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Technical field of the invention

This invention relates to the field of the production of chemicals from renewable sources, in particular algae. It relates in particular to a process for the production of starch from microalgae. The process is subdivided into a stage of autotrophic cultivation and a subsequent stage of heterotrophic cultivation on a substrate such as for example whey with added phenols. Other algal components are also extracted in addition to the starch.

Known art

World production of chemicals is currently strongly dependent on fossil sources and is accompanied by problems of an economic and environmental nature associated above all with the emission of pollutants and dependency on a non-renewable source that is currently becoming exhausted. In the last few years research has been directed towards the utilisation of biological sources for the production of chemical compounds and energy in order to overcome these problems.

These sources have the advantage that they are renewable and can be used in a way involving a lower environmental impact.

The use of biomass is particularly significant in this context, and this can be classified into first, second and third generation biomass.

From biomass it is possible to produce a number of chemicals of great industrial utility such as for example bioplastics, various types of biofuels (biodiesel, bioethanol, biogas), dyes, antioxidants, solvents, etc.

Among the various products which can currently be obtained through the use of biomass, starch accounts for most of the worldwide market in biodegradable materials produced from renewable sources. Starch is currently obtained from dedicated agricultural crops (maize, potato, grain, etc.), that is using first generation biomass. Second generation biomass comprises wastes, mainly derived from agriculture, and has the advantage that it does not compete with the food production system, but the disadvantage that it is in hmited quantity and widely dispersed over the land surface, giving rise to problems associated with collection and transport. Second generation biomass can be used for production of various kinds of polymers such as for example polyvinyl chloride (PVC), polyurethanes (PU), polyacrylic acid (PAA), polyvinyl acid (PVA) and polyesters (PE).

Microalgae are a third- generation biomass and have the advantage that they do not compete with the food production system and have high productivity per unit surface area, prompting increasing interest in industry for the production of chemicals, including polymers.

Despite the fact that great improvements have been made in recent years, the production cost of algal biomass in autotrophic cultivation is still too high for most applications. At the present time the lowest cost which can be achieved in the production of microalgal biomass is approximately

2-2.5 euro/kg (Acien et al. 2012). There are two main strategies that can be used to make a further reduction in these costs - integration of cultivation with the use of organic substrates and the development of biorefineries, that is the production of various products from the same biomass. Various strategies for cultivating microalgae have been developed with this in view, to increase the production of useful components. Various studies have shown that algae cultivated under heterotrophic conditions, using organic substrates, can achieve higher productivities than under autotrophic conditions (Richmond 2004). There is increasing interest in the use of wastewater of various kinds for the cultivation of microalgae (Zhou et al. 2014). These are available at zero or negative cost, contain high concentrations of nutrients, including organic substrates, and can therefore be used for the heterotrophic cultivation of microalgae.

However, when organic substrates are present in cultivation media the risk of contamination by fungi and bacteria, which can compromise algal growth, increases appreciably. Although the use of wastewater on the one hand offers an advantage in terms of reducing the costs of nutrients, on the other hand it increases the risks associated with contamination. Each wastewater in fact contains a characteristic flora of microorganisms, mainly bacteria, suited to growth under the characteristic conditions of the wastewater. It follows that the use of wastewater as such for the cultivation of microalgae without specific pre-treatments is often inappropriate because the contaminants present in the wastewater quickly tend to dominate in the reactor in comparison with them, because doubling times are generally much shorter. There are many scientific works in the literature describing processes for cultivation of microalgae in wastewater. However the latter are generally pre-treated through chemical and physical sterilisation processes to overcome the contamination problem. These processes result in a considerable increase in production costs, and are difficult to apply to industrial production (Wu et al. 2014).

Among wastewaters of natural origin, cheese whey, a waste deriving from the cheese industry, offers a good source of nutrients for the growth of microalgae because of its high content of lactose (40-50 g/L), phosphates and other mineral salts (Prazeres et al. 2012). Various studies have demonstrated that some microalgal species are able to grow on whey, with improved outputs of biomass and bio-oil (Abreu et al. 2012; Espinosa-Gonzalez et al. 2014; Girard et al. 2014). However in these studies good algal growth has only been made possible through the adequate control of contaminants, performed in the laboratory through pre-treatments to sterilise the whey and growth conditions in a sterile environment. Whey is characterised by the presence of a high bacterial load which has the ability to grow using the nutrients present within it (lactose). These are more competitive than microalgae on this substrate and without suitable procedures to control them (such as for example sterilisation) algal growth on whey would be impossible.

Although it is widely known from the literature that microalgae are capable of degrading phenols (Lika & Papadakis 2009; Papazi & Kotzabasis 2007; Lima et al. 2004; EUis 1977; Pinto et al. 2002), these compounds also have an antimicrobial effect upon them. These effects are manifest not only on microalgae but also on other microorganisms such as bacteria and fungi (Dermeche et al. 2013), giving rise to inhibition of their growth. Under the cultivation conditions tested in the literature, when microalgae are cultivated in the presence of phenols, the latter show a negative inhibitory effect on their growth. Mainly synthetic media or oil mill wastewater (OMW) have been tested as sources of phenols. Oil mill wastewater is characterised by the presence of a large quantity and variety of phenol compounds (Leouifoudi et al. 2014). In the case where microalgae are cultivated in oil mill wastewater, the pre-treatments yielding the best results have been those capable of largely removing the concentration of phenols present in them, such as for example activated carbons and treatments with hypochlorite, showing that if the phenols are not removed they have a negative effect on microalgal growth (Markou et al. 2012; Hodaifa et al. 2012). Comparing microalgae grown on OMW under heterotrophic conditions with the same microalgae cultivated under autotrophic conditions it has been found that the microalgae cultivated using OMW have a higher carbohydrate content per cell (Di Caprio et al. 2015). This increase is due to the presence of high concentrations of sugars and organic acids in the OMW, which stimulate the build-up of carbohydrates. It is however significant that although the quantity of starch per cell increases, there is no increase in the output of starch from cultivation in the reactor (quantity of starch produced per unit time and volume) because of the lower cell growth - the cell starch content is higher but the number of cells is lower, and thus overall the output of starch is lower. This is due to the negative effect of the phenols, which arrest microalgal growth at an early stage, reducing the output of carbohydrates. In general therefore analysis of the literature demonstrates that microalgae are capable of degrading phenols, but that this capacity is not generally sufficient to overcome the inhibiting effects which the phenols have on their growth, particularly when wastewaters containing phenols which in themselves contain suites of bacteria adapted to the phenols, which rapidly prevail over the microalgae, are used. Under the conditions tested in the literature the phenols present in wastewaters demonstrate adverse effects on the cultivation of microalgae and represent a problem for their cultivation.

The problem of obtaining efficient cultivation of microalgae has also not been resolved by the following documents.

