140 mLL 0.0175 mLL H2O2 H2O. 1 H2O2 0.0105 mL/L 1 time per 0.0140 mL/L time per 1 time per Time (days H2O2 day for 3d H2O2 day for 3d day for 2 after Untreated 1 time per then every 1 time per then every d then inoculation) Control day other day day other day every other O O.82.O O.82.O O.8 2.0 O.82.O O.82.O O.82.O 3 17 24 O.O.O.O O.O.O.O 4.2 + 1.2 4.2 + 1.2 31.7 - 11.8 4 O.O. O.O 17 O.O 17 O.O 10.0 - 8.7 1O.O 8.7 22.285 5 O.O. O.O 17 O.O 1.724 1O.O 2.4 15.8 5.9 19.2 17.6 6 O.O. O.O O.82.O 3.34.7 923.5 6.7 O.O 25 O.O 7 17 O.O O.O.O.O O.O.O.O 2009.4 13.3 4.7 No Data 8 5.8 1.2 17 24 O.8 1.2 6.7 - 7.0 13.32.4 No Data 9 O.8 1.2 7.58.3 O.8 1.2 21.77.1 S.O 2.4 No Data 10 6.7 2.4 O.O.O.O O.8 1.2 923.5 15.8 12.9 No Data

TABLE 29 % Chytrid infection 0.0175 mLL 0.01.05 mLL 0.0140 mLL H2O2 1 H2O2 H2O2 time per 0.0105 mL/L 1 time per 0.0140 mL/L 1 time per day for 2 Time (days H2O2 day for 3d H2O2 day for 3d d then after Untreated 1 time per then every 1 time per then every every inoculation) Control day other day day other day other

4 14:28.3 O.8 1.2 O.8 1.2 O.O.O.O O.O. O.O O.O.O.O 5 21.7 - 4.7 O.8 1.2 S.O. 24 O.O.O.O O.O. O.O O.O.O.O 6 36.7 16.5 1.7-2.4 2.5 - 1.2 O.O.O.O 17 24 O.8 1.2 7 17.5 3.5 3.3 O.O O.O.O.O 1.724 O.O. O.O No data 8 2O.O 7.1 O.O.O.O O.O.O.O O.8 1.2 O.8 1.2 No data 9 21.77.1 O.8 1.2 2.5-3.5 O.8 1.2 O.O. O.O No data 10 26.7 - 14.1 O.O.O.O 1.724 O.O.O.O O.O. O.O No data

TABLE 30 Culture Dry Weight (g/L 0.01.05 mLL 0.0140 mLL 0.0175 mLL H2O2 H2O2 H2O2 0.0105 mL/L 1 time per 0.0140 mL/L 1 time per 1 time per Time (days H2O2 day for 3d H2O2 day for 3 d day for 2d after Untreated 1 time per then every 1 time per then every then every inoculation) Control day other day day other day other day O O.05 - O.O1 O.OS O.O1 O.OS O.O1 O.05 - O.O1 O.OS O.O1 O.OS OO1 3 No data No data No data No data No data No data 4 O.35 0.03 0.32 O.O1 O.32 O.O1 O.32 O.O1 O.32 O.O1 O.25 O.O2 5 No data No data No data No data No data No data 6 O37 O.O2 (0.35 - O.O8 O29 O.O1 O.30 - O.O3 0.35, O.O1 O.24 O.O3 7 No Data No Data No Data No Data No Data No Data 8 O540.06 042 O.O1 O49 0.04 O.41 O.OS 0.48, O.O1 No Data 9 No Data No Data No Data No Data No Data No Data 10 O39 O.04 0.38 O.O1 O40 O.OO O.35 - 0.04 0.38 O.O1 No Data

As shown in Table 28, percent lysis remained under 10% were pulled back 5 days after inoculation of the parent and for control cultures and those treated with 0.0105 mL/L were likely further along in cyst stage. As shown in Table 29, hydrogen peroxide for 11 days. Those treated with 0.0140 60 mL/L hydrogen peroxide reached 20% lysis by day 7 and chytrid infection reached 20-40% in the untreated controls, those treated with 0.0175 mL/L reached 30% lysis by day 3 but remained less than 10% in all treated flasks. As shown in after only being dosed twice. These flask cultures were dis Table 30, culture dry weights were slightly reduced in flasks carded on day 6. Cultures in this set may have been less treated every day compared to controls and flasks treated tolerant to hydrogen peroxide because they were pulled back 65 every day for 3 days and then every other day. The results from the parent reactor within two days of inoculation while show that lower concentrations of hydrogen peroxide are in the previous experiment (Example 9), culture samples capable of reducing both lysis and chytrid infections, while US 9,347,035 B1 33 34 the high concentrations of hydrogen peroxide were more TABLE 33-continued effective for reducing chytrid infections than lysis. Example 11 Culture dry weight (gL) A second flask experiment was conducted to fine tune 0.01.05 mLL 0.0105 mLL treatment of 0.03 mL/L 35% stock concentration of hydrogen Time 0.0105 mL/L H.O. 1 time H.O. 1 time peroxide (calculated concentration of 0.0105 mL/L) for (days after Untreated H2O2 1 time every other daily for reducing chytrids in an infected culture of Haematococcus inoculation) Control per day day 3 days pluvialis. Culture samples of Haematococcus pluvialis 10 (Strain 2) were collected on the second day of culture from 4 O.355 0.049 O.308 OO11 O.331 - 0.011 O.285 O.O open raceway bioreactor #2410 operating in conditions: reac 6 OSOO, O.O71 O.395 O.OO7 O4SS, O.O21 O.475 O.O3S tor volume of 150,000 L; Daily PAR of 9.77 molm’d'; pH 7 O.475 O.OO7 0.440 O.O28 OS4O 0.042 O.53O O.O14 of 7.3; and paddlewheel speed of 70%. Culture samples were 8 O-360- 0.057 0.490 O.O14 O.51O. O.O14 O.56O O.O28 distributed into flasks for culturing, consisting of duplicate 15 untreated controls and duplicate treatments. Treatments com pared different frequency doses (once daily, once every other As shown in Table 31, cell lysis remained below 10% in day and once daily for 3 days only) of 0.03 mL/L of 35% stock flasks across treatments. As shown in Table 32, chytrid infec hydrogen peroxide (calculated concentration of 0.0105 tion remained below 5% in all treated flasks but increased in mL/L). Samples were taken from the flask cultures daily to the untreated control after day 4 to greater than 90% infection. quantify '% lysis and % chytrid infection and cell dry weight As shown in Table 33, culture dry weight increased in every was quantified every other day for a duration of 11 days. The treatment but noticeably decreased in infected controls after results are shown in Tables 31-33. day 6. These results suggest that a conservative treatment of 0.01.05 mL/L hydrogen peroxide applied every day, every TABLE 31 25 other day or every day for 3 days only would be sufficient for % Lysis preventing chytrids in reddening cultures of Haematococcus pluvialis while also not affect biomass accumulation. 