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Algae Strain Report 9

Hello Friends,

      How are you?

Participating in the conferences in Italy,Japan & London, I am back here with report of some species which are being tested at Albert Einstein Science Institute,both in India & Germany..

  

    Description of the research performed at AESI in the area of microalgal collection and

screening. In addition to performing actual research in this area, AESI personnel were

responsible for coordinating the efforts of the many subcontractors performing similar activities,

and for standardizing certain procedures and analyses. These efforts ultimately resulted in the

development of the AESI Microalgal Culture Collection, which is more fully described in

Chapter II.A.3.

Brief review of algal taxonomic groups and characteristics:

For the purposes of this report, microalgae are defined as microscopic organisms that can grow

via photosynthesis. Many microalgae grow quite rapidly, and are considerably more productive

than land plants and macroalgae (seaweed). Microalgae reproduction occurs primarily by

vegetative (asexual) cell division, although sexual reproduction can occur in many species under

appropriate growth conditions.

There are several main groups of microalgae, which differ primarily in pigment composition,

biochemical constituents, ultrastructure, and life cycle. Five groups were of primary importance

to the ABP: diatoms (Class Bacillariophyceae), green algae (Class Chlorophyceae), goldenbrown

algae (Class Chrysophyceae), prymnesiophytes (Class Prymnesiophyceae), and the

eustigmatophytes (Class Eustigmatophyceae). The blue-green algae, or cyanobacteria (Class

Cyanophyceae), were also represented in some of the collections. A brief description of these

algal groups follows.

? Diatoms. -Diatoms are among the most common and widely distributed groups

of algae in existence; about 100,000 species are known. This group tends to

dominate the phytoplankton of the oceans, but is commonly found in fresh- and

brackish-water habitats as well. The cells are golden-brown because of the

presence of high levels of fucoxanthin, a photosynthetic accessory pigment.

Several other xanthophylls are present at lower levels, as well as β-carotene,

chlorophyll a and chlorophyll c. The main storage compounds of diatoms are

lipids (TAGs) and a β-1,3-linked carbohydrate known as chrysolaminarin. A

distinguishing feature of diatoms is the presence of a cell wall that contains

substantial quantities of polymerized Si. This has implications for media costs

in a commercial production facility, because silicate is a relatively expensive

chemical. On the other hand, Si deficiency is known to promote storage lipid

accumulation in diatoms, and thus could provide a controllable means to induce

lipid synthesis in a two-stage production process. Another characteristic of

diatoms that distinguishes them from most other algal groups is that they are

diploid (having two copies of each chromosome) during vegetative growth;

most algae are haploid (with one copy of each chromosome) except for brief

periods when the cells are reproducing sexually. The main ramification of this

from a strain development perspective is that it makes producing improved

strains via classical mutagenesis and selection/screening substantially more

difficult. As a consequence, diatom strain development programs must rely

heavily on genetic engineering approaches.

? Green Algae-. Green algae, often referred to as chlorophytes, are also abundant;

component. However, N-deficiency promotes the accumulation of lipids in

certain species. Green algae are the evolutionary progenitors of higher plants,

and, as such, have received more attention than other groups of algae. A

member of this group, Chlamydomonas reinhardtii (and closely related species)

has been studied very extensively, in part because of its ability to control sexual

reproduction, thus allowing detailed genetic analysis. Indeed, Chlamydomonas

was the first alga to be genetically transformed. However, it does not

accumulate lipids, and thus was not considered for use in the ABP. Another

common genus that has been studied fairly extensively is Chlorella.

? Golden-Brown Algae-. This group of algae, commonly referred to as

chrysophytes, is similar to diatoms with respect to pigments and biochemical

composition. Approximately 1,000 species are known, which are found

primarily in freshwater habitats. Lipids and chrysolaminarin are considered to

be the major carbon storage form in this group. Some chysophytes have lightly

silicified cell walls.

? Prymnesiophytes. -This group of algae, also known as the haptophytes, consists

of approximately 500 species. They are primarily marine organisms, and can

account for a substantial proportion of the primary productivity of tropical

oceans. As with the diatoms and chrysophytes, fucoxanthin imparts a brown

color to the cells, and lipids and chrysolaminarin are the major storage products.

This group includes the coccolithophorids, which are distinguished by

calcareous scales surrounding the cell wall.

? Eustigmatophytes-. This group represents an important component of the

?picoplankton?, which are very small cells (2-4 μm in diameter). The genus

Nannochloropsis is one of the few marine species in this class, and is common

in the world?s oceans. Chlorophyll a is the only chlorophyll present in the cells,

although several xanthophylls serve as accessory photosynthetic pigments.           