For example, in US 2011/0131869, US 2008/0160593 and US 2014/0045229, which describe processes for the cultivation of microalgae in two autotrophic and heterotrophic stages, controlled closed reactors have been used to reduce the problem of contamination, and even viruses capable of specifically killing the contaminating microorganisms have been used. Auxiliary activities of this kind are essential in order to prevent contaminating bacteria and fungi from prevailing during heterotrophic cultivation. Specific rates of growth which can be 10 times greater than those of the microalgae have in fact been demonstrated under these cultivation conditions.

The main disadvantage of this approach lies in the need to use specific procedures for managing viruses and finding specific viruses for each contaminating species which may potentially be able to grow under the conditions used.

US 2009/0298159, US 2011/0027827 and US 2009/0075353 describe similar one- or two-stage processes for the cultivation of microalgae, which provide for cultivation under heterotrophic or mixotrophic conditions. The methods used to control contaminants are not sustainable from the environmental and economic points of view. Conventional chemical and physical strategies have been used to control contaminants, but these are difficult to apply. Conventional strategies such as the use of high pressure filtration, high temperature heat treatments or chemical treatments appreciably increase costs and energy expenditure, especially if wastewater is used. The latter are in fact very much more difficult to treat using these processes because of the high concentrations of suspended solids and organic compounds of various kinds present in them. The use of antibiotics instead gives rise to appreciable problems of a health nature, due to the growth of antibiotic-resistant bacteria.

The production processes currently used on a large scale for the production of bioplastics deriving from starch are based on the use of dedicated agricultural crops (first generation biomass).

The main disadvantages of this type of bioproduction are:

Competition with the food production system, with consequent socio-economic problems.

The need for specific socio-economic assessments to assess the environmental, social and economic sustainability of first generation bioproduction facilities.

Low productivity in comparison with microalgal cultivation (third generation biomass).

The processes for microalgal cultivation (third generation biomass) reported in the state of the art have the following main disadvantages.

High costs associated with the control of contaminants through procedures requiring particularly high quantities of energy, such as heat treatments and filtration.

The use of chemicals (for example antibiotics) which are also difficult to manage in health terms.

The use of biological agents (such as viruses) requiring complex management procedures and continuous adjustments, being contaminant-dependent.

Poor scalability of the proposed processes, compatible with the economic sustainability of those processes.

The process described here is a process for the production of starch using third generation biomass which overcomes the main disadvantages of production processes from first generation biomass.

The proposed process constitutes a step forward in comparison with other processes based on microalgal cultivation reported in the state of the art in that it solves the problem of controlling contaminants through an economic alternative having reduced environmental impact which is simple to manage. A process in which phenols are not only a negative factor for microalgal growth, but a positive factor, controlling the growth of contaminants, is described here.

Summary of the invention

The invention relates to a method for the production of large quantities of starch from a microalgal biomass using a two-stage cultivation process. In the first stage (autotrophic stage) the microalgae are cultivated under autotrophic conditions in reactors exposed to air (or air enriched with CO2) and light (natural or artificial), while in the second stage (heterotrophic stage) they are cultivated under heterotrophic conditions. Heterotrophic cultivation is carried out in closed reactors without illumination using a source of organic substrate containing no phenols, such as whey. Between the autotrophic and the heterotrophic stages there is an intermediate stage of acclimatising the algae to phenols. Contamination is limited by adding phenols of various kinds, including for example those belonging to the classes of acid phenols, simple phenols and flavonoids, or phenols selectively extracted from the wastes and wastewaters of the olive oil production industry, the winemaking industry, the brewing industry and wastewaters from the cultivation of microalgae.

In particular the process according to the invention comprises the following stages:

A) Selecting a suite of algae which are resistant to phenols and capable of growing under heterotrophic conditions on an organic substrate;

B) Causing the microalgae to grow under autotrophic conditions;

C) Thickening the microalgae and acclimatising them to phenols;

D) Causing the microalgae to grow under heterotrophic conditions gradually adding both the organic substrate and the phenols over the course of the fermentation in a proportion which increases over time (fed-batch). These stages are followed by collection and drying of the algae and extraction of the starch and other products of value, such as oily products which can be used as biodiesel, or from which nutraceutical products such as carotenoids can be extracted.

Another object of the invention is selection of a suite of algae which are resistant to treatment with the phenols used to limit contamination. Yet another object of the invention is the selection of a suite of microalgae capable of accumulating a high starch content (in excess of 20% w/w in comparison with the weight of dry biomass).

Other objects will be apparent from the detailed description of the invention.

Brief description of the figures

Figure 1 shows a block diagram of the process according to the invention.

Figure 2 shows A) the build-up of starch measured for the strain Scenedesmus sp. during the selection stages. B) the change in nitrates in the course of growth during the cultivation of Scenedesmus sp.

Figure 3 shows A) the increase in the biomass concentration of Chlorella sp. cultivated under nitrogen-deficient conditions. B) the change over time in the starch content of the biomass of Chlorella sp. cultivated under nitrogen-deficient conditions.

Figure 4 shows the cell growth of Chlorella sp. during heterotrophic batch cultivation on only whey (triangles) and whey with added phenols (0.5 g/L) selectively extracted from vegetation wastewater (circles).

Figure 5 shows the production of Scenedesmus sp. biomass cultivated in batch and fed-batch heterotrophic reactors. Fed-batch condition: 10 mL of source of substrate containing 10 g/L of glucose, added daily up to the end of day 10, and 0.001 L of phenol stock solution (50 g/L) added daily up to the end of day 10. Batch condition: 1 g/L of glucose and 0.5 g/L of phenols present in the medium since the first day. In the data relating to the fed-batch condition the biomass concentration has been normalised with respect to the final volume.

Figure 6 shows the results of the purification of starch with NaOH solution, for both microalgal biomass as such and for biomass obtained after lipids have been extracted.

Detailed description of the invention

The following definitions apply within the scope of this invention.

Microalgae: microorganisms capable of living using a photosynthetic or autotrophic metabolism. In this document the terms microalgae and algae are used as synonyms. In this document the terms autotrophic and photosynthetic are used as synonyms.

Microalgal suspension or biomass: mixture of algae and growth medium.

Microalgal suite: a microorganism culture comprising various strains of algae belonging to the same species or different species. The suite is selected so as to be used for the purposes of the invention, as described below. In this document a suite of microalgae and strain of microalgae are used as synonyms.

Heterotrophic cultivation: cultivation in a reactor which is not exposed to light and contains organic substrates as a source of energy and carbon.

Autotrophic or photosynthetic cultivation: cultivation in autotrophic reactors in a growth medium containing water and mineral salts.

The reactors are exposed to air (or air enriched with CO2) and light (natural or artificial). In this cultivation CO2 is the only source of carbon and light is the only source of energy.

