Universidade de Aveiro Departamento de Qu´ımica 2018
Margarida Isabel
Penedo Rodrigues
Potencialidades da extrac¸˜ao a frio de compostos de
Universidade de Aveiro Departamento de Qu´ımica 2018
Margarida Isabel
Penedo Rodrigues
Potentialities of cold extraction of value-added
Universidade de Aveiro Departamento de Qu´ımica 2018
Margarida Isabel
Penedo Rodrigues
Potentialities of cold extraction of value-added
compounds from microalgae species
Dissertac¸˜ao apresentada `a Universidade de Aveiro para cumprimento dos requisitos necess´arios `a obtenc¸˜ao do grau de Mestre em Biotecnologia Industrial e Ambiental, realizada sob a orientac¸˜ao cient´ıfica do Doutor Jorge Manuel Alexandre Saraiva, Investigador Auxiliar do Departamento de Qu´ımica e co-orientac¸˜ao cient´ıfica da Doutora Joana Gabriela Laranjeira da Silva, Diretora de Investigac¸˜ao na Allmicroalgae.
O j ´uri
Presidente Doutora Ivone Delgadillo Giraldo
Professora Associada com Agregac¸ ˜ao do Departamento de Qu´ımica da Universidade de Aveiro
Arguente Doutora Elisabete Maria da Cruz Alexandre
Investigadora na Escola Superior de Biotecnologia da Universidade Cat ´olica do Porto
Orientador Doutor Jorge Manuel Alexandre Saraiva Investigador Auxiliar na Universidade de Aveiro
Agradecimentos Em primeiro lugar agradec¸o ao professor Jorge Saraiva e `a doutora Joana Silva, pelo desafio que me colocaram, por todo o apoio e motivac¸˜ao que me deram ao longo deste percurso.
A todos os meus colegas do grupo ”HP”, a vocˆes em especial S´ılvia, Liliana, Mauro e Rui um obrigada enorme, por me terem acolhido e guiado com mestria nos momentos de apuros. Ao meu amigo Carlos, vou-te sempre ser grata por me teres acompanhado e nunca me teres negado a tua ajuda, e mostrares sempre que eu era capaz.
Ao pessoal incr´ıvel da Allmicroalgae, pelo apoio incondicional na aproximac¸˜ao do contexto acad´emico ao mundo empresarial, nunca me senti longe de casa. `A Joana Teles, `a Joana Galante, ao Bernardo, `a N´adia, e a todos os que me davam carinho e bons momentos. Muito obrigada Hugo, que apesar da distˆancia ajudaste-me imenso.
Aos amigos do crossfit MARE, pelas risadas que me trazem diariamente e felicidade nos piores momentos.
Agora, `a melhor amiga do mundo, Guida, como gosto de ti, e a todos vocˆes que me enchem o corac¸˜ao Inˆes, Ricardo, Jorge, Mimoso, J´essica Tavares, J´essica Jacinto, Rita, Lu´ısa s˜ao um m´aximo. Obrigada por fazerem de mim uma pessoa muito feliz. J´a tenho saudades.
Ao meu namorado, Edgar, que me d´as forc¸a e todo o apoio do mundo, fizeste tudo isto ser poss´ıvel. Fazes-me t˜ao bem. N˜ao me deixaste ir abaixo, para sempre tua e para sempre grata a ti.
M˜ae, Pai, Irm˜a e Irm˜ao, Cunhado e Sobrinhos vocˆes s˜ao a minha luz, quem me d´a forc¸a. Sou assim feliz porque sempre me mostraram que ´e este o caminho. ´E tudo para vocˆes. Amo-vos tanto.
Palavras-chave Chlorella vulgaris; Processamento por alta-press˜ao; Extrac¸˜ao por alta-press˜ao ; Sumo de fruta; Pasteurizac¸˜ao
Resumo Os objetivos deste trabalho consistiram em investigar o efeito da alta press˜ao, para o desenvolvimento de um extrato aquoso de uma cultura de Chlorella vulgaris, com otimizac¸˜ao das condic¸˜oes de extrac¸˜ao `a temperatura ambiente. E ainda, enriquecer dois sumos de frutas portuguesas: laranja do Algarve e pera Rocha com o extrato obtido, sendo que a extrac¸˜ao e pasteurizac¸˜ao ocorrem simultaneamente.
Inicialmente, foram otimizadas as condic¸˜oes de extrac¸˜ao, utilizando diferentes press˜oes (350, 400, 600 MPa) e tempos (5, 10, 15, 30 minutos). A atividade antioxidante e a concentrac¸˜ao de prote´ına dos extratos foram quantificados. Obteve-se uma concentrac¸˜ao m´axima de prote´ına de 0,58±0.01 mg/mL, a 300 MPa durante 5 min e atividade antioxidante m´axima de 0,29±0.02mmol de Trolox Equivalent Antioxidant Capacity/g de extrato, a 600 MPa durante 10 min. Com base nos resultados fez-se um compromisso entre os dois parametros tendo-se decidido prosseguir o estudo a 600 MPa durante 5 min. Foram preparados sumos de pˆera Rocha e laranja do Algarve enriquecidos com cultura de C. vulgaris 50/50 (v/v), e sujeitos a um ciclo de press˜ao nas condic¸˜oes anteriormente mencionadas.
Foi feito um estudo de prazo de validade durante 31 dias, com avaliac¸˜ao dos parˆametros f´ısico-qu´ımicos (pH, acidez total, e s´olidos sol´uveis totais), da carga microbiana (mes´ofilos aer´obios totais, Enterobacteriaceae, bolores e leveduras), e an´alise sensorial a cada 7 dias. A concentrac¸˜ao de prote´ına e atividade antioxidante foi avaliada no in´ıcio e no fim deste estudo.
No geral, os parˆametros f´ısico-qu´ımicos foram semelhantes quer na amostra n˜ao processada quer na processada. Os m´esofilos aer´obios totais mostraram menos suscetibilidade ao tratamento, tendo-se obtido uma validade de no m´ınimo 31 dias para o sumo processado. A n´ıvel sensorial, foi preferido o sumo de laranja. A concentrac¸˜ao m´axima de prote´ına obtida foi no in´ıcio do estudo, para o sumo de pˆera, com 0,46% em peso seco.
De acordo com a literatura, este estudo ´e o primeiro que reporta a otimizac¸˜ao da extrac¸˜ao de compostos bioactivos de cultura fresca de C. vulgaris, aplicando alta-press˜ao. Esta t´ecnica provou ser eficiente na
Keywords Chlorella vulgaris; High Pressure Processing; High Pressure Extraction; Fruit Juice; Pasteurization
Abstract The aim of this work was to investigate the use of high pressure to develop an aqueous extract of a Chlorella vulgaris culture, along with extraction conditions optimization at room temperature. Also, enrich two Portuguese fruit juices: orange from Algarve and Rocha pear with the obtained extract, both extraction and pasteurization were accomplished simultaneously.
Initially, the extraction conditions were optimized, by testing different extraction pressures (350, 400, 600 MPa) and time (5, 10, 15, 30 minutes). The extracts were analyzed for their antioxidant activity and protein concentration. It was obtained a maximum protein concentration of 0.58±0.01mg/mL, at 300 MPa during 5 min, and a maximum antioxidant activity of 0.29±0.02mmol of Trolox Equivalent Antioxidant Capacity/g of extract, at 600 MPa for 10 min. According to the obtained results it was made a compromise between the two parameters and the extraction conditions,for this study, were set to 600 MPa for 5 min. Rocha Pear and orange from Algarve juices with C. vulgaris culture were prepared 50/50 (v/v), and were processed at the afore mentioned conditions.
A shelf-life study for 31 days was obtained, with evaluation of physicochemical parameters (pH, titratable acidity and total soluble solids), microbial load (total aerobic mesophiles, Enterobacteriaceae, yeast and moulds) and sensorial analysis, each 7 days. The protein concentration and antioxidant activity were also evaluated at the beginning and the end of this study.