 

*Cyanobacteria-. This group is prokaryotic, and therefore very different from all

other groups of microalgae. They contain no nucleus, no chloroplasts, and have

a different gene structure. There are approximately 2,000 species of

cyanobacteria, which occur in many habitats. Although this group is

distinguished by having members that can assimilate atmospheric N (thus

eliminating the need to provide fixed N to the cells), no member of this class

produces significant quantities of storage lipid; therefore, this group was not

deemed useful to the ABP.

Growth rates.

Six promising strains were analyzed in AESI Type I, Type II, and ASW (Rila) using the

temperature-salinity gradient described previously. These included the diatoms Chaetoceros

muelleri , Navicula , Cyclotella , Amphora 1 & 2, and the chlorophyte Monoraphidium minutum . Navicula and

Cyclotella were actually collected from the Florida keys; the remaining strains were collected in

Colorado and Utah.) All strains exhibited rapid growth over a wide range of conductivities in at

least two media types. Furthermore, all strains exhibited temperature optima of 30?C or higher.

Maximal growth rates of these strains, along with the optimal temperature, conductivity, and

media type determined in these experiments are shown in Table II.A.1. (Higher growth rates     

were determined for some of these strains in subsequent experiments.

       Experiments were also conducted in an attempt to identify the chemical components of AESI

Type I and Type II media most important for controlling the growth of the various algal strains.

Bicarbonate and divalent cation concentrations were found to be important determinants in

controlling the growth of Boekelovia sp. and Monoraphidium . The

growth rate of Monoraphidium minutum 2 .  increased by more than five-fold as the bicarbonate concentration of

Type II/25 medium was increased from 2 to 30 mM, and the growth of BOEKE1 by

approximately 60% over this range. These results make sense, since media enriched in

bicarbonate would have more dissolved carbon available for photosynthesis. An unexpected

finding was that there was a decrease of nearly 50% in the growth rate of BOEKE1 as the

divalent cation concentration increased from 5 mM to 95 mM (in Type I/10 medium containing

altered amounts of calcium and magnesium). The effects of magnesium and calcium

concentration on the growth of Monoraphidium minutum .  were less pronounced. These results indicate that

matching the chosen strain for a particular production site to the type of water available for mass

cultivation will be important.

Lipid content.

The lipid contents of several strains were determined for cultures in exponential growth phase

and for cultures that were N-limited for 7 days or Si-limited for 2 days. In general, nutrient

deficiency led to an increase in the lipid content of the cells, but this was not always the case.

The highest lipid content occurred with Navicula 1, which increased from 22% in exponential

phase cells to 49% in Si-deficient cells and to 58% in N-deficient cells. For the green alga

Monoraphidium minutum ., the lipid content increased from 22% in exponentially growing cells to 52% for cells

that had been N-starved for 7 days. Chaetoceros

muelleri also exhibited a large increase in lipid content in

response to Si and N deficiency, increasing from 19% to 39% and 38%, respectively. A more

modest increase occurred for nutrient-deficient AMPHO1 cells, whereas the lipid content of

Cyclotella was similar in exponential phase and nutrient-deficient cells, and actually decreased in

Amphora 2  as a result of nutrient deficiency.

    These results suggested that high lipid content was indeed achievable in many strains by

manipulating the nutrient levels in the growth media. However, these experiments did not

provide information on actual lipid productivity in the cultures, which is the more important

factor for developing a commercially viable biodiesel production process. This lack of lipid

productivity data also occurred with most of the ABP subcontractors involved in strain screening

and characterization, but was understandable because the process for maximizing lipid yields

from microalgae grown in mass culture never was optimized. Therefore, there was no basis for

designing experiments to estimate lipid productivity potential.

 

Six algal  Strains

 

 (B. braunii, Dunaliella primolecta, Isochrysis sp., Monallanthus salina, Phaeodactylum

tricornutum, and Tetraselmis sueica) were obtained from existing culture collections and

analyzed with respect to lipid, protein, and carbohydrate content under various growth

conditions. For these experiments, all cultures except for B. braunii were grown in natural

seawater that was enriched with N, P, and trace metals. B. braunii was grown in an artificial

seawater medium. Initial experiments to determine productivities of these species were

performed using batch cultures in 9-L serum bottles. Of the strains tested, the highest growth

rates were observed with P. tricornutum (Thomas strain) and M. salina.

