Engineering solutions for open microalgae mass ...

Apel, Weuster-Botz (2015) Engineering solutions for open microalgae mass cultivation and realistic indoor simulation of outdoor environments

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Engineering solutions for open microalgae mass cultivation and realistic indoor simulation of outdoor environments Andreas Christoph Apel, Dirk Weuster-Botz*

Institute of Biochemical Engineering, Technical University of Munich Boltzmannstr. 15, 85748 Garching, Germany *

Corresponding author: [email protected] Phone +49.89.289.15712 This is the accepted version of the manuscript. It may be disseminated freely. Its content is identical to the final publication, the formatting is slightly different. The paper formatted by Springer was published in Bioprocess and Biosystems Engineering, June 2015, Volume 38, Issue 6, pp 995-1008. The final publication is available at link.springer.com doi: 10.1007/s00449-015-1363-1

Abstract

vantages of microalgae in comparison with terrestrial crops are higher areal yields and lipid contents. Furthermore, they

Microalgae could become an important renewable source for chemicals, food, and energy if process costs can be reduced.

can be cultivated photoautotrophically – consuming the greenhouse gas CO2 and using sunlight as energy source for

In the past 60 years, relevant factors in open outdoor mass

photosynthesis – and do not require freshwater, but can be

cultivation of microalgae were identified and elaborate solutions regarding bioprocesses and bioreactors were devel-

grown in saline water or wastewater on non-arable land [2, 3]. Major issues in the commercial implementation of large-

oped. An overview of these solutions is presented. Since the cost of most microalgal products from current mass cultiva-

scale microalgae production processes are scalability of cultivation systems, poor comparability of cultivation sys-

tion systems is still prohibitively high, further development is required. The application of complex computational tech-

tems due to incomplete information in literature (e.g. incompatible yield statements: gross and net yield, short-term

niques for cost-effective process and reactor development

and long-term yield, location and weather dependence), and

will become more important if experimental validation of simulation results can easily be achieved. Due to difficulties

high cost for investment (e.g. cultivation systems, harvesting devices, auxiliary equipment) and operation (e.g. CO2, nutri-

inherent to outdoor experimentation, it can be useful to conduct validation experiments indoors. Considerations and

ents, personnel) which is especially problematic when biofuels are to be produced [4–9].

approaches for realistic indoor reproduction of the most important environmental conditions in microalgae cultiva-

Mass cultivation systems for microalgae

tion experiments – light, temperature, and substance concentrations, are discussed.

Mass cultivation of microalgae has been researched for more than 60 years [10] and a wide variety of reactor designs for

Introduction

mass cultivation systems have been conceived and tested [11, 12]. Based on the degree of exposedness to the atmos-

Microalgae are a promising source for renewable chemicals, human nutrition, animal feed, and biofuels. ‘Microalgae’ can

phere or to environmental conditions, mass cultivation systems for microalgae are usually classified as ‘open’ or

be defined as small uni- and multicellular organisms that can

‘closed’. Any container – be it open or closed – in which a

photosynthesize [1], a definition which includes both eukaryotic algae and prokaryotic cyanobacteria. Important ad-

bioreaction using photosynthesis takes place is technically a ‘photobioreactor’. Nevertheless, many phycologists use this


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term only for closed cultivation systems, while open cultivation systems are generalized by the term ‘open ponds’.

for food, feed, and cosmetics purposes, exceeds the cumulated annual production of all other microalgae species by

Grobbelaar’s criticism [13] of this practice is justified not only due to the technical definition of the term photobioreac-

far [32]. Commercial viability for large-scale biofuel processes, however, remains to be demonstrated [33].

tor, but also because ‘pond’ is connoted with ‘low-tech’. However, there is elaborate engineering even behind seemingly simple large-scale open bioreactor designs such as

Improving cultivation systems and processes

raceways [14–17], sloped channels [18, 19], or the French IFP airlift reactor [20, 21], and especially behind highly