In general, the physicochemical parameters were similar for the unprocessed and processed juices. Total aerobic mesophiles showed less susceptibility to high pressure than the remaining studied microorganisms, it was obtanined a minimum shelf-life of 31 days, for the processed juice. Accordingly to sensorial analysis, it was preferred the orange juice. The maximum protein yield obtained was for the pear juice at the beginning of the study, 0.46% of dry weight.
According to literature, this study is the first one reporting the optimization extraction of C. vulgaris bioactive compounds from fresh culture, using high-pressure. This technique proved to be efficient to
Contents
Contents i List of Figures v List of Tables ix Nomenclature xi 1 Introduction 11.1 Motivation and objetives . . . 1
1.2 Allmicroalgae . . . 2
2 Overview 5 2.1 Microalgae . . . 5
2.1.1 Morphology, Composition and Reproduction . . . 7
2.1.2 Primary composition . . . 9
2.1.3 Production conditions . . . 12
2.1.4 Harvesting and processing . . . 18
2.1.5 Microalgae applications and market . . . 19
2.2 High Pressure . . . 20
2.2.1 History . . . 22
2.2.2 Equipment description . . . 22
2.2.3 Process and principles description . . . 24
2.2.4 Impact of HPP . . . 25
2.2.5 HPP as an extraction method . . . 28
2.3 Fruit Juices . . . 29
2.3.2 Nutritional value of fruits . . . 32
2.3.3 Pear fruit . . . 34
2.3.4 Orange fruit . . . 35
3 Materials and methods 37 3.1 Chemicals and solutions . . . 37
3.2 Microalgae strain and inoculum culture conditions . . . 37
3.3 Scale-up, harvesting and transportation . . . 38
3.4 Growth assessment and nitrates content . . . 38
3.5 High-pressure extraction assays optimization . . . 39
3.5.1 High-pressure treatments . . . 39
3.6 Biochemical assays . . . 40
3.6.1 Protein content: Lowry method . . . 40
3.6.2 Antioxidant activity . . . 41
3.6.3 Pressure conditions . . . 42
3.7 Juices preparation . . . 42
3.7.1 Shelf-life study . . . 42
3.7.2 pH, Titratable acidity and Total soluble solids . . . 43
3.7.3 Elemental analysis . . . 44
3.7.4 Microbiology . . . 44
3.7.5 Sensorial analysis . . . 45
3.8 Statistical analysis . . . 45
4 Results and discussion 47 4.1 High pressure treatment . . . 47
4.1.1 Protein assay . . . 47
4.1.2 Antioxidant activity . . . 49
4.1.3 Pressure conditions . . . 50
4.2 Juices preparation . . . 51
4.2.1 Shelf-life study . . . 51
4.2.2 pH, Titratable acidity and Total soluble acids . . . 51
4.2.3 Elemental analysis . . . 54
4.2.4 Microbial analysis . . . 56
4.2.5 Antioxidant activity . . . 60
5 Conclusions and future work 63
References 65
Annex A Tables 84
List of Figures
1.1 Allmicroalgae production unit, near the plant Cibra-Pataias, from [5]. . . 3 2.1 Schematic illustration of C. vulgaris structure and different organelles. Adapted
from [1]. . . 8 2.2 Absorption spectra of C. vulgaris algae, with: (1) the simulated spectrum, (2)
spectrum of chlorophyll a (3) spectrum of chlorophyllb and (4) spectrum of carotenoids. Adapted from [49]. . . 11 2.3 Green-wall structure implemented in the Allmicroalgae unit. . . 15 2.4 Time course of continuous culture of Chlorella spp. first under heterotrophic,
then mixotrophic condition, in a vertically flat culture bottle with acetic acid as carbon source. From [62]. . . 16 2.5 Illustration of the pressures used to process food. Adapted from [93] . . . 22 2.6 Illustration of the Hiperbaric55 equipment. From [98] . . . 23 2.7 Water temperature increasing during the adiabatic compression. This increase is
a function of the initial temperature. From [91] . . . 24 2.8 Protein solubilisation yield under different conditions of pressure (1 kbar or 2.7
kbar), pH (7 or 12) and cycle time (1 or 2). From [43] . . . 29 3.1 Pictures from experimental procedures: a) polyamide-polyethylene bags with
culture inside prior to high pressure extraction treatment; b) centrifuged tubes with clear phase separation; c) filtration set. . . 40 4.1 Protein concentration in mg/mL, obtained as a function of time (5, 10, 15, 30
min) and pressure (300, 400, 600 MPa). . . 48 4.2 Antioxidant activity inmmol of TEAC/g of extract, obtained as a function of time
4.3 Total titratable acidity of the unprocessed and processed pear juice, during 31 days, expressed in g of malic acid/L. . . 51 4.4 Total titratable acidity of the unprocessed and processed orange juice, during 31
days, expressed in g of citric acid/L. . . 52 4.5 pH of the unprocessed and processed pear juice, during 31 days. . . 52 4.6 pH of the unprocessed and processed orange juice, during 31 days. . . 53 4.7 Total soluble solids of the unprocessed and processed pear juice, during 31 days,
expressed inoBrix. . . 53 4.8 Total soluble solids of the unprocessed and processed orange juice, during 31
days, expressed inoBrix. . . 54 4.9 Protein concentration of the unprocessed and processed pear juice, at the 0 and
31 days, expressed in % of dry weight. . . 55 4.10 Protein concentration of the unprocessed and processed orange juice, at 0 and 31
days, expressed in % of dry weight. . . 55 4.11 Total count of total aerobic mesophiles of unprocessed and processed pear juice,
during 31 days, expressed in log10CFU/mL. . . 56
4.12 Total count of total aerobic mesophiles of unprocessed and processed orange juice, during 31 days, expressed in log10CFU/mL. . . 57
4.13 Total count of Enterobacteriaceae of unprocessed and processed pear juice, during 31 days, expressed in log10 CFU/mL. Discontinuous bars indicate values
lower than 1 log10CFU/mL. . . 58
4.14 Total count of Enterobacteriaceae of unprocessed and processed orange juice, during 31 days, expressed in log10 CFU/mL. Discontinuous bars indicate values
lower than 1 log10CFU/mL. . . 58
4.15 Total count of yeast and moulds of unprocessed and processed pear juice, during 31 days, in log10 CFU/mL. Discontinuous bars indicate values lower 1 log10
CFU/mL. . . 59 4.16 Total count of yeast and moulds of unprocessed and processed orange juice,
during 31 days, in log10 CFU/mL. Discontinuous bars indicate values lower 1
log10CFU/mL. . . 59
4.17 Antioxidant activity of the unprocessed and processed pear juice, at 0 and 31 days, expressed inmmol of TEAC/L. . . 60 4.18 Antioxidant activity of the unprocessed and processed orange juice, at the 0 and
4.19 Overall quality (Q) of the processed pear and orange juices, during 31 days. . . . 62 B.1 BSA calibration curve used to determine the protein concentration of the C.
vulgarisextracts during the optimization assay. . . 89 B.2 Trolox calibration curve used to determine the antioxidant activity of the C.