Additional experiments were performed in plexiglas vessels that were 5 cm thick, 39 cm deep,

2,000-watt tungsten-halide lamp, which was placed behind a water/CuSO4 thermal filter. In

these experiments, the cultures were typically maintained for 40 to 90 days. In the early stages of

an experiment, the cultures were maintained in a batch mode, and then converted to a continuous

or semi-continuous dilution mode. Various culture parameters (including light intensity, dilution

rate, and N status) were manipulated during the course of these experiments to determine their

effects on the productivities and proximate chemical composition of the strains. The results of

these experiments with each species tested are discussed below. These experiments are difficult

to compare because the experiments were all carried out slightly differently (i.e., different light

intensities, different culturing methods [batch, semi-continuous, and continuous], different means

of obtaining N-deficient cultures, and inconsistent use of a CuSO4 heat filter, which resulted in

differences in light quality and culture temperature). Nonetheless, the general conclusions of this

study are of interest.

P. tricornutum):

This strain has been used for several past studies, and was concomitantly being tested in outdoor

mass culture by another subcontractor (the University of Hawaii; principal investigator Dr.

Edward Laws; discussed in Section III). Therefore, this strain was subjected to more extensive

testing than the other strains in this subcontract. In one experiment reported for this strain, the

effects of light intensity on productivity were determined in batch cultures (i.e., in the Plexiglas

culture apparatus described earlier without culture replacement and dilution). The maximum

productivity observed for this strain (21 to 22 g dry weight?m-2?d-1)3 was observed at a total daily

illumination of 63-95 kcal (representing approximately 40%-60% of full sunlight in southern

California during the summer). This value was slightly higher than the productivity observed

with a total daily illumination of 70% full sunlight (17.1 g dry weight?m-2?d-1). Productivities

under N-limiting, continuous growth mode conditions were between 7 and 11 g dry

weight?m-2?d-1. Likewise, productivities under N-sufficient, continuous growth mode conditions

were reduced relative to batch cultures.

In addition to measuring overall productivities, the levels of protein, carbohydrate, lipid, and ash

were determined for cells grown under the various conditions described earlier. Illumination of

the cultures from 40% to 70% of full sunlight did not have a large impact on the cellular

composition. Growth of P. tricornutum cells under N-deficient conditions resulted in a reduction

of the protein content from 55% (in N-sufficient cells) to 25% of the cellular dry weight.

Carbohydrate content increased from 10.5% to 15.1%, and the mean lipid content increased from

19.8% to 22.2%, although these differences in carbohydrate and lipid contents did not appear to

be statistically significant. At one stage of the experiment, however, a time course of N

deficiency led to a consistent rise in lipid content from 19.9% to 30.8% over the course of 7 days.

The actual rate of lipid production did not increase, however, because the overall productivity of

the cultures was reduced under N-deficient growth.

D. primolecta:

The maximum productivity observed for this species (12.0 g dry weight?m-2?d-1 ) occurred during

continuous culture at 60% full sunlight under N-sufficient conditions. Doubling the light

intensity lowered the productivity to 6.1 g dry weight?m-2?d-1. The chemical composition of Nsufficient

cells (as an average percentage of total cell dry weight) was 64.2% protein, 12.6%

carbohydrate, and 23.1% lipid. After 7 days of growth under N-deficient conditions, the

composition was 26.8% protein, 59.7% carbohydrate, and 13.7% lipid. Therefore, this alga

accumulates carbohydrates rather than lipids in response to nutrient deficiency, limiting its

usefulness as a lipid production strain.

M. salina:

This alga reportedly contained high levels of lipids when grown under N-deficient conditions.

The highest productivity (13.9 g dry weight?m-2?d-1) was observed under N-sufficient conditions

at a light intensity of 50% full sunlight, although detailed experiments with regards to the effects

of light intensity on productivity were not conducted. There was little difference in the lipid

3Reporting of productivities in g dry weight?m-2 ?d-1 derives from the goal of mass culturing the algae in

shallow open ponds. The objective would be to maximize biomass produced per area of pond. However, it

is often difficult to compare results between experiments when the data are reported in this manner, as

factors such as culture depth and vessel design would significantly affect productivity of the cultures.

content of cells grown under N-sufficient and N-deficient conditions (20.7% and 22.1%,

respectively).

 

 

T. sueica:

The highest productivity observed for this strain was 19.1 g dry weight?m-2?d-1, which occurred

in N-sufficient batch cultures grown under a light intensity of 60% full sunlight. N deficiency

resulted in a large increase in carbohydrate content (from a mean value of 10.7% to a mean value

of 47.1%). On the other hand, protein content was reduced substantially (from 67.6% to 28.3%),

and the lipid content decreased from 23.1% to 14.6% in response to N deficiency.