0.0105 mLL 0.01.05 mLL Time 0.0105 mL/L H.O. 1 time H2O. 1 time (days after Untreated H2O. 1 time every other daily for 30 Example 12 inoculation) Control per day day 3 days 1 S.O.S.O S.O.S.O S.O.S.O S.O.S.O 2 8.3 O.O 2.5 1.2 3.32.4 3.3 O.O An experiment was conducted to evaluate the effectiveness 3 3.32.4 5.83.5 4.2 + 1.2 7.5 - 1.2 of Salt as a treatment method for reducing chytrids in an 4 3.3 O.O 4.2 + 1.2 3.3 O.O O.8 1.2 infected culture of Haematococcus pluvialis, without the 5 1.724 4.23.5 3.32.4 S.O.O.O 35 addition of hydrogen peroxide. This test was doneside by side 6 O.O.O.O O.O.O.O 3.32.4 4.2 + 1.2 7 O.O 2.4 24 O.O 3.3 O.O 1.724 in flasks (0.1 L) and in reactors located in a greenhouse 8 No data No data No data No data operating in conditions: reactor volume of 230 L; Daily PAR of 20 molm’d'; pH (not measured); and paddlewheel speed 40 of 21 RPM. Flasks were inoculated (100 mL volume) with samples from a culture of green Swimmer stage Haematococ TABLE 32 cus pluvialis (Strain2) cells from open raceway pond outdoor % Chytrid Infection reactor #2410 operating in conditions: reactor Volume of 193,750 L; pH of 7.3; and paddlewheel speed of 70%; and 0.0105 mLL 0.01.05 mLL 0.01.05 mLL 45 brought up to concentration of 2 ppt salt (NaCl). The flask Time H2O2 H2O2 1 time H2O2 1 time (days after Untreated 1 time every other daily for cultures were mixed by shaking at 140 rpm and received inoculation) Control per day day 3 days 300-400 umol/ms of photosynthetically active radiation (PAR) in 12 hour light cycles and constant supply of 2.5% 1 O.O. O.O O.O.O.O O.O.O.O O.O.O.O 2 O.O. O.O O.O.O.O O.O.O.O O.O.O.O CO. The motility of the culture was monitored by visual 3 2.5 3.5 O.O.O.O 2.5 1.2 O.O.O.O 50 observation under a microscope. Once the motility of the cells 4 17 O.O O.O.O.O O.O.O.O O.O.O.O was observed to be below 10%, the level of salt was elevated 5 23.3 4.7 2.53.5 O.8 1.2 O.8 1.2 6 48.3 4.7 O.O.O.O 3.3 O.O O.O.O.O in duplicate to 4-11 ppt and compared to the culture in two 7 92.5 3.5 O.O.O.O O.8 1.2 1.724 flasks remaining at 2 ppt salt. Nine 230 L reactors were also 8 No data No data No data No data seeded with culture from bioreactor #2410 on the same day as 55 the flasks and brought to 2 ppt concentration of salt. Once motility of the cells was reduced to <10% (day 3 of culture for flasks and day 2 for 230 L reactors) the level of salt was TABLE 33 elevated to a point between 4-18 ppt and compared to the Culture weight (gL 60 culture in two reactors remaining at 2 ppt salt. Flasks and 230 L reactors were monitored for percentage of cell lysis via 0.01.05 mLL 0.01.05 mLL Time 0.01.05 mL/L H.O. 1 time H.O. 1 time visual observation under a microscope, culture dry weight (days after Untreated H2O. 1 time every other daily for (biomass accumulation in g/L), and percentage of chytrid inoculation) Control per day day 3 days infection via visual observation under a microscope. Caro 1 O.066 O.O O.066 O.O O.O66 O.0 OO66 O.O 65 tenoid production, a proxy for determining astaxanthin, was 2 0.255 0.021 0.210 - 0.028 0.215 0.007 No data quantified every other day of culture in the 230 L reactors using the UV method. Results are shown in Tables 34-40. US 9,347,035 B1 35 36 TABLE 34

% Cell Lysis in Flasks NaCl concentration (ppt)

Time (d) 2 4 5 6 7 8 9 10 11

9 O O% O O% O O% 4.3% O O% 3 - 2%. O. O%. 11% O (O-6 11 O O% O O%. 11% 54% 73%. 20%. 20%. 3, 1%. 11%

TABLE 35 % Cell Lysis in 230 L reactors NaCl concentration (ppt Time (d) 2 2 4 5 6 7 8 10 18 2 O.00% O.00% 1.67% 3.18% O.00% O.00% 3.18% O.00% O.00% 3 1.67% 0.00% O.00% S.00% O.00% O.00% 3.33% S.00% 3.33% 4 O.00% 1.67% O.00% 3.33% 8.33% 3.33% O.00% 6.67%. 13.33% 5 O.00% O.00% 3.33% 1.67% O.00% 1.67% 3.33%. 36.67% S.00% 6 O.00% O.00% O.00%. 21.67%. 13.33%. 18.33%. 16.67%. 31.67%. 26.67% 7 O.00% 3.33%. 30.00% 10.00% 18.33% 8.33% S.00% 3.33%. 20.00% 8 O.00% 1.67%. 23.33% 1.67% 1.67% 1.67% O.00% O.00% 1.67% 9 O.00%. 30.00% 13.33% S.00% 6.67% 6.67% 6.67% S.00% 8.33% 10 O.00% O.00% 11.67% S.00% 1.67% 6.67% 15.00% 3.33% S.00% 11 1.67% no data S.00% 8.33% 6.67% 11.67% 11.67% 6.67% 1.67% 12 O.00% no data 6.67% 3.33% S.00% S.00% 3.33% S.00% 3.33%

TABLE 36 % Chytrid infection in Flasks NaCl concentration (ppt Time (d) 2 4 5 6 7 8 9 10 11 9 82, 17%. 22, 20%. 18, 16%. 8, 11%. 35%. 11%. 11% OO% OO6 11 96 - 4% 85 16%. 539%. 1927% O O%. 22%. 11% O O96 1724%

