Abstract
Astaxanthin is a red ketocarotenoid, widely used as a natural red colourant in marine fish aquaculture and poultry and, recently, as an antioxidant supplement for humans and animals. The green microalga Haematococcus pluvialis is one of the richest natural sources of this pigment. However, its slow growth rate and complex life cycle make mass culture difficult for commercial purposes. The aims of this research were (i) to standardize and apply a genetic improvement programme to a Chilean strain of H. pluvialis in order to improve its carotenogenic capacity and (ii) to evaluate the performance of a selected mutant strain in commercial-sized (125 000 L) open ponds in the north of Chile. Haematococcus pluvialis strain 114 was mutated by ethyl methanesulfonate. The level of mutagen dose (exposure time and concentration) was one that induced at least 90 % mortality. Surviving colonies were screened for resistance to the carotenoid biosynthesis inhibitor diphenylamine (25 µM). Resistant mutants were grown in a 30-mL volume for 30 days, after which the total carotenoid content was determined by spectrophotometry. Tens of mutants with improved carotenogenic capacity compared with the wild-type strain were isolated by the application of these standardized protocols. Some mutants exhibited curious morphological features such as spontaneous release of astaxanthin and loss of flagella. One of the mutants was grown outdoors in commercial-sized open ponds of 125 000 L in the north of Chile. Grown under similar conditions, the mutant strain accumulated 30 % more astaxanthin than the wild-type strain on a per dry weight basis and 72 % more on a per culture volume basis. We show that random mutagenesis/selection is an effective strategy for genetically improving strains of H. pluvialis and that improved carotenogenic capacity is maintained when the volume of the cultures is scaled up to a commercial size.
Highlights
Astaxanthin is a red ketocarotenoid, highly prized as a pigment for fish aquaculture and, more recently, for human consumption because of proven antioxidant, anti-ageing, anti-inflammatory and immune-stimulating properties (Christiansen and Torrissen 1997; Lorenz and Cysewski 2000; Jin et al 2006; Zhang et al 2009).At present, the global demand for this pigment is satisfied mainly by synthetic astaxanthin produced by DSM in The Netherlands and by BASF in France
The results of this study indicated that astaxanthin biosynthesis primarily depends on the transcriptional control of the gene encoding crtR-b and, to a lesser extent, on the genes encoding ipi, pds, psy and carotenoid oxygenase (crtO)
Despite chemical synthesis providing a stable source of synthetic astaxanthin, there is concern about its biological functions and food safety
Summary
The global demand for this pigment is satisfied mainly by synthetic astaxanthin produced by DSM in The Netherlands (http://www.dsm.com) and by BASF in France (http://www.basf.com). Haematococcus pluvialis is a unicellular green alga able to accumulate large amounts of astaxanthin (4 % dry weight) under stress conditions (Bubrick 1991; Krishna and Mohanty 1998; Boussiba 2000). Astaxanthin biosynthesis is accompanied by morphological changes of the motile vegetative (green) cells into non-motile cysts (red), which represent a resting stage with a heavy resistant cellulose cell wall (Boussiba 2000). Astaxanthin is accumulated in the cytoplasm of cyst cells, providing protection against photo-inhibition and oxidative stress (Yong and Lee 1991; Kobayashi et al 1997)
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