vulgarisextracts during the optimization assay. . . 90 B.3 Trolox calibration curve used to determine the antioxidant activity of the
List of Tables
2.1 Examples of several Microalgae species applications, conducted in different studies 6 2.2 Comparison between the protein, carbohydrate and lipid content present in
several sources and organisms used for food and feed, in percentage of dry matter. Adapted from [16]. . . 10 2.3 Mineral element content from C. vulgaris in mg/100g DW. Adapted from [52]. . 12 2.4 Comparison of extraction yields in dry weight (DW) of different compounds
from Chlorella species, under different growth conditions . . . 17 2.5 Effects observed in different microorganisms under high pressure treatment:
variable pressure, temperature and cycle time. . . 26 2.6 Composition of fruit in water, carbohydrates, protein, fat and fiber in g per 100 g
of edible portion. Adapted from [136]. . . 33 A.1 Protein concentration in mg/mL, obtained as a function of time (min) and
pressure (MPa). Different lower case letters (a and b) and upper case letters (A and B) indicate significant differences (p < 0.05) between extraction pressure and times, respectively. The greek letter sigma (s) indicates that the values are statistically similar (p > 0.05) to the initial value. . . 84 A.2 Antioxidatant activity inmmol of TEAC/g of extract, obtained as a function of
time (min) and pressure (MPa). Different lower case letters (a, b and c) and upper case letters (A, B and C) indicate significant differences (p < 0.05) between extraction pressure and times, respectively. The greek letter sigma (s) indicates that the values are statistically similar (p > 0.05) to the initial value. . . 84 A.3 pH, total titratable acidity and total soluble solids of unprocessed (UP) and
processed (PP) pear juices, during 31 days. Different lower case letters (a, b and c) and upper case letters (A and B) indicate significant differences (p < 0.05) between juices and days, respectively. . . 85
A.4 pH, total titratable acidity and total soluble solids of unprocessed (UO) and processed (PO) orange juices, during 31 days. Different lower case letters (a and b) and upper case letters (A and B) indicate significant differences (p < 0.05) between juices and days, respectively. . . 85 A.5 Protein concentration at 0 and 31 days of unprocessed (UP), processed (PP) pear
juices and unprocessed (UO), processed (PO) orange juices, at 0 and 31 days. Different lower case letters (a and b) and upper case letters (A and B) indicate significant differences (p < 0.05) between juices and days, respectively. . . 86 A.6 Nutritional value of each pulp alone; energy, fat, carbohydrates, protein, salt and
vitamin C content per 100 mL of pulp. . . 86 A.7 Total count of total aerobic mesophiles (TAM), Enterobacteriaceae (ENT) and
yeast and moulds (YM) of unprocessed (UP) and processed (PP) pear juices, during 31 days, expressed in log10 CFU/mL. Different lower case letters (a, b
and c) and upper case letters (A and B) indicate significant differences (p < 0.05) between juices and days, respectively. . . 87 A.8 Total count of total aerobic mesophiles (TAM), Enterobacteriaceae (ENT) and
yeast and moulds (YM) of unprocessed (UO) and processed (PO) orange juices, during 31 days, expressed in log10 CFU/mL. Different lower case letters (a and
b) and upper case letters (A and B) indicate significant differences (p < 0.05) between juices and days, respectively. . . 87 A.9 Overall quality (Q) of the processed pear and orange juices in function of the
time, during 31 days. Different lower case letters (a, b and c) indicate significant differences (p < 0.05) between days of each juice. . . 88
Nomenclature
AA Ascorbic acid
ATP Adenosine Triphosphate
CA Citric acid
CGF ChlorellaGrowth Factor
DW Dry Weight
EFSA European Food Safety Authority ENT Enterobacteriacae
FDA Food and Drug Administration GRAS Generally Recongnised as Safe
GW Green-wall
HPE High Pressure Extraction HPP High Pressure Processing
LC-PUFAs Long-Chain Polyunsaturated Fatty Acid
MA Malic acid
NADPH Nicotinamide Adenine Dinucleotide Phophate PBRs Photobioreactors
PO Processed Orange Juice PP Processed Pear Juice RT Room Temperature TA Titratable acidity TAGs Triacylglycerols
TAM Total aerobic mesophiles
TEAC Trolox Equivalent Antioxidant Capacity TSS Total soluble acids
UO Unprocessed Orange Juice UP Unprocessed Pear Juice YM Yeast and moulds
Chapter 1
Introduction
1.1
Motivation and objetives
One of the biggest challenges regarding microalgae biotechnology is the need to find new strategies to take full advantage of the diverse range of the biomolecules synthesized by these organisms. Chlorella vulgaris is becoming a preferable product for consumers and is being increasingly included in food, feed and other industries as a value-added product with multiple health benefits associated. Different techniques have been developed and applied for an efficient extraction of its intracellular compounds, while new ways are constantly being investigated to increase the extraction yields [1].
High pressure processing (HPP) is a novel non-thermal food processing technology. The process conditions (pressure level, treatment time and the operation temperature) are designed according to the final aim and product. This technique has shown promising results in food pasteurization and extraction processes, considered a suitable method in both cases. It does not involve high temperatures, therefore food characteristics are conserved during the process. Also, it increases membrane permeability hence it can be used to improve the extraction of biomolecules with added value from different sources [2].
Fruits are attractive and nutritional, there is a tremendous variety of species, with diverse bioactive compounds of interest. Fruit juices market has been a leader in the beverage industry, it is associated to a healthy way to consume vitamins and nutrients, also in a practical way for the new consumer lifestyle [3].
There are several objectives for this internship, namely: development of an aqueous extract of C. vulgaris culture using high-pressure; optimization of high-pressure extraction time and pressure conditions, at room-temperature (RT); enrich a fruit juice with C. vulgaris culture for
commercialization processed by high-pressure, along with its shelf-life study, microbiology and chemical parameters evaluation.
For this work, biomass production took place in Allmicroalgae unit, located in Pataias, Leiria. After the harvesting process, the microalgae were subjected to HPP in the University of Aveiro, to obtain an aqueous extract rich in bioactive molecules.
Firstly, the extraction conditions were assessed, in terms of pressure level and dwell time. For each condition it was evaluated the biochemical value of the obtained extract, particularly: antioxidant activity and protein content. Then, the conditions afore decided were applied to obtain an orange and pear juice incorporated with the microalga extract. In the same pressure cycle, the extract is obtained as well as the juice was pasteurized.
At this point, as far as could be verified in the literature, no other study in this matter, using the same strategy, have been conducted. And there is still very little literature about microalgae compounds extraction with high pressure.
1.2
Allmicroalgae
Secil group was founded in 1930. This company produces concrete, cements, aggregates and mortars. Since its foundation, it has had continuous technological improvement and a strong environmental awareness. The group has a very old policy on sustainability, and covers its activities in its Integrated Policy for Quality, Environment and Occupational Health and Safety [4].
In 1950, a new plant Cibra-Pataias was inaugurated, as presented in Figure 1.1. In this plant, both white cement and grey cement are produced, with an annual production capacity of 620 million tonnes [4]. To reduce carbon dioxide (CO2) emissions and the carbon footprint of their
activities, Secil started testing the mitigation of CO2 using microalgae, which gave birth to the
Allmicroalgae unit in Cibra-Pataias. This project was firstly developed to retain and use the carbon emissions, with the main purpose of having a sustainable cement industry. Currently, it produces exclusively biomass of premium quality, and has become the biggest production industry in closed cultivation system in Europe [5].
The unit facilities allow the integration of the entire production process from a small laboratorial scale to the final packaging step. Both, Chlorella vulgaris and Nannochloropsis sp. are produced at industrial-scale. In addition, other species are being produced under taylor-made [5]. Microalgae can grow under autotrophy, mixotrophic and heterotrophic conditions. Recently the unit started to produce under heterotrophic regime, to obtain higher concentrations of
pre-inoculum to go further to the scale up. Allmicroalgae is certified by ISO 9001, ISO 14001, ISO 22000, OHSAS 18001 and Halal. Production begins with seed cultures in axenic Master cell banks. The vial is scaled to a 7 L fermenter. Which will inoculate a 200 L fermenter and then pass to a 5000 L reactor. After 7 days of scaling steps, the 5000L fermenter inoculates the large system of tubular photobioreactors (PBRs). The growth in PBRs is performed in mixotrophic conditions.