TABLE 37 % Chytrid infection in 230 L reactors NaCl concentration (ppt Time (d) 2 2 4 5 6 7 8 10 18 2 1.67% O.00% O.00% 1.67% 1.67% 3.33% O.00% O.00% O.00% 3 O.00% O.00% 1.67% O.00% O.00% O.00% O.00% O.00% O.00% 4 O.00% O.00% O.00% 1.67% 1.67% O.00% O.00% 1.67% O.00% 5 O.00% O.00% O.00% O.00% 1.67% 1.67% 1.67% O.00% 3.33% 6 O.00% O.00% O.00% O.00% 1.67% O.00% O.00% O.00% O.00% 7 8.33% 3.33% 1.67% 8.33% S.00% S.00% O.00% O.00% O.00% 8 16.67% 21.67% O.00% O.00% 3.33% 1.67% O.00% O.00% 1.67% 9 45.00% 10.00% 11.67% 10.00% O.00% 3.33% O.00% 1.67% 3.33% 10 71.67% 100.00% 30.00% 3.33% 1.67% S.00% 1.67% O.00% O.00% 11 81.67% no data 36.67% 13.33% O.00% O.00% 1.67% 6.67% 1.67% 12 100.00% no data 65.00% 28.33% 15.00% 3.33% 3.33% 3.33% 1.67%

TABLE 38 Culture Dry Weight (g/L) in Flasks NaCl concentration (ppt Time (d) 2 4 5 6 7 8 9 10 11 O O.O44 O.044 O.O44 O.O44 O.O44 O.044 O.044 O.044 O.044 11 O49 0.04 0.48, O.OS O-43 - 0.04 0.48, O.O2 O49, O.O2 OSO 0.04 O.45 O.OO 0.42 O.O1 O40 0.04 US 9,347,035 B1 37 38 TABLE 39 Culture Dry Weight (g/L) in 230 L reactors NaCl concentration (ppt Time (d) 2 2 4 5 6 7 8 1O 18 O O.O3O O.O43 O.O37 0.04O O.O4O O.O3S O.O18 O.O4S O.O3S 1 O.042 0.048 0.063 0.07O O.OS8 O.048 0.057 0.06O O.OS2 2 O.065 O.O72 0.080 0.095 O.O85 0.103 0.093 0.088 O.098 3 O.12O O.10S 0.103 0.143 0.133 O.123 O.135 0.14O O.158 4 O-145 0.113 O. 115 0.162 0.140 O-145 0145 0.12O 0.135 5 O.228 O.125 0.12O 0.185 O.178 O.1SO 0.168 0.130 0.173 6 O.173 0.130 O.125 0.213 O.198. O.17O O.193 0.168 0.18O 7 O.185 O.1SO 0.148 0.223 O.218. O.198. O.2OS 0.145 0.16S 8 O.273 0.17O 0.153 0.27O O.26O O.260 0.237 0.145 0.16S 9 O-145 0.218. O.198. O.293 0.260 0.148 O.218. O.16O. O.175 10 O.22O O.1SO 0.143 0.26S O.2SO 0.2OS 0.257 0.173 0.183 11 0.145 no data 0.148 0.250 0.282 0.218 0.220 0.145 0.200 12 0.187 no data 0.150 0.280 0.323 0.245 0.263 0.168 0.175

TABLE 40 2O observed after 24 hours, sporangia and Swimming Zoospores (80-100% motility) were observed after 48 hours, and Swim % Carotenoid by UV Method in 230 L reactors ming Zoospores (90-100%) motility were observed after 72 Time NaCl concentration (ppt hours. From the observation of the control culture, a treatment (d) 2 2 4 5 6 7 8 10 18 that reduces the formation or motility of Zoospores may be 3 1.38 1.32 1.3 1.26 1.71 1.76 1.25 1.48 1.34 further investigated as a viable treatment. 5 2.53 2.14 2.6 3.2 2.67 2.82 202 1.96 is1 In the culture receiving 0.03 mL/L of bleach 12.5% stock 7 3.77 3.69 3.87 S.1 2 4.57 428 4.11 2.82 2.63 concentration (calculated concentration of 0.00375 mL/L). . 3. 3. 6, t t i. sporangia and Zoospores were observed after 24 hours. Spo data rangia and Swimming Zoospores (100% motility) were 12 3.16 no 3.07 5.74 5.63 5.72 5.71 4.38 3.79 30 observed after 48 hours, and swimming Zoospores (100% data motility) were observed after 72 hours. In the culture receiving 0.1 mL/L of bleach 12.5% stock As shown in Tables 34 and 35, cell lysis remained below concentration (calculated concentration of 0.0125 mL/L). 10% in flasks for all treatments, and below 30% in 230 L sporangia only was observed after 1 hour. Only bacteria were reactors across all treatments except 10 ppt. Lysis was highest observed after 24 hours, bacteria and a few zoospores were at 4, 10 and 18 ppt salt. As shown in Table 36 and 37, chytrid observed after 48 hours, and bacteria with no Zoospores and a St. life R increased S. both i. t few sporangia were observed after 72 hours. reactors. Chytrid infection remained below 5-10% for In the culture receiving 0.2 mL/L of bleach 12.5% stock treatments between 7-10 ppt in flasks, and treatments at 7 ppt lculated ion of 0.025 mL/L and above in 230 L reactors. As shown in Tables 38, the 40 concentration (calculated concentration of 0.025 m ). spo biomass accumulation measured by culture dry weight was rangia and Zoospores were observed after 1 hour. Swimming similar for all treatments except the two highest Salinities, Zoospores were observed after 24 hours, and bacteria and which were reduced. As shown in Table 39, biomass accumu- sporangia only (no Zoospores) were observed after 48 hours lation was highest in the 230 L reactors after day 7 for treat- and 72 hours. The lack of Zoospores after 72 hours for the ments between 5-8 ppt. As shown in Table 40, carotenoid 45 0.0125 and 0.025 mL/L bleach treatments indicate that the accumulation was highest for treatments between 5-8 ppt in treatment may be effective against chytrids, but the known the 230 L reactors. Visual observation under a microscope tolerance of Haematococcus pluvialis (Strain 1) is 0.00375 showed that cysts in cultures set to 8 ppt were the healthiest mL/L and thus the higher concentrations would not be viable and cleanest looking (e.g., large, red, and least fouled) in both as a treatment during culturing. flasks and 230 L reactors. These findings suggest that as an 50 In the culture receiving 20 ppt salt (NaCl), clumped spo alternative to hydrogen peroxide treatments, 8 ppt salt can be rangia and Zoospores were observed after 1 hour. Wilting used to reduce chytrid infection while maintaining biomass sporangia and no Zoospores were observed after 24 hours, and astaxanthin accumulation. bacteria with shriveled Zoospores and sporangia were observed after 48 hours, and no Zoospores or sporangia were Example 13 55 observed after 72 hours. The results show that the high level of salt is effective against chytrids, but the level is above