Large scale production of carotenoids

Introduction

• Carotenoids are colored lipid-soluble compounds that can be found in higher plants and algae, as well as in nonphotosynthetic organisms like animals (although they are not able to synthesize carotenoids), fungi, and bacteria.

• Carotenoids are responsible for the red, orange and, yellow colors of plant leaves, fruits, and flowers, as well as for the color of feathers, crustacean shells, fish flesh and skin, etc.

Chemical structure

• The chemical structure of the more than 600 different carotenoids is derived from a 40- carbon polyene chain, which can be considered as the backbone of the molecule.

• The polyene system gives carotenoids their distinctive molecular structure, their chemical properties, and their light-absorbing characteristics. This chain may be terminated by cyclic groups (rings) and can be complemented with oxygen-containing functional groups. The hydrocarbon carotenoids are named carotenes, whereas oxygenated derivatives are known as xanthophylls.

• In the latter, oxygen can be present as OH groups (as in lutein), as oxi-groups (as in cantaxanthin), or in a combination of both (as in astaxanthin

Uses of carotenoids

• carotenoids play multiple and essential roles in photosynthesis.

• light harvesting, maintain structure and function of photosynthetic complexes, quench chlorophyll triplet states, scavenge reactive oxygen species, and dissipate excess energy

• Antioxidant activity• The pigmentation properties of carotenoids have

granted to some of them extensive application in the food and feed industry

Commercial value• According to a recent report by Business

Communications (http://www.bccresearch.com), the worldwide market value of all commercially used carotenoids is estimated at US$887 million for 2004 and expected to rise at an average annual rate of 2.2% to about US$1,023 million

• Astaxanthin sells for approximately US$2,500 kg-1 with an annual worldwide aquaculture market estimated at US$200 million (Cysewski and Todd Lorenz 2004). Projections for 2009 of global astaxanthin market rise to US$257 million (http://www.bccresearch.com).

(Cuurent projection of carotenoid markets. Price of carotenoids 2010)

Large scale production from different sources

• Different systems have been designed for the growth and handling of microalgae on a large scale

• Within the open systems, the best choice seems to be the open shallow pond, made of leveled raceways?? 2–10 m wide and 15–30 cm deep, running as simple loops or as meandering systems. Each unit covers an area of several hundred to a few thousand square meters.

• Turbulence is usually provided by rotating paddle wheels, which create a flow of the algal suspensions along the channels at a rate of 0.2–0.5 m s−1.

• The adequate supply of carbon dioxide is very critical, and it is usually controlled through a pH-stat, so warranting both provision of carbon and optimum pH of the culture simultaneously.

• The open raceway pond reactor has some drawbacks that limit its use to strains that, by virtue of their weed-like behavior (e.g. Chlorella) or by their ability to withstand adverse growing conditions as Spirulina (Arthrospira) or Dunaliella, can outcompete other microorganisms.

• Closed systems?

• Photobioreactors- either flat or tubular and can adopt a variety of designs and operation modes. They offer higher productivity and better quality of the generated biomass (or product), although they are certainly more expensive to build and operate than the open systems.

Types of carotenoids

Carotene

• Astaxanthin

• Leutin

Production of carotene by Dunaliella salina

• The most common carotenoid, β-carotene, is present in green leafy plants (parsley, spinach, broccoli), fruits (peach), and several vegetables (carrot, pumpkin).

• Nevertheless, the most important process for natural production of β-carotene is the culture of the green, unicellular alga D. salina.

• The extent of carotenoid accumulation in oil globules in the interthylakoid spaces of their chloroplast depends on high salinity, a stress temperature, high light intensity, and nitrogen limitation. Under these conditions, up to 12% of the algal dry weight is β-carotene

General nutrients required for Dunaliella culture

• For a 40L culture medium:• 8% NaCl For normal growth• 70ppm Nitrogen• 25%NaCl• 5ppm Nitrogen For carotenoid

prodtn

• 1.5% vol CO2

• Illuminated with continuous cool white fluorescent lamps (400wm-2)

• Dunaliella production facilities are located in areas where solar irradiance is maximal, cloudiness is minimal, climate is warm, and hypersaline water available

• Its cultivation is based on autotrophic growth in media containing inorganic nutrients, with carbon dioxide as the carbon source

• The Australian producers use the so-called ‘extensive• cultivation’ approach, consisting of very large ponds of

up to 250 ha in area. These are unmixed other than by wind and convection, have a suspension depth of up to 0.5 m, and operate without CO2 addition and with minimal control. Economic viability of this system seems to rest on low land costs, as well as on the facts that water is free other than for pumping costs and that climate is close to optimum.

• Production can thus be maintained all year round, • Other Dunaliella producers use channels in the form of

oblong raceways of ca. 3,000-m2 of production surface area, 0.2 m deep, the culture being paddle-wheel driven to give a flow of about 0.2 m s−1, and supplied with CO2.

• In this ‘intensive cultivation’ approach, plants are constructed. of repeated units, with each pond unit being an independent production system. Concentration of β-carotene in the pond at the time of harvesting can reach 15 g m−3, yielding up to 9 kg β-carotene on total harvest per production unit.