To overcome the limitations of current open mass cultivation processes, biological and engineering approaches have

dynamic reactor designs such as Šetlík et al.’s thin-layer cascade system which is accordingly termed ‘open photobi-

been proposed [34–38]. While genetic engineering can yield relevant improvements to microalgae [39], its practical ap-

oreactor’ by its creators [22–25] (Fig. 1).

plicability is limited due to legal restrictions that many juris-

Closed photobioreactors have advantages such as good control of cultivation parameters and reduced contamination

dictions pose on genetically modified organisms in open cultivation systems. Promising biological approaches which

risk. However, due to the complex and expensive technical equipment and poor cleanability, according to many re-

avoid the problems related to genetically modified organisms are isolation of novel strains from natural habitats and

searchers mass cultivation of microalgae for low-value commodity products such as biofuels will only be economi-

improvement of known strains using non-directed mutagenesis and high-throughput screening technologies. This re-

cally feasible in open cultivation systems [26–31].

view, however, will focus on engineering approaches.

Despite considerable research effort in the past decades, today there are still only few industrial-scale microalgal

Engineering of bioprocesses and bioreactors requires consideration of biological, technological, and economic

production processes for high-value products such as nutraceuticals or fine chemicals. Examples include the ca-

factors. During design, it is important to keep in mind that the ultimate parameter to be optimized is cost [28]. There

rotenoids β-carotene from Dunaliella salina (BASF, Australia) and astaxanthin from Haematococcus pluvialis (Al-

are many publications projecting the cost of microalgae mass cultivation in different process routes [28, 40–43].

gatechnologies, Israel; Cyanotech, USA), and the whole

However, due to the lack of actual experience with industri-

microalgae Chlorella vulgaris and Arthrospira platensis (‘Spirulina’). The annual production of Arthrospira and

al-scale cultivation of microalgae, a recurring problem of these projections is that they are based on a multitude of

Chlorella, which are mainly produced in the USA and Asia

assumptions and parameter estimates [42, 44], leading to a

Fig. 1 Schematic drawing of the three open photobioreactor designs which have been intensively studied for microalgae mass cultivation in the past decades (top: central cross section; bottom: plan view). Left: Raceway Pond (designed for level ground) Center: Thin-layer cascade (for slopes of ca. 1 - 3 %) Right: Sloped channel (for slopes of ca. 1 – 10 % and higher) Dashed lines: Pumps and pumping circuits. Vertical scale exaggerated.


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significant uncertainty in the results. A general conclusion from these studies is the identification of relevant cost fac-

Simple approaches to minimize animate contaminants are use of chemical agents or cultivation of strains which

tors. Total capital cost is heavily influenced by photobioreactor and harvesting equipment cost. Major operational

require selective environments such as high pH (e.g. Arthrospira platensis) or high salinity (e.g. Dunaliella salina).

cost factors are nutrients, CO2, electricity, and labor. There-

However, contamination can be controlled even in open

fore, these cost factors should be main areas of attention during process and reactor design. Interrelated effects must

cultivation systems with non-selective environments by deliberate design of bioreactors and process control strate-

be considered: A technological innovation leading to a novel bioreactor which allows for, e.g., higher growth rates would

gies to ensure presence of desired culture conditions [53, 54]. Examples of process and reactor engineering solutions

be of no use if this novel bioreactor was prohibitively expensive. An analysis of the entire process must be performed

to contamination problems include variations of cultivation conditions (e.g. temporary pH reduction), concentration-

to avoid being misled by singular improvements which are

based control of volume or timing of inoculation and har-

evaluated out of context [9, 45]. Novel reactor and process designs therefore need to address the shortcomings of cur-

vest, installation of restraining devices (e.g. nets, screens, sand pits), design for cleanability, and multi-stage processes

rent open mass cultivation systems while evaluating the influence of envisaged modifications on cost.