Autotrophic reactors: reactors designed to allow autotrophic cultivation, being constructed of transparent material (e.g. glass, polyethylene, polycarbonate) which allows light to pass through. Various geometries such as for example those of tubular, column or panel photobioreactors or open tanks may be used. In the case of open tanks there is no need to use transparent material.

Starch: polymer produced by the metabolism of microalgae and comprising glucose units bonded together by means of alpha-glycoside bonds having the generic formula (CeHi206)n (n > 20).

Organic substrate: by this term is meant any organic molecule which can be used by microalgae as a source of carbon and energy. In particular the following classes can be used: carbohydrates, organic acids, alcohols and polyalcohols, proteins and amino acids.

Nutrient deficiency: a condition of nutrient deficiency is considered to occur when nutrients reach a concentration of less than 85% of the initial concentration.

Growth medium: Growth medium is defined as being the sum of what is added to a microalgal inoculum in the reactor in the aqueous phase during the cultivation of microalgae. Growth medium is generically an aqueous solution in which salts and synthesised organic substrates and/or substrates originating from wastewater are dissolved.

Glucose equivalents: concentration of reducing sugars determined using the phenol, sulphuric acid and glucose method as a reference standard (Dubois et al. 1956).

This invention relates to the cultivation of microalgae through a two-stage process, mainly but not restricted to the production of starch. The process comprises a stage of autotrophic cultivation followed by a stage of acclimatisation to phenols and a stage of heterotrophic cultivation. It is characterised by the fact that the heterotrophic cultivation stage is carried out by adding phenols to the growth substrate, which can derive from wastewaters or wastes which are byproducts from the agri-food sector which do not contain or are substantially free from phenols, or which contain phenols in quantities < 50 mg/L. For example the substrates may be whey, by-products from the production of cane sugar and sugar beet, starch- containing wastes, wastes from various biotechnological types of cultivation including microalgae.

As will be described in greater detail below, microalgae which are specific for the production of starch under plant conditions are selected to maximise the output of starch during the heterotrophic cultivation stage.

Contrary to what has been described in the known art, in which costly techniques (chemical and physical) are used to sterilise cultivation media, the inventors have found that adding phenols according to an optimised protocol described below makes it possible to control the growth of contaminants under heterotrophic conditions without compromising microalgal productivity.

Thus antimicrobial agents of the phenolic type, of synthetic or plant origin, are added to the source of organic substrate which substantially does not contain phenols in order to reduce the growth of contaminants of bacterial and fungal origin.

The term phenols indicates molecules containing at least one aromatic ring attached to at least one hydroxyl group.

Phenols of any type may be used in the scope of the invention, and among the preferred are those belonging to the classes of acid phenols, simple phenols and Uavonoids (Tsao 2010). If these phenols are not pure synthesised products it is essential that they should be purified from their original matrices to prevent the joint inclusion of contaminants capable of growing in the presence of phenols in the growth medium.

Of the acid phenols the following are preferred: o-coumaric acid, p-coumaric acid, m-coumaric acid, ferulic acid, chlorogenic acid, sinapic acid, caffeic acid, salicylic acid, parahydroxybenzoic acid, vanillic acid, isovanillic acid, syringic acid, protocatechuic acid, gentisic acid, gallic acid, veratric acid, syringaldehyde, vanillin, 3 -hydroxybenzoic acid, 3,5- dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, pyrocatechuic acid, anisic acid, hydroxybenzaldehyde, acetovanillone, acetosyringone, methyl vanillate, methyl syringate, 4- hydroxyphenylacetic acid.

By the term "simple phenols" are meant phenols comprising a single aromatic ring substituted in various ways. Among these are preferred: tyrosol, hydroxytyrosol, homovanillic alcohol, hydroquinone, cresol, m-cresol, guaiacol, floroglucinol, phenetole, catechol, resorcinol, phenol, pyrogallol, hydroxyhydroquinone, 3,4-dihydroxyphenol ethanol.

Among the flavonoids the following subgroups are preferred: flavones, flavonols, flavanones, flavononols, neoflavones, isoflavones, chalcones, flavans, flavan-3-ol proanthocyanidine, and anthocyanidine. The following are preferred in particular: luteolin, rutin, luteolin- glycosides, esperidin, quercetin, quercetin-glycoside, apigenin, apigenin-glycoside, cyanidin, cyanidin-glycoside, daidzein, formononetin, glycitein, genistein, biocanin, xanthumol, phloretin, tangeretin, nobiletin, kaempferol, myricetin, isorhamnetin, taxifolin, naringenin, hesperitin, catechin, catechin gallate, gallocatechin, gallocatechin gallate, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, procyanidin Bl, procyanidin B2, procyanidin A2, procyanidin Cl, theaflavin, delphinidin, pelargonidin, malvidin, peonidin, petunidin, leucoanthocyanidin, tangeritin, kaempferidin, fisetin, rhamnazin, eriodictyol, homoeriodictyol, dihydroquercitin, dihydrokaempferol, anthocyanidin, equol, nivetin, dalbergin, coutareagenin, dalbergichromene. All the phenols indicated in this description can also be used as mixtures of each other.

Of the phenols listed above, those particularly preferred are tyrosol, caffeic acid, vanillic acid, gallic acid, cinnamic acid, hydroxybenzaldehyde, coumaric acid, hydroxybenzoic acid, chlorogenic acid, syringic acid, ferulic acid, synaptic acid. The selection of antimicrobial agents of this type for the specific use described in comparison with the techniques used for example in US 2009/0298159 makes it possible to overcome major environmental and health problems.

Phenols extraction stage

The phenols which can be used in the process according to the invention need not be pure but can be extracted from alternative sources or industrial wastes (both solid and liquid fractions), such as oil mill wastewater.

The phenols extraction stage is essential, in that if they derive from a solid fraction, such as for example residual algal biomass, solid wastes from the processing of olives or solid vegetable wastes, they have to be extracted in a liquid phase which can be diluted in the reactor. If instead they derive from a liquid phase, such as an agri- industry effluent, such as for example wastewater from the olive oil, winemaking or brewing industries, it is essential that they should first be selectively separated from this so as to eliminate the bacterial flora that are capable of living in the presence of the phenols and which would prevail over the microalgae under heterotrophic conditions.

Any method known to those skilled in the art which is capable of extracting phenols selectively with respect to bacteria and other biological contaminants can be used as a method for extracting the phenols (Azmir et al. 2013; Ahmaruzzaman 2008; Scoma et al. 2012). Resins, organic solvents or combinations of these may be used. Extracted phenols should be conserved in a stock solution comprising an aqueous solution at acid pH (< 6), preferably pH 3. Their concentration in the stock solution should be such that it can be added to the heterotrophic growth medium by dilution by a factor of at least > 10, preferably > 50.