the Experiments were performed to determine chytrid toler- threshold that has been shown to negatively affect some ance to bleach, salt, Lufenuron (chemical biocide), and Rid strains of Haematococcus during culturing. Fungus (blend of natural organic herbs) for evaluation as a In the culture receiving 40 ppm of Lufenuron, Swimming treatment for chytrids. A pure chytrid culture in a well plate 60 Zoospores were observed after 1 hour. Swimming Zoospores was treated every 24 hours and observed under a microscope were again observed after 24 hours, and heavy bacteria with to determine the effect. An untreated culture was used as a sporangia and Zoospores were observed after 48 hours and 72 control for comparison purposes. Qualitative assessments hours. The resulting proliferation of Zoospores demonstrates and quantitative estimates were made by visual observation that Lufenuron is not effective as a chytrid treatment. under a microscope. 65 In the culture receiving 0.01% of Rid Fungus, swimming In the control culture, sporangia and Zoospores without Zoospores were observed after 24 hours. Bacteria only were tails were observed after 1 hour. Swimming Zoospores were observed after 48 hours and 72 hours. US 9,347,035 B1 39 40 In the culture receiving 0.1% of Rid Fungus, sporangia and with chytrids were used as comparisons for the treatments. Zoospores were observed after 1 hour. Bacteria and Zoospores The cultures were treated with bleach at or less than the were observed after 24 hours, bacteria and a few zoospores tolerance level of Haematococcus: 0.01, 0.02 and 0.03 mL/L with no motility were observed after 48 hours, and bacteria of 12.5% stock concentration bleach (calculated concentra tion 0.00125, 0.0025, and 0.00375 mL/L), or different con only were observed after 72 hours. The results show that Rid 5 centrations of Lufenuron (0.01, 0.1 and 1% of culture vol Fungus may be effective for treating chytrids, however the ume). The cultures were observed under a microscope during effect on Haematococcus cells needs to be determined if Rid the experiment to determine effectiveness. Fungus is to be used as treatment during culturing. In the first control culture, the cells were observed to be healthy red cysts at the time of inoculation. Healthy red cysts Example 14 10 and bacteria were observed after 24 hours. Healthy red cysts and some clumping were observed after 48 hours. Healthy red Several concentrations of salt and Rid Fungus (blend of cysts were observed after 72 hours. organic herbs) were further investigated as a chytrid treat In the second control culture, Swimming Zoospores and ment, as in Example 13. A pure chytrid culture in a well plate sporangia on red cysts were observed at the time of inocula was treated and visually observed under a microscope to 15 tion. Swimming Zoospores and positive infection of the red determine the effect. An untreated culture was used as a cyst cells were observed after 24 hours. Bacteria, swimming control for comparison purposes. Zoospores, and positive infection of the red cyst cells were In the control culture, motile dense Zoospores were observed after 48 hours. Heavy bacteria, few swimming observed after 1 and 4.5 hours. From the observation of the Zoospores, and decreased infection of the red cyst cells were control culture, a treatment that reduces the formation or observed after 72 hours. motility of Zoospores may be further investigated as a viable In the culture receiving 0.01 mL/L of bleach 12.5% stock treatment. concentration (calculated concentration of 0.00125 mL/L, In the culture receiving 5 ppt salt (NaCl), a less dense mass which is a concentration below the tolerance level of Haema of Zoospores with some being motile but sluggish were tococcus), Swimming Zoospores and positive infection were observed after 1 hour. A mix of non-motile and motile 25 observed after 24 and 48 hours. Some Swimming Zoospores, Zoospores were observed after 4.5 hours. high bacteria, and dead sporangia were observed after 72 In the culture receiving 10 ppt salt (NaCl), a mass of hours. Zoospores at the same density as the 5 ppt treatment but with In the culture receiving 0.02 mL/L of bleach 12.5% stock all being non-motile were observed after 1 hour. Non-motile concentration (calculated concentration of 0.0025 mL/L, Zoospores only were observed after 4.5 hours. 30 which is a concentration below the tolerance level of Haema In the culture receiving 20 ppt salt (NaCl), a mass of tococcus), Swimming Zoospores and positive infection were Zoospores less dense than mass of the 5 and 10 ppt treatments observed after 24 and 48 hours. Some swimming Zoospores but with all being non-motile were observed after 1 hour. and dead sporangia were observed after 72 hours. Non-motile Zoospores only were observed after 4.5 hours. In the culture receiving 0.03 mL/L of bleach 12.5% stock The results show that the high level of salt is effective against 35 concentration (calculated concentration of 0.00375 mL/L, chytrids, but the level is above the threshold that has been which is a concentration at the tolerance level of Haemato shown to negatively