Heme production from microalgae

Ulfat J Lithi

Nearly one-third of the global population experience severe health consequences like anaemia due to diet-derived deficiencies in iron consumption. Three-quarters of functional iron in the human body comes from heme. In emerging economies, dietary iron from animal and plant-based sources is not enough to meet daily iron demand. Iron supplements exist but are generally not very bioactive. Hence, alternative sources of bioavailable heme are required. Microalgae could be that alternative, as the heme is present in the chlorophyll and phycobilin pigment biosynthetic pathway. There are competitive branch points that direct protoporphyrin IX and/or heme to downstream biosynthetic pathways, suggesting that manipulation of these could lead to an accumulation of heme in the microalgal cell. The main aim of this project was to investigate non-GMO approaches to manipulating these branch points and determine their effect on microalgal heme yield. The overall aim was achieved through investigating two approaches: • inhibition of heme oxygenase (HO) enzyme in phycobilin-producing microalgae, and; • the manipulation of light-dark cycles to regulate the competitive chlorophyll-heme pathway in microalgae that do not produce phycobilins. Before proceeding with optimization studies, it was crucial to establish a robust analytical method for quantifying heme in microalgae. Thus, standard spectrophotometric and HPLC methods (originally developed for heme-rich blood products) were assessed for the suitability of microalgal heme quantification. The HPLC method worked best for microalgae due to its higher sensitivity and lower limit of detection (0.1 pmole). In addition, the HPLC method also offers advantages in terms of mitigating potential interferences, reducing extraction steps, and improving occupational health and safety (OHS) outcomes for analysts. The enhanced heme analysis methodology was used to survey heme content in 6 microalgae that are commonly used for various food and nutraceutical applications, 3 of these for the first time. Arthrospira platensis MUR-126 and Chaetoceros muelleri were found to have the highest natural heme content. With the developed heme quantification method in hand, heme enhancement studies could be reliably performed. In the first heme enrichment study, the cyanobacterium Arthrospira platensis was selected for its ability to accumulate a large amount of phycocyanin, a pigment derived from heme by the action of heme oxygenase (HO) enzyme. Therefore, inhibition of HO enzyme activity could enhance the heme content in this organism. Heme enrichment in A. platensis was studied using Zn-protoporphyrin IX (ZnPP) to inhibit the heme-oxygenase enzyme activity. As a result, a 38% increase in heme content in Arthrospira was observed after a single application of ZnPP at a concentration of 0.4 μmole.g-1 of microalgae. The heme-enriched microalgae exhibited an anticipated decrease in phycocyanin content, providing evidence that the HO enzyme was, in fact, blocked by the application of ZnPP and that this concept works to increase heme content in the biomass. This study is the first instance of heme enrichment in Arthrospira achieved by blocking the HO enzyme. During the Arthrospira platensis heme optimization study, it became apparent that extraction of the biomass to provide a sample for quantification using the traditional heme extraction method was excessively time-consuming. Hence, efforts were made to modify the traditional heme extraction process to expedite the entire heme analysis procedure. Consequently, a rapid, reliable, simple, optimized extraction protocol was developed, utilizing acidified N,N-dimethylformamide. This new extraction technique reduced the extraction time and solvent consumption by approximately 90% and improved heme extraction yield by ~ 50% compared to the traditional extraction protocol. This new heme extraction method, coupled with the newly developed HPLC protocol was tested in a second heme enrichment study. In the second heme enrichment study, the non-phycobilin-producing Chaetoceros muelleri was chosen as earlier results indicated that it contained relatively high amounts of heme. This study examined the change in heme content in C. muelleri by modifying light-dark cycles to increase protoporphyrin-IX distribution towards the heme pathway rather than chlorophyll biosynthesis. Changing the light-dark period from 12:12 to 3:21 resulted in a 34% and 53% increase in biomass and cellular heme content, respectively. Additionally, chlorophyll and other light-harvesting accessory pigments of C. muelleri, e.g. fucoxanthin, significantly increased when exposed to this increased dark period. However, the application of repeated, prolonged dark periods resulted in a reduction in the growth of C. muelleri. Overall, the outcomes of this project provide a more detailed understanding of the effects of optimization strategies to exploit microalgal heme synthesis, suggesting that microalgae could be grown as a source of bioavailable iron.

Heme production from microalgae

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