PBRs are made of transparent plastic tubes with a total length of 300 km. The transparent material allows the solar light to penetrate in the form of radiation while preventing mass transfer to the outside and the culture is able to grow in autotrophy. This configuration is more advantageous than open-pond systems, since is less susceptible to contaminants, provides higher productivities, allows to obtain high quality biomass, and better CO2sequestration. Mixotrophic
strategy potentiates higher biomass concentrations, is preferable to grow C. vulgaris at industrial scale, and reduces the scale-up time, as explained in Section 2.1.3.3 [6].
Allmicroalgae plant was carefully designed for maximum productivity and sunlight exposition, see the production unit in Figure 1.1. After large-scale production, the culture is harvested using cross-flow filtration with membranes. The concentrated microalgal solution is pasteurized before being subjected to the spray-drying process, thus guaranteeing a high-quality product [6].
In conclusion, this project has been built on quality and sustainability, each batch produced is controlled in real-time by Allmicroalgae, and validated by external laboratories. Nowadays, Allmicroalgae has a total production volume of 1300 m3, and a production capacity of 100 tons of dried biomass per year [7].
Chapter 2
Overview
2.1
Microalgae
Algae are ancient organisms with a footprint that dates around three billion years ago. They are a diverse group of aquatic, eukaryotic organisms able to produce a wide range of bioactive compounds, which allowed them to adapt and progressively colonize, throughout the time, all kind of harsh environments. They can either be found in fresh or marine waters, as well as, on tropic or polar areas [1, 8], and, classified as macroalgae (multicellular organisms that can reach 60 meters in length) or as microalgae (unicellular organisms with size ranging from 0.2 to 2 mm). Microalgae are eukaryotic photosynthetic microorganisms that grow rapidly, their ability to colonize diverse habitats is due to their unicellular or simple multicellular structure in some cases. Eukaryotic microalgae include green algae (Chlorophyta) and diatoms (Bacillariophyta) [9]. Over the past decades, the interest in microalgae biotechnology has been rising, and its use includes different purposes and industries.
Microalgae capture massive amounts of CO2 using solar energy and produce approximately
50% of the atmospheric oxygen on earth [1]. The CO2 fixation efficiency by these organisms
is ten times higher compared to terrestrial plants, also since they do not have roots or stems they grow much faster than them [10]. Microalgae, when cultivated, require simple growing conditions and grow in autotrophic, mixotrophic or heterotrophic mode [8]. These organisms produce various substances in large amounts within short periods of time, such as proteins (6-71%), lipids (7-23%), carbohydrates (5-64%), pigments, vitamins and minerals, according with the algae species and growth conditions [10–14]. For instance, especially after nitrogen starvation some of them are capable of accumulate lipids used as feedstocks for biodiesel production [15].
Microalgae have gained multiple interests in this area, since their oil production would not affect the food supply chain or agricultural lands in defiance of first and second-generation fuels. They also exhibit high plasticity since the same process can be used for different applications [16]. All these facts turn microalgae into a potential game changer, turning these single-celled organisms more and more tailored as sustainable economic solutions. Not only can they mitigate global warming [12], but also have the potential to reinvent cosmetics [9], food [10, 17], feed [18, 19], fuels [20] and even aquaculture and pharmaceutical industries, as seen by the examples described in Table 2.1.
Table 2.1: Examples of several Microalgae species applications, conducted in different studies
Specie Aim Applications References Chlorella sp. Protein and Feed [21]
biomass production Health Food
Chlorella sp. Nitrogen (N) , Phosphate (P) removal Bioremeditation and [22] Methyl ester and biomass production Biodiesel industry
Haematococcus pluvialis Astaxanthin Pharmaceuticals [23] production Additives
Dunaliella salina b-carotene, lutein Food and [24, 25] and zeaxanthin production Pharmaceutical industry
Scenedesmus obliquus N and P removal Bioremediation [26] Tetraselmis suecica
Harvest of Chlorella sp. and
Bio-flocculation Nannochloropsis sp. Biodiesel [27] Lipid extraction Tetraselmis marina Biomass, Aquaculture
carotenoid Health food [28] and lipid production Biodiesel
However, there is still a big barrier to overcome so that microalgae can be considered a potential profitable market. Most of the studies are based in laboratory-scale simplistic growth models, that show overestimated productivity yields. The structural and chemical robustness nature of the cell wall is also a big drawback, thus the quality of the product obtained depends on its digestibility. Therefore, usually a pre-treatment step is required to rupture cells and optimize the extraction process, increasing the chemical or energy input [14, 17, 29].
Nevertheless, multiple companies such as Terravia in USA or Dulcesol Group in Spain are starting to integrate microalgae biomass in innovative healthier food products. In fact, it is expected that the annual rate growth for algae products (from 2016 until 2023) may be higher than 5% [17]. The market is becoming competitive and driven by the increase interest of
consumers for natural ingredients and functional foods. Some of the most studied microalgae and commercially produced at large scale are Chlorella spp. [30].
Chlorella vulgaris, is a green, eukaryotic, single-celled microalga, first discovered by Martinus Beijerinck, a Dutch researcher, in 1890. Its high protein content, usually above 55% of dry weight (DW), was noticed in the early 1900s in Germany [1]. In 1950’s, studies conducted in the USA, Germany and Japan showed promising results for the cultivation of Chlorella spp. in open ponds using the photosynthetic pathway. This single-cell organism have been given the GRAS (generally recognised as safe) status by Food and Drug Administration (FDA) of the USA. In Europe, Chlorella sp. is commonlly sold as food supplement and is approved by the Europeen Food Safety Authority (EFSA). The market introduction using the whole microalgae or products that include them are subjected to food safety regulations that are applied evenly to all food products [31]. Nowadays, each year, the USA, Japan, China, Taiwan and Indonesia produce over 2500 tons of dried Chlorella spp. [32, 33], being, Japan the world leader consumer of this microalga.
C. vulgaris has long served as a model organism, exhibits a spherical or ellipsoidal shape with a diameter around 2-10 mm, inhabits usually in fresh waters, and is structurally similar to plants. It is imperative to understand its compartmental organization and metabolism to optimize the production of valuable compounds.
2.1.1
Morphology, Composition and Reproduction
This specie contains a well-defined nucleus, chloroplasts, mitochondria and other organelles suspended in a cytoplasm, as described in Figure 2.1. The cytoplasm is a gel-like substance, composed of water, soluble proteins and minerals that host all the organelles [1]. It has a major role, since it establishes the osmotic balance between the extracellular media and the remaining organelles. About 44% of the metabolites and 55% of the reactions are located in the cytoplasm, while the chloroplast contains 30% of all metabolites and 25% of the reactions. According to the growth conditions, the cytoplasm can be more or less active, if the cell is under light or in the dark, respectively. In fact, in the dark, mitochondria, cytoplasm and chloroplast have similar activity [34].
Figure 2.1: Schematic illustration of C. vulgaris structure and different organelles. Adapted from [1].
Each mitochondria contains the respiratory apparatus, providing CO2at all times, and genetic
information. The cell contains a single chloroplast with a double membrane [35]. This organelle has considerable metabolic activity, starch granules and some lipid globules can accumulate inside, especially under stress conditions. In the presence of light, NH4 is imported to this
organelle to be used in amino acid and nucleotide biosynthesis, instead in heterotrophy conditions is released into the cytoplasm to be used in other pathways [34]. Chlorophyll and carotenoid pigments are also located in the chloroplast, the most abundant pigment in C. vulgaris is chlorophyll. Carotenoids act as an accessory pigment for better light catch, which is trapped and transferred to the photosystem. A carotenogenesis process can be induced in response to stress conditions like nutrient starvation or environmental conditions [36]. Besides these pigments, C. vulgaris is enriched in other bioactive compounds, namely phenolic compounds. Together they are resposible for the radical scavangening capacity of microalgae. Phenolics comprise a structurally diverse group of components, including simple phenols, phenolic acids, flavonoids, tannins, and lignans [37]. During the quenching mechanism of radicals, one polyphenol molecule is able to quench two radical molecules. Another promising product obtained from Chlorella spp. is the Chlorella Growth Factor (CGF) which is a water-soluble extract that contains free amino acids, peptides, glycoproteins, vitamins, minerals, nucleic acids and other components [38]. Studies concerning the beneficial effects of CGF, show its ability to promote tissue regeneration, cell growth, enhancement of immunity and enhance the lifetime of tested animals [39, 40].