affect some strains of Haematococcus coccus), Swimming Zoospores and positive infection were during culturing. observed after 24. Swimming Zoospores, positive infection, In the culture receiving 0.10% of Rid Fungus, a mass of and lysed/dead Haematococcus cells were observed after 48 Zoospores less dense than the control but highly motile were 40 hours. Some Swimming Zoospores, high bacteria, and active observed after 1 hour. Motile Zoospores were observed after infection were observed after 72 hours. Based on the results, 4.5 hours. the concentrations of bleach below the tolerance level of In the culture receiving 0.25% of Rid Fungus, a mass of Haematococcus were ineffective at treating chytrids. Zoospores less dense than the control but less motility than the In the culture receiving 0.01% of Lufenuron, swimming 0.1% treatment were observed after 1 hour. Some motile 45 Zoospores and positive infection were observed after 24 and Zoospores and possibly dead sporangia were observed after 48 hours. Some Swimming Zoospores, Ochromonas (single 4.5 hours. celled, motile, golden-brown alga), and active infection were In the culture receiving 0.50% of Rid Fungus, some motile observed after 72 hours. Zoospores were observed after 1 hour. Some motile Zoospores In the culture receiving 0.10% of Lufenuron, dead sporan and dead sporangia were observed after 4.5 hours. The results 50 gia, Some infection, and active Zoospores were observed after show that the higher levels of Rid Fungus may be effective in 24 and 48 hours. Some Swimming Zoospores and active infec reducing chytrid Zoospores, but the effect on Haematococcus tion were observed after 72 hours. cells needs to be determined if Rid Fungus is to be used as In the culture receiving 1.00% of Lufenuron, dead sporan treatment during culturing. gia and Zoospores attached to cyst cells were observed after 55 24 hours. Ochromonas, bacteria, dead sporangia, Zoospores Example 15 attached to cyst cells, and few Swimming Zoospores were observed after 48 hours. Ochromonas, no active infection, Several concentrations of bleach and Lufenuron were fur and no zoospores were observed after 72 hours. Based on the ther investigated for their effectiveness as chytrid treatments results, concentration of 1% Lufenuron may be an effective for Haematococcus pluvialis during culturing. Cultures of 60 treatment for chytrids in a Haematococcus culture, however Haematococcus pluvialis (Strain l) red cyst cells infected the effect on biomass and carotenoid accumulation needs to with chytrids were treated in well plates (0.5 mL volume) be determined. with bleach or Lufenuron to evaluate the effect on the Hae matococcus cells if bleach or Lufenuron were to be used as a Example 16 contamination treatment. A first control culture of Haemato- 65 coccus red cyst cells absent of chytrids and a second control A solution comprising 50% sodium hydroxide (NaOH) culture of healthy Haematococcus red cyst cells inoculated was evaluated as a treatment for chytrids that could be applied US 9,347,035 B1 41 42 to empty reactors in order to clean them for Subsequent In some embodiments, the level of chytrids in the culture batches. A pure culture of chytrid Zoospores and sporangia may be maintained below the level of chytrids at the time of was treated with a 1% dose of a solution comprising 50% contact with hydrogen peroxide while culturing the Haema NaOHattemperatures of 25, 40, and 50° C. The cultures were tococcus pluvialis cells in reddening conditions to produce then observed under a microscope for a reduction in the cells in the red cyst stage for the accumulation of carotenoids. chytrid Zoospores or sporangia number, or a reduction in the In some embodiments, the chytrid level after contacting the integrity of the chytrid cells. The results showed that there culture with hydrogen peroxide may be 20-95% less than a was not a reduction in chytrid Zoospores or sporangia number control culture not receiving treatment with hydrogen peroX or integrity for any of the treatments. ide. 10 In some embodiments, the cells may be contacted with the Example 17 hydrogen peroxide multiple times. In some embodiments, the cells may be contacted with the hydrogen peroxide every 6-24 Experiments were conducted to evaluate the use of biologi hours. In some embodiments, the cells may be contacted with cal agents for the control of chytrids in a culture of microal the hydrogen peroxide every 6-12 hours. In some embodi gae. Ochromonas is a single-celled, motile, golden-brown 15 ments, the cells may be contacted with the hydrogen peroxide alga known to ingest bacteria and Small eukaryotes, and was every 6-8 hours. In some embodiments, the cells may be evaluated for its properties as a chytrid Zoospore predator. contacted with the hydrogen peroxide every day over the Janthinobacterium is a gram-negative soil bacteria known to course of 1-14 days. In some embodiments, the cells may be prey on chytrid Zoospores and was evaluated for its properties contacted with hydrogen peroxide every other day over the as Such. A pure culture of chytrids was inoculated into well course of 3-15 