[14, 16, 18, 38, 54–59]. Mixing and biomass concentration

Bioprocess and bioreactor engineering for improvement of open mass cultivation systems

Mixing of the cultivation medium is important to prevent

Open mass cultivation of microalgae can be seen as a branch

photoinhibition, photodamage, sedimentation, temperature gradients, and concentration gradients of nutrients, CO2, and

of agriculture and is accordingly affected by similar problems as other monocultures. Major issues in most open mass

O2. Terry et al. [60] accurately stated that “the history of mass culture growth unit design reflects, more than any

cultivation systems are contamination, poor and uneven mixing, low biomass concentration, low CO2 utilization

other single factor, a history of system design for the turbulent or ordered mixing of water in layers of a few centime-

efficiency, and the strong influence of weather on the culti-

tres to about one metre in depth spread over large surface

vation.

areas” and that during reactor design, tradeoffs must be made between good mixing and consequential good cultiva-

Contamination

tion results on the one hand, and energy expenditure and consequential cost on the other hand. In engineering, mixing

As open systems are exposed to the environment, their susceptibility to contamination is evident. Contaminants are of

is usually evaluated based on the Reynolds number. However, in cultivation of photosynthetic microorganisms, the

a very diverse nature. A rough distinction can be drawn

importance of the Reynolds number is reduced in favor of

between animate contaminants such as bacteria, fungi, viruses, unwanted strains of algae, grazers/predators, and

vertical mixing patterns [61, 62]. Based on different decisions regarding the aforemen-

animals; and inanimate contaminants such as dust, sand, stones, leaves, and dead animals or parts thereof [6, 15, 16,

tioned tradeoff, cultivation systems with very distinct mixing approaches have been developed. The extremes are

46–48]. While significant bacterial contamination only occurs under non-optimal cultivation conditions [16, 49], con-

covered by unmixed ponds [63] and intensively-mixed thin layer cultures [25], while raceway ponds and the sloped

tamination by other algae is more common. A well-

meandering channel developed in the Peruvian-German

performing algae strain identified in laboratory-scale experiments might not persist in an open bioreactor because in-

microalgae research project [64, 65] are placed in between. As a general rule, mixing and biomass concentration are

vading algae from the environment might replace the desired strain over time [50]. This is especially the case if the pro-

positively correlated. Biomass concentration influences harvesting cost [31], and open mass cultivation systems are

duction strain was chosen for its high lipid content as these

often criticized for the low achievable biomass concentra-

algae usually grow slowly, therefore being at a disadvantage against fast-growing invading algae [28, 51, 52].

tion. This is true for raceway ponds or sloped meandering channels, where biomass concentrations are typically 0.1 - 1.0 g L-1, rarely up to 1.5 g L-1 [8, 66–68]. Large-scale


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closed photobioreactors reach cell densities of 1.5 - 10 g L-1 [69–71]. However, the highest reported cell densities in

Influence of weather

photoautotrophic microalgae mass cultivation have consistently been reached in the open thin-layer cascade cultivation

Weather conditions exert strong influence on open cultivation systems. For large open cultivation volumes, many

system developed by Šetlík et al. [22] in Třeboň. Engineer-

researchers consider temperature control not economically

ing aspects of this system have been improved over several decades, as can be inferred from the many detailed publica-

feasible [94–97]. A process design solution for cultivation in locations of higher latitude is to adapt the process to the

tions by the research group and the progress of achievable cell density from 3 g L-1 [72] to 15 g L-1 [45] until recently

cyclic changes of environmental conditions by cultivating a ‘summer strain’ and a ‘winter strain’ [11, 98]. Depending on

up to 45 g L-1 [73, 74].

the choice of mixing device, the cultivation medium can be heated due to its operation, resulting in better growth and

CO2 utilization efficiency

offsetting the higher cost for mixing [99].

In rare cases, atmospheric CO2 is sufficient for mass cultiva-

Water exchange with the atmosphere is another weather-related problem [100]. It results in a varying cultivation

tion of microalgae [75]. Most often, however, an additional CO2 supply is required [76]. Possible low-cost sources for

volume and hence changing concentrations and growth conditions. Rainfall dilutes the culture volume, and evapora-

large amounts of CO2 include combustion of fossil fuels, natural reservoirs, anaerobic digestion, ethanol fermentation,

tion concentrates it. As the location for microalgae mass cultivation is usual-

and synthesis of chemicals [77]. Experimental studies using

ly chosen for abundant sunlight, rainfall could be mistaken

flue gas as CO2 source have shown promising results [78– 81]. Nevertheless, current large-scale microalgae production

for a negligible problem. However, in these locations there can be torrential rain, especially during wet season, resulting

processes employ pure CO2, possibly due to flue gas-related problems such as low CO2 concentration and necessary

in drastic changes of the cultivation conditions if this possibility was not taken into account during reactor and process

additional gas purification processes. Major bioprocess development challenges regarding

design [101]. Reactor design solutions include spillways and closed tanks for temporary storage [16, 22]. A process de-