Any source of natural origin, such as wastewaters from the olive oil, brewing and winemaking industries, and wastes from the production of microalgae may be used as a source of phenols.

The use of microalgae that are resistant to phenols, the use of an inoculum having a particularly high algal concentration (usable values between 0.3 and 25 g/L and preferred between 1 and 3 g/L), the preliminary acclimatisation stage, the use of fed-batch feeding, that is gradual feeding during cultivation, and the addition of purified phenols to wastewater characterised by contaminants which are little suited to growth in the presence of phenols, are all conditions which make it possible to avoid the costly and difficult prior sterilisation of wastewaters and at the same time to overcome problems associated with the contamination of the cultures.

The microalgae are cultivated in a heterotrophic reactor until they achieve an optimum starch content which is at least higher than 20% w/w in relation to the weight of dry biomass, preferably > 40%, more preferably > 50%.

Two fractions are obtained from the microalgal biomass produced. The lipid fraction (containing triglycerides and carotenoids) is first collected; starch is instead purified from the remaining fraction through basic digestion processes to produce a starch which can be used for various applications, including, preferably, the production of bioplastics.

The process according to the invention comprises the following stages:

Stage A) Choice or selection of microalgae

The microalgal strain or suite used for cultivation in this process is preselected as follows for growth under both autotrophic and heterotrophic plant conditions. In particular the selection criteria are: ability to grow under autotrophic plant conditions (resistance to rotifers, to changes in pH, light, temperature, etc.), ability to grow under heterotrophic conditions, ability to accumulate a good quantity of starch, lipids and carotenoids, and resistance to the phenols used during the heterotrophic growth stage.

Sampling of the microalgae directly in situ in plant is to be preferred in that it provides strains which have evolved and adapted to the specific environmental conditions of the area.

Different types of algae may be used to produce starch by means of the process described. Algae belonging to the divisions Chlorophyta, Dinophyta, Cryptophyta, Rhodophyta and Cyanophyta may be used. Among the Chlorophyta algae of the genera Chlorella, Scenealesmus and Chlamydomonas may for example be used. Ability to accumulate starch, preferably in concentrations which can exceed 20% and amount to 50% or more in relation to dry weight, is a first selection criterion. Any microalgal strain which has characteristics known from the literature as being suitable for cultivation under the process conditions may be used for this process. However a strain or suite which has such characteristics can be selected using any means known to those skilled in the art (Wu et al. 2014; Anderson 2005). The preferred method is the procedure described below.

The strain or suite is selected by means of a series of steps or stages performed in series:

i. Sampling and isolation of the microalgal strain or suite. ii. Growth in the presence of phenols under heterotrophic conditions making use of the selected organic substrate.

iii. Growth of the microalgal strain or suite under autotrophic plant conditions through cultivation on the same medium as is used in the plant (non-sterile) under variable pH, lighting and temperature conditions.

iv. Ability of the microalgal strain or suite to accumulate starch through cultivation under nitrogen and/or phosphorus-deficient conditions, preferably below 85% with respect to the dose typically used under growth conditions (0.015 - 0.35 g/L of nitrogen, 0.1 - 10 mg/L of phosphorus).

Sampling according to stage (i) should preferably be carried out in situ or on the wastewater used as the source of organic substrate, and may be performed using any technique known to those skilled in the art (Anderson 2005). It may be performed either starting from solid matrices (e.g. soil) or liquid matrices (e.g. wastewater). The sample obtained in situ in plant is plated using the method of plating on Petri dishes containing wholly synthetic medium and exposed to artificial lighting at ambient temperature (autotrophic cultivation). The lighting may be maintained at values of between 10 and 400 μΕ m ^{- 2} s ^{~ 1} , with optimum values of 80 μΕ m ^-2 s ^{- 1} , the temperature at values of between 15 and 40°C, with an optimum value of 27°C. BGl l (Richmond 2004) may be used as the growth medium, but any medium for the autotrophic cultivation of microalgae is suitable. The N concentration may vary between 0.3 g/L and 0.001 g/L, with an optimum concentration of 0.05 g/L.

In order to carry out stage (ii) the microalgal strains or suites which have grown under the cultivation conditions in stage (i) are transferred to a different plate for growth under heterotrophic conditions in the presence of phenols. This cultivation is carried out on Petri dishes containing an easily assimilable organic substrate in the medium (e.g. sugars) in addition to phenols. The medium is prepared using the same medium as is used for autotrophic cultivation, with the addition of phenols and organic substrate. The predominant substrate or the mixture of predominant substrates present in the growth medium which will be used in the heterotrophic stage of the process, for example lactose in the case where whey is used, should be used as the organic substrate. This can be added in a concentration which may vary between 0.1 and 50 g/L, with an optimum range of 0.5-10 g/L and an optimum value of 5 g/L (expressed as glucose equivalents in the case of sugars). The phenols, expressed as tyrosol equivalents (following determination carried out using the Folin-Ciocalteu method (Atanassova et al. 2011)), may be added over a range varying between 0.01 and 5 g/L with an optimum concentration of 0.5 g/L. The preferred option is use of the same mixture of phenols as is used in heterotrophic cultivation. The plates are left in the dark at a temperature of between 15 and 40°C, with an optimum value of 27°C.

In stage (hi), the microalgal strains or suites which have succeeded in growing in the cultivation described in stage (ii) undergo autotrophic cultivation under plant conditions. The microalgal strains or suites are initially transferred onto plates known as "maintenance" plates, characterised by the same cultivation conditions as described in stage (i). From these plates the algae are taken and suspended in the liquid media of the autotrophic reactors. The autotrophic reactors are filled with the growth medium used for cultivation during the plant stage. This is a non-sterile aqueous medium comprising local tap water with added missing nutrients (mainly nitrogen, phosphorus and potassium) as will be mentioned below. In these reactors the algae are inoculated at an initial concentration which varies between 0.005 and 0.3 g/L, with an optimum value of 0.01 g/L. Cultivation may be carried out either in reactors installed under closed conditions with controlled lighting and temperature, or in reactors located in the open. The second option is the recommended one. Illumination, temperature, gas feed, nutrient concentration values, etc., are those mentioned in the section on "autotrophic growth stage". They are therefore the same as those for plant conditions. During this stage the change in biomass concentration must be evaluated over time. This may be determined using any effective method known to those skilled in the art, such as for example direct determination by filtering the suspension or measuring optical density. The maximum productivity and the maximum concentration achieved over time by the microalgae must be determined during this cultivation and a check needs to be made to ensure that values of more than 0.05 g/L/day and 0.5 g/L respectively are achieved.