days. plates. A control was left untreated and compared to cultures In some embodiments, the biomass yield of the Haemato treated with Ochromonas and Janthinobacterium. Chytrid coccus pluvialis cells contacted with the hydrogen peroxide Zoospore density was quantified after three days by visual may be equivalent to or greater than a control culture not observation under a microscope. Results showed that the receiving treatment with hydrogen peroxide. In some culture treated with Ochromonas had an average number of 25 embodiments, the biomass yield of the Haematococcus plu Zoospores that was approximately half of the culture treated vialis cells contacted with the hydrogen peroxide may be with Janthinobacterium and approximately one third of the 0.01-0.25 g/L greater than a control culture not receiving control. Further testing would have to be performed to deter treatment with hydrogen peroxide. mine the effect on Haematococcus cells before use as cultur In some embodiments, the carotenoids yield of the Hae ing treatment. 30 matococcus pluvialis cells contacted with the hydrogen per oxide may be equivalent to or greater than a control culture ASPECTS OF THE INVENTION not receiving treatment with hydrogen peroxide. In some embodiments, the carotenoid yield of the Haematococcus In one non-limiting embodiment of the invention, a method pluvialis cells contacted with the hydrogen peroxide may be of culturing Haematococcus pluvialis, may comprise: cultur 35 0.10-1.50% greater than a control culture not receiving treat ing a population of Haematococcus pluvialis cells in growth ment with hydrogen peroxide. conditions in a liquid culture medium to obtain a culture of In some embodiments, the method may further comprise Haematococcus pluvialis cells in which the cells are prima transferring the culture of Haematococcus pluvialis cells to a rily in a green Swimmer stage; contacting the primarily green new culturing vessel after contacting the culture with the Swimmer stage culture with hydrogen peroxide to form a 40 hydrogen peroxide. calculated concentration in the range of 0.005-0.020 mL of In one non-limiting embodiment of the invention, a method hydrogen peroxide per L of culture medium (mL/L); and of culturing Haematococcus pluvialis may comprise: cultur culturing the Haematococcus pluvialis cells in reddening ing a population of Haematococcus pluvialis cells in redden conditions to form cells in the red cyst stage for accumulation ing conditions in a liquid culture medium comprising 1-5 ppt of carotenoids. 45 of salt to obtain a culture of Haematococcus pluvialis cells in In some embodiments, the calculated concentration of which the cells are primarily in a cyst; and contacting the hydrogen peroxide may be in the range of 0.005-0.010 mL/L. primarily cyst stage culture with hydrogen peroxide to form a In Some embodiments, the calculated concentration of hydro calculated concentration in the range of 0.005-0.020 mL of gen peroxide may be in the range of 0.010-0.015 mL/L. In hydrogen peroxide per L of culture medium (mL/L). In some Some embodiments, the calculated concentration of hydrogen 50 embodiments, the cyst stage may comprise at least one peroxide is in the range of 0.015–0.020 mL/L. selected from the group consisting of green cysts and red In some embodiments, the growth conditions may com cysts accumulating carotenoids. prise a photosynthetically active radiation intensity in the In some embodiments, the salt may be sodium chloride. In range of 30-60 molm'd', nitrate concentration in the range Some embodiments, the Sodium chloride may be present in of 20-50 ppm in the culture medium, and less than 1 ppt of 55 the liquid culture medium at a concentration of 1-3 ppt. In sodium chloride in the culture medium. In some embodi Some embodiments, the Sodium chloride may be present in ments, the reddening conditions may comprise the present of the liquid culture medium at a concentration of 1-2 ppt. 1-5 ppt sodium chloride in the culture medium. In some embodiments, the chytrid level after contacting the In some embodiments, the method may further comprise culture with the hydrogen peroxide may be 10-95% less than determining a level of chytrids in the culture of Haematococ 60 a control culture not receiving treatment with hydrogen per cus pluvialis cells as a percentage of infected cells out of the oxide. In some embodiments, the biomass yield of the Hae total cells in a culture. In some embodiments, the culture of matococcus pluvialis cells contacted with the hydrogen per Haematococcus pluvialis cells may be contacted with the oxide may be 0.01-0.30g/L greater than a control culture not hydrogen peroxide when the level of chytrids is less than receiving treatment with hydrogen peroxide. 