CO2 for microalgae mass cultivation are the design of effi-

sign solution by Galiby [102] was to operate the cultivation

cient CO2 supply devices, and the reduction of CO2 losses to the atmosphere. The value to be optimized during design is

process in the upper range of tolerated salinity at the beginning of the wet season, allowing continuation of cultivation

the CO2 utilization efficiency, a parameter derived from the CO2 mass balance that relates the amount of CO2 fixed as

as the rain would dilute the medium to the lower range of tolerated salinity. The fact that, due to density differences,

carbon in microalgae to the amount of supplied CO2 [82, 83]. A multitude of different CO2 supply devices have been

freshwater from rainfall would form a layer on top of the high-salinity layer containing the cultivated microalgae, was

developed [6, 25, 67, 72, 84–89]. No device could yet be

elegantly used by Borowitzka et al. [103] by removing the

determined as the ‘best of all’, therefore different devices should be evaluated during process engineering to select an

freshwater layer after the rain before it could mix with the brine.

appropriate one based on the chosen reactor design and process conditions.

While episodes of rainfall are singular events, water loss through evaporation is a permanent process. Due to the

CO2 loss to the atmosphere depends on the mass transfer coefficient and the concentration difference between

enthalpy of vaporization, evaporation serves the important purpose of protecting the cultivation medium from overheat-

atmospheric and dissolved CO2. Several options are availa-

ing. Evaporation rates depend on meteorological conditions,

ble to reduce this loss. Variation of temperature, culture depth, and turbulence can influence the mass transfer coeffi-

reactor design, and operational parameters. Reference values for raceway ponds, sloped ponds and thin-layer cascades are

cient [83, 90, 91]. Approaches aimed at reducing the concentration difference include the design of high pH process-

5 - 6 L m-2 day-1 [25, 104]. In very dry climates such as Kuwait or Western Australia, raceway ponds and thin-layer

es, so carbon is mainly present as bicarbonate or carbonate,

cascades reached evaporation rates of up to 20 L m-2 day-1

and deliberate arrangement of carbonation stations in the reactor [73, 92, 93].

[105, 106]. A theoretical calculation of evaporation rates for given environmental conditions is possible [107].


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Table 1 Summary of the presented engineering solutions

Challenge

Solution

Challenge

Solution

Contamination

Selective environment

Weather (temperature)

Cultivation of summer and winter strains

Chemical agents

Heating by mixing device

Variation of culture conditions Concentration-based control of volume or timing of inoculation and harvest

Weather (rain)

Closed tanks

Restraining devices

Anticipatory salinity adaption

Design for cleanability

Freshwater layer removal

Multi-stage processes CO2 utilization efficiency

Variation of medium temperature, culture depth, and turbulence

Spillways

Weather (evaporation)

Blowdown Adapted nutrient feeding

Design of high-pH processes Deliberate spacing of carbonation stations “Mixing and biomass concentration” not included in this table as it does not refer to a solution applicable to any given cultivation system, but rather to a basic choice of a cultivation system

To keep the cultivation volume constant, evaporated

A summary of the engineering solutions presented in

water needs to be replenished regularly or continuously. The

this section is given in Table 1. A general overview of cur-

concomitant increase of substance concentrations in the cultivation medium – especially salinity when cultivating

rent major bioprocess engineering challenges is given in Fig. 2. Despite the presented multitude of elaborate engineering

with seawater or brackish water [105] – necessitates a total harvest followed by a culture restart using fresh medium

solutions for process and reactor design conceived in the past 60 years, microalgae mass cultivation for commodity

when a critical concentration of a medium component has been reached. By sophisticated process design using blow-

products is still to become a commercial reality. Therefore, further research and development is necessary.

down and adapted nutrient feeding, the time until a necessary culture restart can be extended significantly [101, 104, 108, 109].