In order to carry out stage (iv) the microalgal strains or suites which have demonstrated the ability to grow under the selection tests described in (i) and (ii), and have demonstrated that they have sufficient biomass production capacity under the conditions described in stage (hi) are finally tested for their ability to accumulate starch. For this check the microalgal strains or suites must be cultivated under nutrient-deficient conditions (nitrogen or phosphorus). This condition can be achieved by cultivating the microalgae under autotrophic plant conditions or under other conditions (stage (hi)) until a condition of nutrient deficiency (N or P) is reached, and holding them under these conditions for several days (5-15 days). The starch content within the biomass will be monitored daily during this stage. This content should increase over time and reach a value which is higher than at least 20% w/w with respect to dry biomass over a period of 15 days.

The microalgal strains or suites selected using the procedure described may be kept cultivated under either the growth conditions described in stage (hi) or the conditions described in stage (i). The algae cultivated in this way are subsequently used to initiate the autotrophic growth stage described below. Different strains or suites may have been selected at the end of the selection stage. The most productive for cultivation in plant is selected by comparing their maximum productivity, maximum concentration and maximum starch contents.

Stage B) Autotrophic growth stage

The selected algae or those obtained from the strain selection stage are initially cultivated in the laboratory in order to produce a volume of algal suspension sufficient for inoculating the plant reactors. When a sufficient volume of microalgal suspension has been produced (equivalent to at least 1/10-1/5 of the useful volume of the autotrophic reactor), with a microalgal concentration of between 0.1 and 5 g/L, the strain is inoculated within the autotrophic reactor together with the growth medium in such a way as to have an initial microalgal concentration of between 0.5 and 0.01 g/L with an optimum value of 0.1 g/L. The growth medium may be prepared using mains water available in the area of the plant and should contain the mineral salts necessary for growth of the microalgae. The chemical composition of the tap water will be characterised in order to assess what nutrients have to be added. The main nutrients which have to be added to ordinary tap water are nitrogen, phosphorus, potassium and iron, as is known to those skilled in the art.

The optimum nutrient as a source of nitrogen is the nitrate ion. Other sources, such as for example ammonium, amino acids or urea may also be used. The optimum concentration range varies between 0.015 and 0.35 g/L of nitrogen, with an optimum value of 0.05 g/L. Phosphorus may be provided in different forms such as PO4 ³ ", HPO4 ² ", H2PO4", H3PO4 in concentrations varying between 0.1 and 10 mg/L, with an optimum value of 5 mg/L. Potassium is provided as K ⁺ in concentrations varying between 0.2 mg/L and 25 mg/L, with an optimum value of 13 mg/L. Iron may be added in concentrations varying between 0.5 mg/L and 20 mg/L, with an optimum value of 1.5 mg/L.

Algae are inoculated in the reactor maintaining a dilution ratio of 1/10 (inoculum/inoculum + growth medium). The growth medium does not undergo any sterilisation treatment before being placed in the reactor. The reactors are fed with a source of CO2. This may comprise air (approximately 0.04% of CO2), air enriched with CO2 (in a percentage varying between 1 and 10%), pure CO2 or gases rich in CO2 deriving from industrial plants. The preferred configuration comprises a control system which regulates the composition of the feed gas as a function of the pH measured in the reactors. In this configuration a plant is fed either with pure air or with air enriched with 3 - 5% of CO2. If the pH in the reactors exceeds a value of 8 they are fed with an air-C02 mixture, and if the pH is below that value they are fed with air.

During this stage of cultivation growth may take place within a pH range of between 4.5 and 12, the preferred pH range being between 6 and 8.

The reactors are illuminated; either artificial lighting or sunlight may be used, the latter being the preferred configuration. In the case of artificial lighting this may be maintained for a minimum of 10 hours per day to a maximum of 24 hours out of 24, preferably 24 out of 24. Lighting may be maintained at average values of between 10 and 400 μΕ m- ² s- ^l .

Microalgae can grow under temperature conditions varying between

5 and 40°C. The optimum temperature range is between 25 and 35 degrees. The preferred temperature is 27 degrees.

The microalgae should be harvested when they reach a concentration of between 0.5 and 10 g/L, preferably between 1 and 3 g/L during the exponential stage, or at most at the start of the stationary stage (1 - 3 days).

Stage C) Thickening and acclimatisation to phenols

When during the autotrophic growth stage the biomass reaches suitable conditions for harvesting (stage B) it is thickened. The thickening stage may be carried out using any separation system known to those skilled in the art, such as for example centrifuging, sedimentation, flocculating or flotation. The thickening process yields an algal suspension that has been thickened by between 3 and 300- fold, the preferred thickening being approximately 10-fold. The algal suspension so obtained is transferred to the heterotrophic reactor.

Before adding the growth medium containing organic substrate an acclimatisation stage is performed. In this stage phenols in a concentration varying between 0.05 and 5 g/L are added to the thickened microalgae. The optimum concentration is 0.1 g/L. Nitrogen is also added during this stage, from any source which can be used by the microalgae, the preferred manner being in the form of nitrates. The nitrogen is added in a concentration between 0.15 and 3.5 g/L, with an optimum value of 0.5 g/L.

The microalgae are left constantly stirred with air with a throughput of between 0.01 and 1 L/L/min, with an optimum value of 0.1 L/L/min. This stage is continued for a time of not more than 24 hours, preferably between 12 and 3 hours, the optimum condition being 6 hours.

The temperature should be kept within a range of 10-40 degrees, preferably 20-30, with an optimum temperature of 27 degrees.

Thickening is a necessary step for reducing the volume of the reactors during heterotrophic cultivation, thus reducing the costs of plant without reducing the productivity of the process. Acclimatisation is necessary in order to pre-adapt the algae to the presence of phenols, before the organic substrate is added, so as to make them more competitive with the ubiquitous contaminants present in the waste or wastewater used as a source of fermentable organic substrate.

The use of high algal concentrations (0.3-25 g/L post-dilution with heterotrophic medium) is a further factor which will reduce the risk of the growth of contaminant species.

Stage D) Heterotrophic growth

During the heterotrophic cultivation stage the aqueous suspension of microalgae is placed in a closed non -illuminated reactor in the presence of the growth organic substrate. This reactor is different from the one used for autotrophic growth, being in fact characterised by a smaller surface/volume ratio.

While reactors with surface/volume ratios of the order of 0.2-0.8 cm ⁴ are used for cultivation under autotrophic growth conditions, for heterotrophic growth reactors having ratios of the order of 0.005-0.02 cm- ¹ may be used.

Any source containing organic molecules which can be easily metabolised by the microalgae but which do not contain phenols can be used as a source of organic substrate. The organic substrates can be derived from biomass which has been hydrolysed by means of saccharification processes, from by-products from various industries, especially the agri-food industry, such as for example sugar production or from wastewaters of various kinds originating from biotechnology and food processes. Whey, which contains lactose in a concentration of approximately 40 - 50 g/L, is particularly preferred.