20%. In some embodiments, the culture of Haematococcus 65 In one non-limiting embodiment of the invention, a method pluvialis cells is contacted with the hydrogen peroxide when of culturing Haematococcus pluvialis may comprise: cultur the level of chytrids is at least 5%. ing a population of Haematococcus pluvialis cells in redden US 9,347,035 B1 43 44 ing conditions in a liquid culture medium to obtain a culture may be 1-80% less than a lysis level in a control culture not of Haematococcus pluvialis cells in which the cells are pri receiving treatment with hydrogen peroxide. marily in a cyst stage; detecting a presence of chytrids in the In some embodiments, the method may further comprise culture; and contacting the culture comprising chytrids and determining a live bacteria count in the culture of Haemato red cyst cells with 5-20 ppt salt. In some embodiments, the coccus pluvialis cells. In some embodiments, the live bacteria cyst stage may comprise at least one selected from the group count may be reduced 10-15x10 CFU/mL after contact with consisting of green cysts and red cysts accumulating caro the hydrogen peroxide. In some embodiments, the live bac tenoids. teria count may be maintained below 107 CFU/mL following In some embodiments, the salt may be sodium chloride. In contact with the hydrogen peroxide. Some embodiments, the concentration of Sodium chloride in 10 In one non-limiting embodiment, a method of preventing the culture medium may be in the range of 5-10 ppt. In some lysis in a culture of Haematococcus pluvialis may comprise: embodiments, the concentration of sodium chloride in the culturing a population of Haematococcus pluvialis cells in a culture medium may be in the range of 10-15 ppt. In some liquid culture medium in growth conditions to obtaina culture embodiments, the concentration of sodium chloride in the of Haematococcus pluvialis cells in which the cells are pri culture medium may be in the range of 15-20 ppt. 15 marily in a green Swimmer stage; determining a level of cell In some embodiments, the culture of Haematococcus plu lysis for the Haematococcus pluvialis cells; contacting the vialis cells may be contacted with the salt when a level of cells primarily green Swimmer cell stage culture with hydrogen infected by chytrids is less than 20% of the total cells. In some peroxide prior to the formation of cysts when the lysis level of embodiments, the culture of Haematococcus pluvialis cells Haematococcus pluvialis cells is less than 5%; continuing to may be contacted with salt when a level of cells infected by culture the Haematococcus pluvialis cells in growth condi chytrids is at least 5% of the total cells. tions; and Verifying that the level of lysis of Haematococcus In some embodiments, the level of chytrids in the culture pluvialis cells is less than 5% after contact with the hydrogen may be maintained below the level of chytrids at the time of peroxide. contact with the salt while culturing the Haematococcus plu In one non-limiting embodiments, a microalgae culture vialis cells in reddening conditions to form cells in the red 25 composition may comprise: a population of Haematococcus cyst stage for the accumulation of carotenoids. pluvialis cells in a liquid culture medium; and a calculated In one non-limiting embodiment of the invention, a method concentration of hydrogen peroxide in the range of 0.005 of preventing a chytrid infection in a culture of Haematococ 0.025 mL of hydrogen peroxide per L of culture medium cus pluvialis may comprise: culturing a population of Hae (mL/L), wherein hydrogen peroxide has been added to the matococcus pluvialis cells in a liquid culture medium; deter 30 culture medium in the previous 120 minutes. mining a number of Haematococcus pluvialis cells infected All references, including publications, patent applications, with chytrids in the culture; contacting the culture with hydro and patents, cited herein are hereby incorporated by reference gen peroxide when the percentage of Haematococcus pluvia in their entirety and to the same extent as if each reference lis cells infected with chytrids is less than 10% of the total were individually and specifically indicated to be incorpo cells; continuing to culture the Haematococcus pluvialis 35 rated by reference and were set forth in its entirety herein (to cells; and Verifying that a percentage of Haematococcus plu the maximum extent permitted by law), regardless of any vialis cells infected with chytrids is less than 10% of the total separately provided incorporation of particular documents cells after contact with the hydrogen peroxide. made elsewhere herein. In one non-limiting embodiment, a method of culturing The use of the terms 'a' and “an and “the' and similar Haematococcus pluvialis may comprise: culturing a popula 40 referents in the context of describing the invention are to be tion of Haematococcus pluvialis cells in a liquid culture construed to cover both the singular and the plural, unless medium in growth conditions to obtain a culture of Haema otherwise indicated herein or clearly contradicted by context. tococcus pluvialis cells in which the cells are primarily in a Unless otherwise stated, all exact values provided herein green Swimmer stage; contacting the primarily green Swim are representative of corresponding approximate values (e.g., mer cell stage culture with hydrogen peroxide to form a 45 all exact exemplary values provided with respect to a particu calculated concentration in the