Microalgae cell walls are very complex, and their exact composition may vary during growth and between species. The cell is usually surrounded by a thick and rigid wall that
preserves its integrity and acts as a protective barrier. However, many biochemical variances are observed within Chlorella intraspecies. Under stress conditions, the wall gets more rigid and the chloroplast starts to go back to the proplastid stage with accumulation of lipid bodies in the cytoplasm at the same time. The dominant polymer is the amino sugar glucosamine, in 10 to 15% [41]. The sugar composition is a mixture of rhamnose, galactose, glucose, arabinose, mannose, xylose, fructose and galactose, of around 25%. Protein make up 6 to 10% and uronic acids constitute 15 to 20%. The wall constitutes a barrier to compounds extraction and has been considered an obstacle [29]. Thus, numerous researchers have employed cell-disruption techniques prior to extraction, to break the cell wall. The main challenge is to find a technique that overcomes its intrinsic variability.
2.1.2
Primary composition
2.1.2.1 Proteins
Proteins play a very important role for the microalgae potential since, they can act as an alternative protein source [42]. C. vulgaris is a high-quality protein source, capable of synthetizing essential and non-essential amino acids [33]. The total protein content of algae is difficult to measure because of the problems encountered in protein extraction from the cells. It can vary during the growth phases, proteins from Chlorella spp. present water-holding capacity, foaming capacity and foaming stability, amog other proprierties. The morphology and chemical composition of the microalgae also influence protein solubilisation that can be enhanced under alkaline conditions [43]. Thus, besides having applications in human nutrition, health and feed they are also promising in chemical industries [32].
2.1.2.2 Lipids
Lipids are soluble in non-polar solvents and not soluble in water. It is a very large and diverse group of molecules, including, among others, neutral lipids (triaclyglycerols (TAGs)), polar lipids (phosphoglycerides, glycosylglycerides) and nonpolar lipids (wax, esters, sterols, acylglycerols). In normal conditions, fatty acids are synthesized, for esterification into glycerol-based lipids. They can be saturated or unsaturated. Unsaturated fatty-acids play a very important role for food and feed. The major membrane lipids are polar lipids, in addition to a structural function some polar lipids play an important role in cell signalling pathways [1]. In microalgae, under unfavourable conditions, lipid biosynthesis pathways shifts to the formation of mainly TAGs that can accumulate up to 58%, with no structural role, according to the specie, but
with energy storage role, which can be easily catabolised to provide metabolic energy [44]. After their synthesis, they are deposited in the form of lipid bodies in the cytoplasm [45]. The fatty acid profile changes according to the current growth conditions. Besides TAGs, these organisms are also important sources of long-chain polyunsaturated fatty acids (LC-PUFAs). They can be used as feedstock for biofuels, edible oils and other applications.
2.1.2.3 Carbohydrates
Starch is the most abundant polysaccharide in C. vulgaris, working as an energy storage function, being located in the chloroplast. This specie is rich in other sugars that have a structural function and are present in the robust cell wall, like rhamnose, arabinose, glucose, galactose and mannose. b-1,3-glucan is another polysaccharide present in this microalga with multiple health benefits [16].
Table 2.2 provides a comparison between the protein, carbohydrate and lipid content present in several sources and organisms used for food and feed.
Table 2.2: Comparison between the protein, carbohydrate and lipid content present in several sources and organisms used for food and feed, in percentage of dry matter. Adapted from [16].
Commodity (%) Protein (%) Carbohydrate (%) Lipid
Bakers’ yeast 39 38 1 Meat 43 1 34 Milk 26 38 28 Rice 8 77 2 Soy bean 37 30 20 Dunaliella salina 57 32 6 Scendesmus obliquus 50-56 10-17 12-14 Spirulina maxima 60-71 13-16 6-7 Chlorella vulgaris 51-58 12-17 14-22 2.1.2.4 Pigments
During photosynthesis, microalgae are exposed to high concentration of oxygen and free-radicals. Its protective antioxidant systems, namely the photosynthetic pigments and phenolic compounds (previously discussed in Section 2.1.1) . The pigments classified under three groups: carotenoids, phycobilins and chlorophylls. All of the three have multiple therapeutic proprieties and are currently used as natural dyes. In high light intensity, chlorophyll
a and other pigments are damaged and decrease, while carotenoids serve as photoprotetive agents, increase and can accumulate [46]. Carotenoids are a large family of pigments, with high antioxidant capacity and among the over 600 known not all of them are being commercially used. Certain carotenoids can act as provitamin A, which can be converted to vitamin A [47]. These pigments have applications in pharmaceutical industries, medical research, cosmetic industry, food and feed. They are present in about 0.1 to 0.2 % of DW. Phycobilins occur covalently bound to proteins with molecular masses of 30 to 35 kDa, these complex are known as phycobiliproteins, and also have interesting proprieties such as anti-inflammatory activity, and are applied in cosmetic and food industry. They have high fluorescence quantum yield, making them very sensitive to fluorescent reagents and interest for research in immunology laboratories [16]. Chlorophylls are green pigments, with chlorophyll a being the most abundant, widely used in food industry as colorants, and with increasing interest in medical field [48]. In Figure 2.2, there is the absorption spectrum of C. vulgaris, obtained through the sum of the individual spectrum of each extracted pigments, adapted from the study conducted by Mahmoud, G. et al., 2016 [49].
Figure 2.2: Absorption spectra of C. vulgaris algae, with: (1) the simulated spectrum, (2) spectrum of chlorophyll a (3) spectrum of chlorophyllb and (4) spectrum of carotenoids. Adapted from [49].
2.1.2.5 Minerals and vitamins
Vitamins are necessary in different metabolic steps for humans. There are 14 vitamins known: vitamin A (retinol), B complex [B1 (thiamine), B2 (riboflavin), B3 (niacin), B5 (pantothenic
acid), B6 (pyridoxine), B9 (folate/folic acid), biotin, choline and B12 (cyanocobalamine)] and vitamins C, D, E and K [1, 16]. C. vulgaris vitamin content varies during growth phases, culture conditions and between strains. In general, it contains high quantities of folates, and vitamins from the B complex [1], like cobalamin (vitamin B12) and folic acid [50].
Minerals are also very important in the human daily intake, they participate in transportation reactions, keep the balance between cells, play a very important role in protein synthesis, nerve impulses and muscle contraction. Generally, the minerals content are determined after incinerating the biomass and then analysed by atomic absorption spectrophotometry [10, 51]. This microalga is rich in Sodium (Na), Potassium (K), Calcium (Ca), Magnesium (Mg), Iron (Fe) and Phosphorus (P), with trace amounts of Chromium (Cr), Copper (Cu), Zinc (Zn), Manganese (Mn), and Selenium (Se), as presented in Table 2.3. The mineral content from C. vulgaris is sufficient to meet the recommended daily intakes for an adult, for example, 40 g of this microalga are required to fufil the reccomended dietery allowences for P. Whereas, the daily consumption of 4 g is enough to meet the recommended dose of Fe [52].
Table 2.3: Mineral element content from C. vulgaris in mg/100g DW. Adapted from [52].