After the acclimatisation stage the heterotrophic reactor will contain a volume of inoculum from 1/2 to a minimum of 1/15 of the reactor volume (VR). The preferred condition is 1/10.

The organic substrate and the phenols are gradually added during an initial stage or during the entire course of the fermentation on the basis of a daily dose (fed-batch) within the range of 0.1 - 10 g of organic substrate per day per litre of reactor (optimum value 1 g per day per litre of reactor), together with 0.005 - 1 g of phenols expressed as tyrosol equivalents per day per litre of reactor (the optimum being 0.05 g per day per litre of reactor).

The total fermentation period may vary from a minimum of 2 days to a maximum of 20, the preferred time being between 5 and 15 days and the optimum 10 days.

The substrates and phenols may be fed during the first two days of fermentation or be extended over its entire duration.

Fermentation is carried out at temperatures varying between 5 and 40°C. The optimum range is between 25 and 35 degrees. The preferred temperature is 27 degrees.

The pH may be held within a range between 4 and 12, the preferred range being between 6 and 8.

The reactor will be constantly stirred with air at a throughput of between 0.01 and 1 L/L/min, with an optimum value of 0.1 L/L/min.

Preferred organic substrates are sugars such as glucose, fructose, saccharose, lactose, organic acids such as acetic acid, alcohols, polyalcohols and amino acids. It is important that the source of organic substrate should have a negligible phenol concentration when leaving the production process, below 50 mg/L. Wastewaters which have been pre-treated using chemical, physical or biological processes for dephenolisation should not be used in order to prevent biological contaminants which are capable of growing in the presence of phenols being added during the stage of heterotrophic fermentation of the microalgae.

Wastewaters which do not originally contain phenols and which can be used are whey, residual molasses from the processing of sugar and sugar products. Whey is the preferred source of organic substrate, added at a flow rate varying within the range 0.0025 - 0.5 L/L/day (optimum 0.025 L/L/day). Phenols of any kind may be added for the purposes of the invention, and among these the preferred are those belonging to the class of acid phenols, simple phenols and flavonoids. Particularly preferred within this class are tyrosol, caffeic acid, vanillic acid, gallic acid, cinnamic acid, hydroxybenzaldehyde, coumaric acid, hydroxybenzoic acid, chlorogenic acid, syringic acid, ferulic acid, and synaptic acid.

If phenols from wastewater are used, these are selectively extracted as previously described in order to minimise the addition of contaminants deriving from phenol-containing wastewater to the reactor as described in the previous section.

The purpose of the heterotrophic growth stage is to maximise the accumulation of starch. Content is monitored during cultivation. The starch may be analysed using any method known to those skilled in the art. The preferred method, because of its simplicity, provides for saccharification of the biomass (Moxley & Zhang 2007).

The starch content of the microalgae tends to increase over time. In accordance with the invention microalgal strains or suites which achieve a starch content of 20% by weight or more with respect to the dry weight of the biomass are used. When the starch content is between 20 and 70%, preferably between 40 and 70%, the reactor is emptied and the algae are harvested. Under the specific conditions detailed above it is possible to achieve a significant increase in the output of starch in comparison with the known art. Unlike what has been reported in the literature (Di Caprio et al. 2015), in which mention is made of quantities of starch produced having concentrations between 0.1 and 0.16 g/L, using OMW medium, by operating in accordance with the procedure described here the output of starch can be doubled (0.3 -0.4 g/L).

Stage E) Harvesting and dry ins the algae

The algae are harvested from the heterotrophic reactor using a system of thickening and separation from the growth medium. Any effective system known to those skilled in the art such as for example sedimentation, centrifuging, filtration, flotation and flocculation may be used for this purpose. The harvested algae can be dewatered using various drying systems known to those skilled in the art. Direct drying at high temperature (> 40°C), lyophilisation, or drying in a flow of gas may be used. Temperatures of 105°C must not be exceeded during the drying stage.

Stage F) Starch extraction

The starch contained in the algae is extracted and purified by separating it from the two other main components of the biomass - lipids and proteins.

Prior to the extraction treatments the algal biomass may undergo pre-treatments to reduce particle size, if necessary. This reduction may be achieved through mechanical treatments such as for example the use of mills. Lipids are separated from the biomass by solvent extraction.

Extraction may be performed using any solvent or mixture of solvents having a low polarity. Among the preferred solvents we have hexane, chloroform, methanol, ethanol, supercritical CO2 and ionic liquids. The preferred configuration provides for extraction with an apolar solvent, for example chloroform or hexane, and a polar organic solvent, such as for example ethanol or methanol, in a ratio of 2:1 or 3: 1. The reaction may be performed in a temperature range varying between 25 and 70°C, preferably 65°C. Extraction is preferably performed until colour disappears from the solvent and/or the biomass, over a time varying between 2 and 10 hours. After the extraction stage the solvent containing lipids is evaporated and recovered by distillation and the lipids are recovered in the form of oil. The lipid fraction may be intended for various uses such as for example the production of biodiesel or the production of nutraceutical products, the recovered algal oil also contains the carotenoids produced by the algae within it.

The residual biomass fraction, which is instead essentially composed of starch and protein, undergoes digestion with basic solution to remove the proteins and concentrate the starch. Digestion may be carried out using a solution having a pH varying between 12 and 14, preferably between 12.5 and 13.5, for a time varying between 1 hour and 10 hours, preferably between 2 and 3 hours, or until the proteins have completely separated out. Digestion is performed in mechanically stirred reactors containing 5 litres of basic solution for every kg of biomass. A liquid/solid ratio varying from 1 to 20 litres of solution per kg of biomass may however be used.

After digestion the starch is separated out from the basic solution and washed with water.

After the washing stage the starch produced may undergo dewatering through methods known to those skilled in the art. The starch produced may be used for the production of bioplastics.

Advantages of the proposed invention

The invention is based on the use of natural antiseptics (phenols) as a method for limiting contamination in the heterotrophic cultivation of microalgae on phenol-free substrates, such as for example whey. Techniques known in the state of the art for controlling contaminants in the heterotrophic cultivation of microalgae provide for costly energy-consuming treatments which cause problems for the environment, particularly in the case where wastewaters are used. These treatments provide for high temperatures, filtration at high pressures, chemical treatments with the use of antimicrobials (such as ozone and chlorine), or more specifically antibiotics. The use of phenols alone described in the invention below instead makes it possible to manage the process in a simplified way, these being economical, widely available (in that they are present in many wastes of plant origin), without any contraindications of a toxicological and health nature, and effective against a wide spectrum of possible contaminants.