• The current productivity of β-carotene under intensive largescale cultivation is claimed to reach around 200 mg β-carotene m−2 day−1 on a yearly average

Use of 55-l closed tubularPhotobioreactors to culture

Dunaliella• When operating under semi-continuous regime under

reduced incoming irradiance, with the tubular loop reactor covered with a polyvinyl chloride sunshade screen, productivity values over 2 g dry biomass and 100 mg β-carotene m−2 day−1 were achieved.

• The carotene cellular level could be further enhanced to reach 10% of the dry weight by allowing exposure of the system to full impinging sunlight.

• Working under semi-continuous regime, at higher cell densities, and carefully controlling nitrogen availability (to maintain nitrate concentration in the suspension between 0.5 and 3 mM),

• the generated Dunaliella biomass exhibited an average β-carotene level of around 14%.

• In addition to offering a higher productivity, the carefully controlled closed culture in the photobioreactor makes possible to produce a biomass with improved carotenoid Profile??

• With regard to harvesting of the carotene-rich Dunaliella biomass, industrial extensive producers are presently using cell surface adsorption, whereas the intensive growers employ centrifugation

Commercial producers of Dunaliella

Company Location β-carotene production

(tonnes per year)

Culture system

Betatene Australia 13-14 Extensive

unmixed ponds

Western Biotechnology

Australia 4-6 Extensive

unmixed ponds

Nature Beta Technologies

Israel 3-4 Raceways ponds

Parry Agro Industries

India -- Raceways ponds

1. Hermatizable vessel2. Water cooled quartz flask3. Rotor4. Sensors5. Windows6. Water jacket7. Separate rack containing the thermostat8. Centrifugal force9. Lamp10. Water jacket

Recombinant DNA technology in carotenoid production

• Carotenoid synthesis can be considered in two different parts, the early stages and the later stages. Carotenoids are isoprenoid compounds. There are seven biochemical conversions required to transform the precursor of all isoprenoids, acetyl Co-A, into the CIS molecule farnesyl diphosphate.

• The genes for all these seven enzymes have been isolated and characterized. The genes for the enzymes to convert farnesyl diphosphate to the compound geranylgeranyl diphosphate and then on to the first specific carotenoid precursor, phytoene, have been isolated and characterized.

• A number of different carotenoid biosynthetic genes encoding enzymes that convert phytoene into lycopene, neurosporene, and 6-carotene have been isolated and characterized as have the genes encoding enzymes that convert these compounds into p-carotene and a-carotene.

• Finally genes that encode enzymes to convert p-carotene into zeaxanthin, canthaxanthin, astaxanthin, capsorubin, and capsanthin have been isolated and characterized

• Industrial research groups, including the Amoco Biotechnology, Grin Research, and ICI/Zeneca Seed Research groups, were among the first to recognize the importance of the isolation and characterization of the genes for the carotenoid biosynthetic enzymes, and they led the way in the initial research

References (Available with me)• Rodney L. Ausich, Pure &App/. Chem.,

Vol. 69, No. 10, pp. 2169-2173,1997.

• José A. Del Campo & Mercedes García-González & Miguel G. Guerrero, Appl Microbiol Biotechnol (2007) 74:1163–1174

Production of Astaxanthin by Haematococcus

• Only two sources of microbial origin compete with synthetic astaxanthin, which presently dominates the market: the heterobasidious yeast Phaffia rhodozyma and the microalga H. pluvialis. P. rhodozyma has been widely studied for more than two decades under the viewpoint of astaxanthin production.

A lot of works: s major conclusions:1) to induce astaxanthin accumulation,

growth should be slowed down or stopped;

2) the astaxanthin accumulation rate increases with irradiance; and

3) the kinetics of astaxanthin accumulation varies with the nature of the stress condition imposed.

• Haematococcus can be grown autotrophically, with inorganic carbon or hetero-trophically, with an organic carbon source, such as acetate

• So far, the successful producers raise Haematococcus autotrophically in closed systems, but the Swedish company BioReal seems to use a mixotrophicbasedindoor system

• Haematococcus is first grown under nutrient-sufficient conditions and low average irradiance, as to achieve a high biomass yield. Later, such green biomass is subjected to environmental and nutrient stress in the second stage. The cells are exposed to deprivation of nutrients (mainly nitrogen and phosphorous), temperature increase or salt addition, and high average irradiance, to induce haematocyst (aplanospore) formation and astaxanthin accumulation.

• Since no selective environment has become available for H. pluvialis, open systems are virtually excluded for outdoor operation. Nevertheless, some companies perform the second stage (induction of astaxanthin accumulation) in the open, although for a short period (5–6 days) only.

• Haematococcus haematocysts contain 1.5–3% of the dry biomass, although some authors claim even higher values

Harvesting the biomass

• Harvesting of Haematococcus is accomplished by settling and subsequent centrifugation, as the big and sticky haematocysts sediment easily. Then, this biomass is dried and cracked to fracture the thick and hard cell wall of the cysts, thus ensuring maximum bioavailability (but again, companies do not provide details about this step).