Specie Na K Ca Mg Fe P Cr Cu Zn Mn Se
Chlorella 1346.40± 49.92± 344.30± 593.70± 259.10± 1761.50± 0.02± 0.06± 1.19± 2.09± 0.07±
vulgaris 0.18 0.09 0.07 0.12 0.04 0.02 0.01 0.10 0.07 0.15 0.03
2.1.3
Production conditions
C. vulgariscan grow on heterotrophic, autotrophic and mixotrophic modes, however there is specificity among strains. In fact, some strains of this microalga can only grow in the dark, while others can only grow in the light [53]. At the end of this section, a comparison between different extraction yields from several Chlorella species, under different growth conditions is presented in Table 2.4.
2.1.3.1 Heterotrophy
Heterotrophic growth takes place in stirred fermenters, in the dark with organic carbon sources to stimulate growth, like glucose, acetate, glycerol or hydrolysed carbohydrates. In heterotrophic systems the need of light is overcome, scale-up is easy, cell density is higher than in autotrophic cultures and increases in a short-period of time, so harvesting becomes cheaper. Also, the culture conditions can be properly controlled. A higher cell density and productivity can be
achieved with fed-batch or continuous strategies [54]. However, the price and availability of organic carbon sources increases the overall value of the process, for instance the cost of glucose can contribute about 80% of the total cost of the medium. Overall, Chlorella spp. fermentation is only economically favourable for high-value products but not commodity low-cost products [55]. However, new strategies have been studied to determine the feasibility of large-scale cultivation under heterotrophic conditions. Xiufeng and colleagues, in 2007 [56], were able to obtain a lipid content up to 55.2% using C. protothecoides under heterotrophy, this value was three times higher when compared to the value obtained from autotrophic conditions. Besides, they also suggest different ways to lower the cost of this cultivation method, highlighting the possibility to perform genetic modifications on critical enzymes and strains, and also to continue the optimization strategy in a bioreactor for Chlorella spp.. Hence, this strategy could be a promising way to establish biodiesel production from heterotrophic Chlorella spp. as a novel technique.
2.1.3.2 Photoautotrophy
During autotrophy through efficient photosynthesis, solar energy is converted to chemical energy, and atmospheric CO2 is fixated. The light-dependent reactions, produce Adenosine
Triphosphate (ATP) and Nicotinamide Adenine Dinucleotide Phophate (NADPH), that are used for CO2fixation. C. vulgaris can use CO2or carbon dioxide in the form of H2CO3. In large-scale
production to maintain a high-cell density, (since the atmospheric air contains only 0.04% of CO2), usually a supply of air enriched in CO2at about 1-5% is provided. During the culture, the
levels of CO2must be under strict control at all times, because higher levels can cause a decrease
in pH causing growth inhibition [57]. Photosynthesis efficiency also depends on the light intensity. C. vulgaris exhibits a light saturation level and above that limit there is photoinhibition and photosynthesis decreases with time [36, 58]. In response to high light intensity chlorophyll a and other pigments decrease, while carotenoids that serve as photoprotetive agents, increase. The limit is estimated to be around 80 to 400 mE.m-2.s-1 on a per cell basis, and even though solar incident intensity is higher (2500mE.m-2.s-1), most of the light is lost as heat, and in fact the maximum efficiency of the photosynthesis is not achieved. In autotrophic cultures is difficult to reach a high density of biomass since light penetration is inversely proportional to the cell concentration, and mutual shading of cells can also cause light insufficiency [53]. Even though, autotrophic growth still provides several advantages, since microalgae can grow at the expense of inexpensive natural resources, which contributes to global CO2reduction. Chlorella spp. can
2.1.3.2.1 Open pond system
Open pond systems are the most common way of large-scale production and also the cheapest. They can be natural ponds when there are suitable climatic conditions and enough nutrients or artificial ponds. Artificial ponds localization is well thought out, usually these large-scale production systems are built near heavy industries with carbon dioxide production that can be absorb by the cultures. Open ponds include circular ponds and raceway ponds. Circular ponds are the oldest culture system and are widely used for Chlorella spp. mass cultivation in Asia [46]. However, they require high-energy consumptions to guarantee efficient stirring and expensive concrete structures. Raceway ponds are more famous worldwide for large-scale production. They consist of a closed loop with recirculation channels, stirring is assured with a paddlewheel in continuous operation. These systems are cheaper to build and operate, and usually used for production of low-value products, they can be construct in concrete or glass fiber. In any of these systems, the cultures are kept no more than 30 cm from the surface to allow sunlight penetration [57]. However, open pounds are susceptible to microorganism contaminations, weather variations, high evaporation rates, low productivity rates, and CO2 and
temperature control is very difficult to perform [59]. Even though open ponds are cheaper to construct and maintain, they present clear disadvantages that are overcome in closed systems. 2.1.3.2.2 Closed systems
Closed systems like photobioreactors (PBRs) are reactors in which microalgae are kept in a closed-culture environment. They are made of transparent materials, so that microalgae have access to sunlight, with a large ratio of surface area to volume. The cultures are kept longer since the danger of contamination is much lower and all the conditions are better controlled here, thus biomass productivity increases, and harvesting costs are lower, contamination is easier to prevent, and this way more species can be produced than open ponds [46, 57, 60]. This type of system, under controlled environment can give consistent high-purity products [33]. PBRs can be classified into tubular, vertical column, and flat reactors [1]. Tubular design is the most popular one, consisting of an array of transparent tubes that can be horizontal to each other, vertical or in loop.
Allmicroalgae during the restrict autotrophic scale-up, uses a green-wall (GW) system, see Figure 2.3, before the tubular PBRs. The unit has the capacity to cultivate simultaneously 12 GW panels of 1000 L each. GW consists of a transparent bag located on a rigid frame. These type of flat reactors have automatic temperature and sunlight control, they are vertically disposed with a
large surface area exposed to illumination. After the GW the cultures are transferred to horizontal tubular PBRs. The unit has a capacity of 19 PBRs all design for maximum productivity per unit area [6].
Figure 2.3: Green-wall structure implemented in the Allmicroalgae unit.
2.1.3.3 Mixotrophy
The third possible growth is mixotrophic that combines both autotrophic and heterotrophic, so the intake of both CO2 and carbon sources in the presence of light are perform. C. vulgaris
grows better in mixotrophic conditions (Figure 2.4), if the light intensity does not surpass the light saturation level, growth is stimulated, and with an external carbon source higher biomass concentration is reached, but also higher compounds productivity [53]. In the study conducted by Yanna et al. [53], using the C. vulgaris #259 strain, the mixotrophic strategy led to better results in cell density and lipid productivity compared with other growth modes. In fact, both glucose and glycerol had inhibitory effects at high concentrations. The maximum biomass density was achieved under light for 6 days with 1% (w/v) of glucose.
As afore mentioned in the begginig of these section, there is a vast specificity among Chlorella spp. cultivation mode and not all strains of C. vulgaris are mixotrophic. Also, since high concentrations of the carbon source can have inhibitory effects, is imperative to evaluate each strain individually. To keep the concentration of the organic carbon low in the medium usually a fed-batch operation is preferred, the carbon source is provided independently of the other inorganic nutrients.
they become highly susceptible to bacterial and fungal contaminants, and the high cost of the carbon source is also to be considered [57]. Therefore, closed PBRs are preferred to open ponds, with acetate feeding being employed to avoid contamination [61]. The addition of acetic acid is also advantageous since is less expensive [62]. To reduce the price of the carbon source, crude glycerol that is a by-product of the biodiesel industry can also be an alternative, and is now being used as carbon energy [63]. Allmicroalgae adopted a mixotrophic strategy [6].
Figure 2.4: Time course of continuous culture of Chlorella spp. first under heterotrophic, then mixotrophic condition, in a vertically flat culture bottle with acetic acid as carbon source. From [62].