range of 0.005-0.025 mL of lar factor or measurement can be considered to also provide a hydrogen peroxide per L of culture medium (mL/L) prior to corresponding approximate measurement, modified by the formation of cell cysts; and continuing to culture the “about where appropriate). All provided ranges of values Haematococcus pluvialis cells in growth conditions. In some are intended to include the end points of the ranges, as well as embodiments, the calculated concentration of hydrogen per 50 values between the end points. oxide may be in the range of 0.005-0.010, 0.010-0.015, The description herein of any aspect or embodiment of the 0.015–0.020, or 0.020-0.025 invention using terms such as “comprising”, “having.” In some embodiments, the method may further comprise “including,” or “containing with reference to an element or determining a level of lysis in the culture of Haematococcus elements is intended to provide Support for a similar aspector pluvialis cells as a percentage of the total Haematococcus 55 embodiment of the invention that “consists of, "consists pluvialis cells in the culture. In some embodiments, the cul essentially of, or “substantially comprises” that particular ture of Haematococcus pluvialis cells may be contacted with element or elements, unless otherwise stated or clearly con the hydrogen peroxide when the level of lysis is less than tradicted by context (e.g., a composition described herein as 20%. In some embodiments, the culture of Haematococcus comprising a particular element should be understood as also pluvialis cells may be contacted with the hydrogen peroxide 60 describing a composition consisting of that element, unless when the level of lysis is less than 5%. otherwise stated or clearly contradicted by context). In some embodiments, the level of lysis in the culture may All headings and Sub-headings are used herein for conve be maintained at or below the level of lysis at the time of nience only and should not be construed as limiting the inven contact with the hydrogen peroxide while continuing to cul tion in any way. ture the Haematococcus pluvialis cells in growth conditions. 65 The use of any and all examples, or exemplary language In some embodiments, the lysis level of the Haematococcus (e.g., “such as”) provided herein, is intended merely to better pluvialis culture after contact with the hydrogen peroxide illuminate the invention and does not pose a limitation on the US 9,347,035 B1 45 46 Scope of the invention unless otherwise claimed. No language 5. The composition of claim 1, wherein the calculated in the specification should be construed as indicating any concentration of hydrogen peroxide is in the range of 0.020 non-claimed element as essential to the practice of the inven 0.025 mL/L. tion. Any claimed embodiment of the invention does not 6. The composition of claim 1, wherein the population of necessarily include all of the "aspects” or "embodiments of 5 Haematococcus pluvialis cells are primarily in a green Swim the specification. mer Stage. The citation and incorporation of patent documents herein 7. The composition of claim 1, wherein the population of is done for convenience only and does not reflect any view of Haematococcus pluvialis cells are primarily in a green cyst the validity, patentability, and/or enforceability of such patent Stage. documents. 10 This invention includes all modifications and equivalents 8. The composition of claim 1, wherein the population of of the Subject matter recited in the claims and/or aspects Haematococcus pluvialis cells are primarily in a red cyst appended hereto as permitted by applicable law. Stage. What is claimed is: 9. The composition of claim 1, wherein the population of 1. A microalgae culture composition, comprising: 15 Haematococcus pluvialis cells comprises a combination of a. A population of Haematococcus pluvialis cells in a liq cells in at least two cell stages selected from the group con uid culture medium; and sisting of green Swimmer stage, green cyst stage, and red cyst b. A calculated concentration of hydrogen peroxide in the Stage. range of 0.005-0.025 mL of hydrogen peroxide per L of 10. The composition of claim 1, further comprising a con culture medium (mL/L), wherein hydrogen peroxide has centration of 1-5 ppt of sodium chloride present in the liquid been added to the culture medium in the previous 120 culture medium. minutes. 11. The composition of claim 10, the sodium chloride is 2. The composition of claim 1, wherein the calculated present in the liquid culture medium at a concentration of 1-3 concentration of hydrogen peroxide is in the range of 0.005 ppt. 0.010 mL/L. 25 12. The composition of claim 11, the sodium chloride is 3. The composition of claim 1, wherein the calculated present in the liquid culture medium at a concentration of 1-2 concentration of hydrogen peroxide is in the range of 0.010 ppt. 0.015 mL/L. 4. The composition of claim 1, wherein the calculated 13. The composition of claim 1, further comprising a live concentration of hydrogen peroxide is in the range of 0.015 bacteria count below 107 CFU/mL. 0.020 mL/L. k k k k k