Isochrysis sp.(Tahitian strain T-ISO):

This strain is commonly used as a feed organism in aquaculture production systems. A

productivity of 11.5 g dry weight?m-2?d-1 was typical for batch cultures of this species, which was

approximately 33% higher than the value recorded during semi-continuous growth (dilution of

0.15 L/d). Productivity was lowered during N-deficient growth to 5.5-7.6 g dry weight?m-2?d-1.

This strain accumulated carbohydrate in response to N deficiency (from a mean value of 23.1%

to 56.9%). Lipid content also increased slightly (from 28.5% to 33.4%), whereas protein content

was reduced from 44.9% to 27.3%. The higher lipid content of N-deficient cells did not translate

to higher lipid productivities, however, because of the lower overall productivity of the stressed

cultures.

B. braunii:

Some very limited experiments were conducted with this species, which is known to accumulate

hydrocarbons. A culture grown under a light intensity of 60% full sunlight had a productivity of

only 3.4 g dry weight?m-2?d-1. The lipid content of these cells was 29% of the cellular dry

weight; the N status of the cells was not reported, but it is assumed that the cells were grown

under N-sufficient conditions.

Overall Conclusions

Of the species examined, P. tricornutum and T. sueica had the highest overall productivities.

These species also had the highest lipid productivities, which were 4.34 and 4.47 g lipid?m-2?d-1,

respectively. For both species, the maximal productivities were obtained in batch cultures, as

opposed to semi-continuous or continuous cultures. Although the lipid contents of cells were

often higher in response to N deficiency, the lipid productivities of all species tested were

invariably lower under N deficiency because of an overall reduction in the culture growth rates.

For the species tested under continuous or semi-continuous growth conditions, lipid

productivities were reduced from 14% to 45% of the values measured for N-sufficient cultures.

The results also pointed to the importance of identifying strains that are not photoinhibited at

light intensities that would occur in outdoor ponds. Finally, this work highlighted the fact that

some microalgae accumulate carbohydrates during nutrient-deficient growth; such strains are

clearly not acceptable for use as a feedstock for lipid-based fuel production.

    The work is going on....

Will share you more when the reports will come..

 Please give your views..

 


 

Thu November 04 2010 09:50:50 AM by Vivek 3590 views

Comments - 9

  • Sun November 07 2010 03:15:29 PM

    interesting.

    Vote Up! 1 Vote Down! 0

  • Vivek wrote:
    Fri November 12 2010 12:09:31 PM

    Thanks....

    Vote Up! 0 Vote Down! 0

  • Sat November 27 2010 01:54:31 PM

    Mr. Vivek,

    I admire your work with algae. 

    In your Algae Strain Report I think you teased out some very important conclusions.  I would like to comment on what was especially interesting to me.

     

    1.  In a solar powered enviroment for raising algae it seems that the algae responded the best at solar light levels of somewhere between 40% and 60% of full sunlight.  Is this true? 

    If it is true then I also think that more than 50% of fossil fuel is consumed between 30 and 50 degree latitudes.  This would include the 'temperate' zones. 

    The question is: What part of the daylight hours gives the "happy time" of 40 to 60% of full sun light? Is it 9 to 10am and 3 to 4pm????      Cloudy days??     What about in the desert when almost every day is full sun?

    2.  You said, "Although the lipid contents of cells were often higher in response to N deficiency, the lipid productivities of ALL species tested were invariably lower under N deficiency because of an overall reduction in the culture growth rates."   

    This is a very important statement! 

     And I agree with it 100%.   Our goal should be to produce the Maximum amount of biomass per unit of time and space.  I think a lot of time and talent is being wasted on the search for the algae with the most oil content when we should simplify and broaden the search for the algae, bacteria,etc. that can consume the most CO2 the fastest and under 'perfect' conditions produce the most biomass.

    This biomass can then be processed through a 'pressure cooker' like U.of Michigan has developed. The output of this process is +90%  conversion of ALL the biomass into bio-liquids.....let the oil refiners take over from there, its what they do best.