Table 2.4: Comparison of extraction yields in dry weight (DW) of different compounds from Chlorella species, under different growth conditions
Specie Product Extraction/Culture conditions Yield References (%DW)
Protein content
Cultivation in closed system, harvested after phase of
78.30 [32] C. maximum growth, cell disruption performed from
pyrenoidosa ultrasonication. Three-phase portioning used to separate, purify and concentrate proteins.
Protein content Semi continuous culture system, 48.00
[52] C. always aerated with a air pump without
vulgaris
Lipid content additional CO2supply, 13.32 and continuous light at 20oC.
C. Protein content 38.00
[64] vulgaris Lipid content Autotrophic conditions in 5.00
(green) Mineral content in airlift bioreactors, at low 24.00 Total pigments light conditions, and 25oC 1.20
C. Protein content carotegenesis process. 12.30
vulgaris Lipid content Harvest trough flocculation 28.00 (carotenogenic) Mineral content and centrifugation. 35.00
Total pigments 1.30
Chlorella sp.
Lipid content Mixotrophic cultivation using spent media rich in 58.00 [65] MJ 11/11 VFA from acetogenic dark fermentation. Air-lift reactors.
2.1.4
Harvesting and processing
The high-energy input during harvesting of the biomass is the main challenge to turn microalgae economically viable. Their production is still very costly so they are economically viable when used for high-value applications, becoming unfeasible for low-value applications.
To separate the biomass from the culture medium, different strategies can be applied. It must be considered the microalgae small size, their low concentration in the medium, equipment and energy consumption of the process and finally the cultivation mode [14].
It is not an easy task, usually one or more solid-liquid steps are applied for a more efficient harvesting, they include steps for biomass concentration, followed by drying. The most used techniques for concentration are centrifugation, flocculation and filtration.
After separation, the biomass must be processed quickly, in the most economically and efficient way. Because microalgae biomass has a very high content of water the evaporation process is quite expensive and stands for 75% of the total processing costs. Even though, the choice of the proper processing is highly specific and the main methods used include spray-drying; freeze-drying (lyophilisation) and sun-drying [66].
2.1.4.1 Centrifugation
Centrifugation is a well-established technique between the main Chlorella spp. producers worldwide. Is efficient, fast, and feasible for large volumes.
2.1.4.2 Flocculation
When dealing with microalgae, the usually used chemical flocculants would affect the quality of the biomass. Auto flocculation results from interactions between the product and other microalgae or bacteria [67], thus is a time consuming strategy. It can be accelerated by adding a base. Until the limit of pH 11, cell lysis is not considerable since the percentage is lower than 15 and the loss of useful bioproducts is minimal. This mechanism depends also on high concentrations of Mg2+ from hydrolysed Mg(OH)2, which precipitates attracting with it the
negatively charged microalgal cells [68, 69]. 2.1.4.3 Filtration
Conventional filtration is not suitable to separate the biomass due to its very small dimension; therefore ultrafiltration or microfiltration is more efficient, and preferable. The choice between
them relies on the dimensions of the biomass, the type of filter used, the pressure employed, and the growth-phase of the culture, since that will affect the density and velocity of the flow. Membrane filtration is also becoming promising, using the correct membrane pore size, since is very efficient in retaining the biomass and culture contaminants, thus the medium can be recycled without further treatment, and even though it requires qualified technicians the energy consumption of the process is lower than overall spent in centrifugation [70]. In Allmicroalgae the harvest is done using cross-flow filtration with membranes, a novel and promising technique well adapted to industrial scale [6].
2.1.4.4 Spray-drying
Spray-drying is in general the preferable method, consisting of generating droplets through atomization that are in contact with hot air in a large vertical chambers. Large droplet surface area and small droplet size ensure high surface contact, so that drying is achieved in seconds. A cyclone separates the gas phase from the solid biomass particles, that are furtherly collected and packed in a controlled atmosphere. This dewatering process has many advantages, such as the continuous operation mode, low residence time, low effective drying temperature and since the product come as powder, it requires no further size reduction. In Allmicroalgae Chlorella sp. is spray-dried [6, 46, 66].
2.1.4.5 Freeze-drying
In freeze-drying, or lyophilization, the biomass is frozen and afterwards sublimed by a slight increase of the temperature without defrosting. This process, prevents biomass degradation, however, the process is very expensive for industrial use. This approach is more suitable for drying microalgae in research laboratories, for low-volume products [71].
2.1.4.6 Sun-drying
Sun-drying is by far the oldest technique applied and the cheapest, relying on the use of solar energy to dry the biomass. However, this approach is completely dependent of the weather conditions so it is not feasible for producing biomass for human diet [46, 72].
2.1.5
Microalgae applications and market
Microalgae are being more and more used in very distinct areas, and the concept of biorefinery of microalgae developed. The biorefinary takes advantage of the various components
in the biomass to improve its value and optimize the process. From the total lipid content, w-3 fatty acids and other highly unsaturated fatty acids are of extreme importance for many marine animals and find their applications in aquaculture, animal feed and nutraceutical industry [73–75].
Biofuels derived from microalgae represent the third generation of biofuels and are being considered an alternative to the previous generations of biofuels [76].
Microalgae production does not cause any type of overexploitation of the environment, like deforestation nor affects the food supply. In fact, around 30% of the current world production of algae goes to feed industry [73, 77]. However, incorporation of powdered microalgae, or in other processed form is usually avoided for its high economical value, and fresh algae incorporation is being developed and studied in replacement.
Proteins after extraction are also used in animal feed, food or pharmaceutical industry. They have increased potential in diverse industries, since they show unique features such as film and foaming capacity, gel forming ability and antimicrobial activity [78]. The pigments synthesized by microalgae are also a high-value product, with antioxidant power, and anti-inflammatory proprieties, therefore suitable as nutraceuticals and in pharmaceutical industry. Carotenoids are very strong antioxidants, preventing cellular oxidative damage. Studies conducted reveal the possibility of these pigments to also have activity against cancer, aging, heart attacks and coronary artery disease [79]. Astaxanthin is a high-value carotenoid, with an antioxidant activity ten times higher than other carotenoids, is the primary pigment responsible for the pink colour of salmon and other marine animals, has a protective activity against UV light, and acts on improving the immune system. Its clear benefits either as strong pigment and nutraceutical increase its value and commercialization [80]. Some microalgae extracts are being incorporated in the skin care market, their extracts are found in face and skin products, and also in products of UV protection [81].
2.2
High Pressure
The natural extracts of microalgae, in the market, come in a variety of forms such as powdered, tablets, capsules, or liquid form. Water extracts are complicated to classify, they are rich in different intracellular compounds, thus have increased value. Different extraction techniques can be applied, however not all of them are favourable and efficient in terms of yield, environmental impact and costs. Different extraction technologies, such as super-critical CO2,
used to enhance extraction yields [82, 83]. Nowadays, the techniques chosen and applied are also exposed to the consumers opinion and increase concerns about health benefits and risks. Extractions technologies are continuously being developed and studied, aiming short operating times and better extraction efficiency. The ideal extraction method should be quantitative, non-destructive and time-saving. Heat treatments when applied to sterilize or pasteurize food usually compromise food sensory and nutritional value. Excessive heat could in fact lead to protein denaturation, non-enzymatic browning, loss of minerals, vitamins and volatile compounds. Non-thermal alternative technologies have been investigated during the last decade [84].