The proposed process has a double advantage in environmental terms - it allows wastewaters and wastes to be doubly co-purified both by using them as a source of organic substrate and by using wastes and wastewaters for extracting the phenols used to control contaminants. In this way polluting wastes such as whey and phenols derived from recalcitrant wastewaters (which cannot be treated using conventional biological treatments) may be treated in an algal growth plant and purified. Finally fixation of CO2 produced by gas-production plants or thermal recovery plants can be incorporated as a source of carbon in the phototrophic stage.

It is known from the literature that phenols under the process conditions conventionally used for the production of microalgae are a problem in that they have adverse effects on their growth. In this invention conditions are described in which not only do the phenols have no negative effects but are positive for microalgal growth. Under the conditions described they control contaminants, encouraging algal growth, and no longer represent a problem which has to be eliminated but have added value which makes it possible to avoid the use of costly chemical or physical sterilisation techniques.

A further advantage lies in the use of a suite sampled from nature and selected by means of the protocol described in the invention. The use of autochthonous suites makes it possible to have strains which are better adapted to plant conditions and avoid environmental contamination by alien species in the case of accidental releases of growth medium, and make it possible for the wastes treated in a plant to be discharged into surface waters after treatment.

Simplification of the process for controlling contaminants makes it possible to cultivate microalgae on a large scale under heterotrophic conditions on various natural substrates that are free of phenols, such as whey and molasses deriving from the production of sugars; heterotrophic cultivation makes it possible to achieve high starch productivity using low-cost reactors.

The production of starch using microalgae represents an advantage in terms of productivity (up to an order of magnitude greater) than the cultivation of plants currently used for this purpose.

The production of starch by means of microalgae also has the advantage over the cultivation of plants that it does not compete with agricultural land intended for satisfying food requirements.

This makes it possible to achieve productivities that are higher than the outputs obtained from terrestrial plants. For example, while in the case of maize (one of the main present sources of starch) starch productivities of the order of 3-9 t/Ha/year are obtained, in the case of microalgae, having regard to an achievable biomass productivity of approximately 170 t/Ha/year (Mata et al. 2010) and a starch content of between 20 and 50% by weight with respect to weight of dry biomass, it can be estimated that a starch productivity of the order of 30-90 t/Ha/year can be achieved. Microalgae also do not need fertile ground for their cultivation, thus enabling them to be cultivated on land which is not intended for agricultural use.

In the context of obtaining starch from microalgae this invention has a further advantage over the known art in that through fed-batch heterotrophic fermentation in the presence of phenols it is possible to obtain twice the output of starch in comparison with literature data (from 0.16 to 0.3-0.4 g/L of starch).

The following examples are provided by way of illustrating the invention and are not to be considered as restricting its scope.

Examples

Strain isolation and selection

The procedure used to select microalgal suites directly on the plant site (in situ) is illustrated in the following example. Through this procedure a strain of Chlorella sp. and one of Scenedesmus sp. having suitable characteristics for use in the process described here were selected using this procedure.

Isolation of strains in situ

Samples were obtained in situ and diluted 1 to 5 by three serial dilutions with distilled water.

Each sample was inoculated onto a Petri dish using the smearing technique. In order to prepare the Petri dishes mineral salts were added to distilled water to obtain the composition of BGl l medium (Richmond 2004). 1.5% agar was added to the medium and all of this was caused to solidify after heating to boiling for 10 minutes.

The plates were incubated at 27 ± 3°C, in an air atmosphere, with constant lighting (24 hours out of 24) at 80 ± 10 μΕ m ^{~ 2} s ^{" 1} .

The individual colonies were isolated and maintained through culture on Petri dishes.

The isolated strains underwent the following procedure.

Selection of phenol-resistant strains

The BGl l medium was prepared by adding mineral salts to distilled water. Tyrosol was added to the growth medium to achieve a concentration of 0.5 g/L and lactose or glucose were added to a concentration of 1 g/L in both cases. The agar was added to 1.5% and allowed to solidify after heating to boiling.

The plates were inoculated with algal suspensions of both the strains, preparing different serial dilutions. 3 serial dilutions of 1 to 5 were performed.

After inoculation the plates were placed in an incubator at 27 ± 3°C in the dark, exposed to air and checked daily.

The selected strains, those capable of growing under the conditions tested, underwent the next procedure.

Strain selection in autotrophic cultivation under plant conditions for ability to accumulate starch

The algae were transferred from the Petri dishes to 10 mL test tubes containing BGl l medium in liquid form and exposed to illumination of 80 ± 10 μΕ m ^{- 2} s ^{- 1} for 24 hours out of 24. The test tubes were shaken every day. After 10 days cultivation in the test tubes the algae were transferred to 300 mL flasks kept under the same cultivation conditions. In the flasks inoculation was carried out maintaining a dilution factor of 1/10 between the volume of inoculum and the volume of BGl l medium. The flasks were kept constantly stirred and exposed to air. When the algal suspension obtained reached a concentration of over 0.5 g/L it was transferred into 4 L column reactors. In this case the growth medium was prepared from tap water available on the plant site. NaNOe and K2HPO4 were added to this water in corresponding concentrations of 300 mg/L and 30 mg/L and the solution obtained was used as a growth medium. The algae in the column reactor were fed with a mixture of air/C02 (5%) and illuminated for 24 hours out of 24 with 80 ± 10 μΕ m- ² s ^{" 1} . The biomass concentration in the reactors was monitored daily. During this stage of cultivation both the Chlorella sp. strain and the Scenedesmus sp. strain yielded adequate results with productivity values of 0.2 g/L/day, and achieved maximum concentrations in excess of 1 g/L.

In order to check starch accumulation capacity microalgae of the strains Chlorella sp. and Scenedesmus sp. were cultivated under nitrogen -deficient conditions. These algae were cultivated under autotrophic conditions for a number of days, measuring both the concentration of biomass and the change in starch content within the algal biomass over time during this period. In order to do this an aliquot of microalgal suspension was collected from the growth medium at various time intervals. Scenedesmus sp. was cultivated in 300 mL flasks, in BGl l growth medium; in the course of this cultivation nitrates were consumed until a condition of deficiency corresponding to the accumulation of starch (Figure 2) was achieved. Chlorella sp. was inoculated at a 1/10 ratio into BGl l without nitrates in 300 mL flasks. The microalgae were allowed to grow for various days achieving a concentration of 0.34 g/L (Figure 3). As illustrated in Figure 3, the Chlorella sp. strain was able to accumulate starch during this stage, with an increase in concentration rising from initial 20-30% to 54%, after which it decreased.