    Warm regards,

    Alan Schaefer

    Vote Up! 1 Vote Down! 0

  • Erika wrote:
    Sat November 27 2010 07:41:26 PM

    What a great report, thank you for sharing Vivek!
    I agree with Alan, N deficiency to increase lipid content resulting in reduced culture growth rates may not be as valuable in the long run as overall biomass productivity.
    I am also very curious about the variance in light intensity.  It seems your report shows some increases in productivity with reduced sunlight levels, and as Alan suggests, this may open the door for the temperate zones to be considered for algae biomass production.
    Alan, you and I have had a brief discussion in the past regarding reduced light intensity for algae production.  I live in a coastal temperate zone, subject to cooler temperatures, coastal summertime fog, heavy winter rain... but it rarely reaches freezing temperature.  There is a coastal bay (Humboldt Bay) which produces enormous quantities of many different macro and micro algae, evidently uninhibited by low sunlight. This is an ideal area to develop an algae biomass industry because we have an excess of water available (60 MGD of water available for industrial use), yet it seems that many algae biotech companies (at least the ones I have spoken to) are hesitant to develop in this area due to the low light intensity.  Dr. Benneman came to do an initial feasibility study and concluded that coastal fog and winter cloud cover won't produce sufficient biomass with the RAF system.  
    Vivek, I am curious about the Rhodophyta, which weren't mentioned in your report.  While the Rhodophytes may not make a good biofuel source due to their complex lifecycles, I am curious about their overall reactions under controlled conditions of light intensity, nutrient availability, etc.  Do you have any information regarding red algae?
    Regarding my hometown bay:  The local university has two separate long-term studies ongoing.  One is to measure local light intensity of the bay region for solar-power studies, and the other is a very thorough measure of macro-algae presence in the bay.  Unfortunately, the algae biomass measurement, while being tediously thorough, is taken only once a year!  However, analysis of light intensity data in the month(s) leading up to the algae measurement (usually performed in March) could yield some interesting data.  Too bad I don't enjoy statistics more.  Is anyone looking for a project? :)
    Best,
    Erika

    Vote Up! 1 Vote Down! 0

  • Shankar wrote:
    Mon November 29 2010 09:54:53 PM

    Erika 
    Do you know that Dr Bennemen has a nick name as Dr NO :-)
    He is supposed to have said " Abandon all hope of Co locating algae culture with a coal plant. "Am sorry Vivek if I am digressing.

    Vote Up! 2 Vote Down! 0

  • Vivek wrote:
    Tue November 30 2010 04:21:34 PM

    Hello Erika,

       Thanks For giving your comment....

    Actually work is still on ....

      I will clear all the doubts of you all....

         I am always here for that....

    And Sure you can give the project to my Research Institute...

     Best regards...

    Vivek

    Vote Up! 0 Vote Down! 0

  • Vivek wrote:
    Tue November 30 2010 04:26:32 PM

    Hello Alan,

       Thanks for your comment....

    I would Love to hear from you regularly...

       You are right about light intensity... Actually we are still working on that..

      When a final report comes then will share it with you...

           You can mail me at -scientistvivek01@gmail.com.

    Best Regards

    Vivek

    Vote Up! 0 Vote Down! 0

  • Ldvitug wrote:
    Tue December 07 2010 06:38:28 PM

    Hi Mr. Vivek
    I'm Lawrence, from the Philippines. Your algal report amazes me, it is very insightful and it interest me a lot. I just wanted to comment on with regards to the following paragraph.
    "However, these experiments did not provide information on actual lipid productivity in the cultures, which is the more important factor for developing a commercially viable biodiesel production process. This lack of lipid productivity data also occurred with most of the ABP subcontractors involved in strain screening and characterization, but was understandable because the process for maximizing lipid yields from microalgae grown in mass culture never was optimized. Therefore, there was no basis for designing experiments to estimate lipid productivity potential."
    This is what actually I am doing in my research optimizing the culture conditions of b.braunii and f. brevistriata with regards to different parameters in order to increase lipid production and growth similar to what is in your report. I was surprised by your conclusion  that there is lack of info regarding lipid productivity in ABP and it was never optimized. 
    Thanks to your report Now that I'm confident that im in the right track of pursuing my research on microalgae and it has a significant impact.I will update you soon with regards to my research and share my findings in my OILGAE BLOG.
    Do you have a hard copy of this report ? May I please request to have a copy for sake of sharing reasearch knowledge and I would also give u copy of my research after I finished for mutual benefits.
    Pls email: ldvitug@yahoo.com
    Cheers,
    Lawrence Graduate Research StudentUST Graduate School

    Vote Up! 4 Vote Down! 0

  • Vivek wrote:
    Wed December 08 2010 02:43:53 PM

    Hello Lawrence,

       Thank you very much for commenting,It was nice to read your comments,ya you are right about the conclusions as we are working and we have not came to a strong conclusion...

        When we come to a strong conclusion we will tell you...

    I appreciate your comment...

      You can mail me at scientistvivek01@gmail.com,there we can have more beneficial chat..

     

    Best Regards

    Vivek

    Vote Up! 0 Vote Down! 0

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