Consumers want fresh, healthy, minimally processed food, with all its bioactive proprieties conserved, including flavour and taste, as well as prolonged shelf-life [85, 86]. For this, food industry needs to be in continuous research. HPP is an emerging non-thermal food pasteurization technique. Liquid and solid foods, with or without packaging are exposed to high hydrostatic pressure, ranging from 100 to 1000 MPa, during a certain period of time, at cold or even at elevated temperatures, see Figure 2.5. Commercial exposure times can range from seconds to more than 20 minutes. HPP shows great potential, since has reduced effects on taste, texture, nutritional and quality parameters in comparison to conventional thermal processing [85, 87]. Also, pressure at a given position and time is the same in all directions, transmitted uniformly, independently of the size, shape and food composition [84, 88]. In addition, it can be used to create ingredients with novel or improved functional properties [89]. HPP has been widely used not only for food processing, but also in ceramics, pharmaceuticals and civil engineering [87]. The use of HPP for the extraction of bioactive ingredients is a promising and novel technique [90]. During the process, according to the mass transfer theory, cells have their permeability increased, solubility also increases, as well as the diffusion of secondary metabolite, therefore the intracellular metabolites become more accessible in the medium [91, 92].
Figure 2.5: Illustration of the pressures used to process food. Adapted from [93]
.
2.2.1
History
From previous works with microorganisms, Hite had demonstrated that microorganisms were affected by pressure, and in 1899 subjected milk to high hydrostatic pressure, and found that the souring process was retarded. Also, a 4-log reduction in microbial total count was found, only with a treatment at 700 MPa for 10 minutes, at RT. A shelf-life extention of raw milk, for another four days after one hour of treatment at 600 MPa was observed, also at RT. In 1914, high pressure applications were extended for preservation of fruits and vegetables. The biotechnological boost of HPP technology happened in Japan in 1990, when the first food product exclusively processed by HPP was sold, consisted in an acidic fruit jam. Since then HPP has been a continuously studied and developed technique [89, 94].
2.2.2
Equipment description
The prototype used by Hite was very primitive in comparison to the technology available nowadays. Through the years high pressure equipment has increased their volume and pressure capacity [89]. The process initiates when the food product is placed in a pressure vessel that will support the pressure applied, the volume of the vessel may vary from less than 100 mL to more than 500 L. The wall thickness of the vessel is determined by the maximum working
pressure and the number of cycles for which the vessel was designed for. There is a closure to sealing the vessel and a device for holding the closure in place while the vessel is under pressure. Pressure fill systems fill the chamber with a pressure-transmitting fluid and are responsible to also remove the air when the system is closed. The liquid is chosen accordingly to its viscosity under the pressure and temperature applied. Usually the medium is water, but can also contain ethanol, glycol or other substances. In addition, there is a pressure-generating device to build up the pressure and a product-handling system to transfer it to and from the pressure vessel. Sometimes is present a heating and cooling system. Industrial processing can be either a batch or semi-continuous process. However, in food processing industries batch production is preferable. Therefore, the product is placed in the loading basket, once in the pressure chamber the vessel is closed and filled with the pressure-transmitting medium, after the pressure reaches the set conditions, they are maintained until the end of the cycle. After the set holding time is over, the system is depressurized, the vessel opened and the product unloaded. The total time of pressurization, holding time and depressurization is the cycle time. [95, 96].
Hiperbaric, Avure Technologies and MULTIVAC are the suppliers of HPP equipment. Hiperbaric is the leading manufacturer of HPP equipment for the food industry [97], with industrial units operating all over the world. Hiperbaric has six equipment types; the 55, 135, 300, 420, 525 and 1050 (tandem unit). In the present work was used the Hiperbaric 55, with a vessel volume of 55L, maximum pressure of 600 MPa, a maximum filling capacity of 50% and with a production rate of 255 kg/h, illustrated in Figure 2.6.
Figure 2.6: Illustration of the Hiperbaric55 equipment. From [98]
2.2.3
Process and principles description
Two principles stand beyond the effect of pressure on food products: Le Chatelier’s principle, and the Isostatic principle. The first one, states that when a system in equilibrium is perturbed, it tends to return to another equilibrium state, i.e., any phenomenon that results in a volume decrease will be accompanied by an increase in pressure, and vice-versa. The Isostatic principle of pressure diffusion states that pressure is instantaneously and uniformly transmitted to the product in the vessel, regardless of its shape and size [87, 99]. During the process, inevitably, temperature will increase because of the work derived from compression through adiabatic heating, around 3 oC per 100 MPa, see Figure 2.7. Water temperature increases and so does the food, depending on its composition. Foods cool down to the initial temperature during decompression if no heat is lost or gained from the walls of the pressure vessel during the holding time under pressure. Thus, it is essential to guarantee an uniform temperature at the beginning of the process to achieve a uniform temperature increase during the compression process [100] (Figure 2.7). The material of the package used during the processing needs to be suitable to the working pressures, since is under a considerable stress and distortion [101].
Figure 2.7: Water temperature increasing during the adiabatic compression. This increase is a function of the initial temperature. From [91]
2.2.4
Impact of HPP
2.2.4.1 Effects of pressure on microorganisms
Pressure higher than 150 MPa are usually considered lethal for most microorganisms, however these effects largely depend on the type of strains, culture media, cycle time, working pressure and others. High pressure interacts differently with biological systems, acting simultaneously on tissues morphology, cell organelles, proteins, and membrane structure and permeability [102]. However, with evolution, organisms developed specialized strategies to perform its functions under various environmental conditions. The membrane is the first site of pressure damage. The perturbation induced affects the phase changes of phospholipid bilayers. The melting temperature of lipids increases, in a reversible manner, by more than 10oC per 100
MPa. The final membrane state and phase transition depends on the water content and processing conditions [103]. Physiological processes mediated by membrane proteins are also compromised even by non-lethal levels of high pressure, since the membrane structure is perturbed [103]. Around 30-50 MPa, gene expression and protein synthesis can be affected [102]. Usually, because of the presence of peptidoglycan in the layer of the gram-positive cell wall they are considered more resistant to pressure than gram-negative bacteria. Bacterial spores inactivation can be very difficult, because they are resistant to high pressure [104]. Sometimes the first cycle only triggers spore germination, spores can withstand pressures higher than 1000 MPa. To eliminate the majority of them usually, a two-stage process is proposed. The first pressure cycle induces spore germination, and the second one kills them [105]. Table 2.5 shows examples on how different treatments, applied to different microorganisms produce distinct results.
There are evidences that the resistance of cells during stationary-phase is higher than during exponential growth phase. On the study developed by Ma˜nas, Pilar et al., 2004 [106], the pressure for the onset of rapid inactivation of Escheichia coli was between 100 and 200 MPa for exponential growth phase, also under this conditions there was complete loss of plasmolysis, loss of RNA and clear perturbations on membrane structure. Whereas, only at 500-600 MPa the cells in stationary phase were inactivated, also there was just a partial loss of the plasmolysis, and the other effects observed before where absent. Also, the chemical composition of the substrate can have a significant effect on the organism response under pressure, for example, a higher cation concentration can have a protective effect, as well as a lower water activity [104].
Table 2.5: Effects observed in different microorganisms under high pressure treatment: variable pressure, temperature and cycle time.
Organism Life Conditions Results Reference
Dominance P(MPa) T(oC) Time(min)
Sargassum
Eukarya 300 25 5-5.5 Better extraction yield of [90]
muticum polysaccharides and bioactivity
Eukarya
100 25 10 Nuclear membrane decomposition
[107]
300 25 10 Release of metal ions
Saccharomyces
400 25 10 Mitochondria, endoplasmatic reticulum
cerevisiae and vacuole deformation or disruption
500 25 10 Cellular disruption
200 -20 180 Severe alterations in the cell’s inner structure Salmonella
Bacteria 340 23 10 Inactivation in log4 cycles [108] senftenberg
Bacteria 400 - 10
Damage the membrane of the majority
[109] bacterial population.
Listeria Increase in the intracellular volume,
monocytogenes appearance of bud scars
on the surface of the cells. Enzymatic reduction.
Bacteria
400 25 5
From rod-like morphology to nodulation
[110] of cells. Empty cavities between the
Lactobacillus cytoplasmatic membrane and outer cell wall. viridescens
600 25 5
Majority inactivation of the population. Fibrillar regions of denatured DNA .