Autotrophic cultivation

The microalgae selected (Chlorella sp. and Scenedesmus sp.) using the above procedure were inoculated in 4 L column reactors at an initial concentration of 0.1 g/L. Inoculation was carried out by diluting the concentrated suspension ten times with cultivation medium prepared from tap water. This cultivation medium was prepared by adding 0.31 g/L of NaN0 ₃ and 0.03 g/L of K2HPO4 to tap water. The reactors were constantly fed with a mixture of C02/air (4%) at a flow rate of 0.5 L/min. The reactors were illuminated 24 hours out of 24 hours using neon fluorescent lamps providing a constant illumination of 80 ± 10 μΕ m ^~2 s ^_1 . The temperature of the reactors during cultivation was maintained constant at 27 ± 3 °C. During this stage of cultivation the pH was stable at a value of 6.0 ± 0.1. During this cultivation the microalgae achieved a concentration of 1 g/L in the case of Chlorella sp. and 0.7 g/L in the case of Scenedesmus sp. over 10 days. Phenol extraction and purification

Phenols were extracted and purified from oil mill wastewater (OMW) using XAD16 resin (Scoma et al. 2012). The wastewater was first caused to sediment out for two hours in order to remove sedimentable solids. 7 g of dry resin were activated by washing in suspension in acidified ethanol (0.5% 0.1 M HC1). The activated resin was stirred with 100 mL of wastewater for 1 hour. After having been separated from the treated wastewater by filtration the resin underwent desorption of the phenols in 100 mL of acidified ethanol (0.5% 0.1 M HC1) for 90 minutes. The ethanol solution was separated from the resin by filtration and placed in a Rotavapor flask at 60 - 70°C. The ethanol was recovered by this means and the phenols were resuspended in 10 mL of acidified distilled water (pH 3). In all 60% of phenols were extracted from the treated oil mill wastewater and concentrated in 5 mL of acidified water at a concentration of approximately 60 g/L. The solution containing the selectively extracted phenols was then used for the cultivation tests using whey.

Biomass thickening and acclimatisation

The biomass collected from the autotrophic reactor was first thickened by sedimentation for 12 hours. The thickened fraction was then centrifuged at 3000 rpm for 5 minutes. When various methods of thickening were compared it was found that an approximately tenfold thickening of the biomass was achieved with sedimentation, while approximately 350-fold was achieved with the subsequent centrifuging. The biomass thickened by sedimentation had a sufficiently high concentration for the inoculum in the heterotrophic reactor. Acclimatisation was carried out by collecting the Chlorella sp. algae by sedimentation after autotrophic cultivation in a closed reactor, without exposure to light, adding 0.1 g/L of phenols selectively extracted from oil mill wastewater (OMW) (using resin) and leaving them stirred for 6 hours. After this stage growth medium comprising BGl l with added glucose (1 g/L) and phenols selectively extracted from the OMW (0.5 g/L) were added to the algae in a volume such as to have an initial concentration of 0.1 g/L.

When the results of growing algae cultivated under identical conditions were compared, but without undergoing any initial acclimatisation stage, it was found that while the acclimatised algae had a latency time of 2 days, those which were not acclimatised had a latency time of 5 days. Acclimatisation is therefore necessary in order to obtain algae which are more suitable for heterotrophic cultivation with phenols.

Heterotrophic cultivation

Microalgae of the Chlorella sp. strain were inoculated in heterotrophic reactors at an initial concentration of 0.6 million cells/L, adding whey diluted with BGl l in such a way as to have a lactose concentration of 10 g/L in the reactor. No thermal and/or chemical treatments were applied to the whey to eliminate contaminants. The concentrated solution of phenols selectively extracted from oil mill wastewater (OMW) was added to the growth medium to achieve a phenol concentration of 0.5 g/L (expressed as tyrosol equivalent). The phenols used in this example are therefore those characteristic of oil mill wastewater, and are the following: caffeic acid, vanillic acid, tyrosol, luteolin, hydroxytyrosol, verbascoside, ligstroside. Cultivation was performed in 300 mL reactors fed with air (0.1 L/L/min). The reactors were stirred magnetically. During this cultivation stage the pH was held at a value of 6 - 8 and the temperature at a value of 30 ± 5°C. The reactors were sealed with aluminium to ensure heterotrophic growth conditions. Figure 4 shows the growth curves for microalgae cultivated under the condition described and under identical conditions but without the added phenols. Comparison between the two tests shows that in the tests with phenols the algae achieved a maximum average concentration of 1.6 million cells/mL, against 0.9 million cells/mL in the absence of phenols. By inhibiting the contaminating bacteria present in whey the added phenols encouraged algal growth. In another example Scenedesmus sp. was cultivated under heterotrophic conditions in two different ways: batch and fed-batch. Heterotrophic reactors of 1 L were inoculated with 100 mL of microalgae in a concentration of 0.1 g/L. Under batch conditions 900 mL of BGll growth medium with added glucose (1 g/L) and phenols selectively extracted from wastewater (0.5 g/L) were initially added. Under fed-batch conditions 0.79 L of BGll were initially added, and 0.01 L of a source of organic substrate containing 10 g/L was added every day for ten days together with 0.001 L of stock solution (50 g/L) containing selectively extracted phenols every day. As shown in Figure 5, with the gradual addition of phenols and organic substrates under fed-batch conditions the output of algal biomass could be appreciably increased. The maximum biomass concentration doubled, from 0.43 to 0.92.

Collection and drying of algae

After heterotrophic cultivation the microalgae were collected by centrifuging. The thickened algal suspension obtained was then dried to constant weight at 105°C. The dry biomass was ground up using a mechanical grinder to obtain a biomass of fine uniform particle size. Starch extraction

Starch was extracted from the ground dry biomass. A lipid fraction was first extracted from the biomass by solvent extraction using a Soxhlet extractor. 2 g of biomass were weighed out and placed in the extraction chamber of the extractor. A mixture of 40 mL of methanol and 90 mL of chloroform was used as a solvent. Extraction was carried out over 7 hours at the boiling point of the solvent mixture. After extraction the solvent was separated from the residual biomass by evaporation under vacuum and 0.43 g of lipids were recovered.

In order to purify the starch a residual biomass underwent basic digestion with 0.05 M NaOH using a volume of 5 mL per gram of biomass. Extraction was carried out at ambient temperature, with magnetic stirring, over a period of two hours. The suspension was centrifuged at 3000 rpm for 5 minutes, the supernatant was removed and the biomass washed 3 times with distilled water using a volume of 15 niL per gram of biomass for each wash. A second wash was performed using the same solid liquid biomass/water ratio but with the addition of 1 N hydrochloric acid (2 mL for each gram of biomass). The biomass was finally separated from the solution by centrifuging and dried at 40°C for three days in order to remove residual water. This purification of the starch was also performed on the biomass "as such", that is the dried ground biomass. The starch content was determined before and after basic digestion. The results are shown in Figure 6. The procedure proved effective on both biomass as such and that obtained after extraction of the lipids.

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