Fig. 2 Current major bioprocess engineering challenges in open mass cultivation of microalgae. Further research is necessary to increase cultivation productivity and reduce losses.


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Model-based bioprocess and bioreactor development In the design of processes and reactors, model-based development (modeling, simulation, and validation) can be a very useful tool to reduce cost and time of the development process. Modeling improves understanding, and models can be used for experimental design and process optimization. Effects of design decisions can be evaluated in silico, important parameters can be identified, and the number of expensive experiments can be reduced. Process simulation is well-established in chemical engineering, and its application in bio-engineering is increasing [110, 111]. In mass cultivation of microalgae, due to low biomass concentrations and consequently large photobioreactors, modeling is especially advantageous [112]. Model-based development is an iterative approach of alternating model refinement and experimental validation. Model refinements can be variations of, e.g. scope, assumptions, equations, parameters, or algorithms [113, 114]. Model validation is the determination of the degree of accuracy to which a model represents reality [115]. Work flow of model-based development

Fig. 3 Flow chart for model-based development of bioreactors and bioreaction processes. The colors describe the affiliation to biology (green; contains kinetics for, e.g., growth rate, light inhibition, lipid accumulation), engineering (blue; contains reactor and process design parameters such as geometry, hydrodynamics, mass balances, pH control ranges), or both models (black).

An overview of the work flow is shown in Fig. 3. There are two loops: a ‘validation loop’ (left) and a ‘virtual design and

tial, some models were published without validation or were

evaluation loop’ (top right). The validation loop ensures that the models accurately describe reality: To determine param-

of such a complex design that correct validation is almost impossible.

eters for the biology model, experiments are conducted while reactor and process design remain unchanged, and the

Options for model validation

loop is repeated until the desired accuracy is achieved and the biological model has hence been validated. Thereafter,

In model-based development of cultivation systems for

the design loop is used to evaluate process and reactor modi-

photoautotrophic outdoor mass cultivation of microalgae

fications (e.g. water depth, raceway geometry [17, 116]) in silico. If the simulation suggests that a certain modification

using sunlight as energy source, there are three options for model validation: no validation, outdoor validation, and

could be advantageous, a validation experiment is conducted, e.g. using a mockup [21]. If the experimental results

indoor validation. Obviously, the first option should not be chosen. The second option, outdoor validation, is the most

differ from the model predictions, the model is further refined in the validation loop. Once the accuracy is as desired,

realistic validation strategy and in larger scales the only possible strategy. However, there are certain drawbacks.

both biological and engineering model have been validated

Outdoor validation is dependent on weather conditions, and

and an improved reactor or process design has been found using a model-based approach. More information regarding

due to the slow growth of microalgae a few days of bad weather can severely disturb a lengthy experimental cam-

various aspects of model-based development of bioprocesses can be found in specialized literature [21, 110, 114, 115,

paign if the model to be validated does not allow for varying weather conditions. Another problem is logistics: Personnel

117, 118].

and equipment must be brought to the location of the experiment, which can be – depending on the scale of the experi-

Huesemann et al. [119] gave a short overview of published models for photoautotrophic microalgae cultivation, noting that despite experimental model validation is essen-

ment – in isolated areas with poor infrastructure (e.g. road


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conditions, power grid, water supply network). Administrative obstacles in outdoor validation include land acquisition,

‘environmental photobioreactor’ for realistic reproduction of outdoor fluctuations of photosynthetic photon flux density

obtainment of building permits, and familiarization with local legal obligations. As experienced by Grobbelaar [8],

and medium temperature. Important environmental parameters are discussed in detail in the following section.

further problems can be bureaucracy, construction management, staff training, and protection of the premises. Miscommunication could also be more likely to happen if pro-

Realistic indoor reproduction of outdoor light, temperature and substance concentrations

cess development and experimental validation are conducted in different locations. If during the experiments the chosen

To be able to reproduce light, temperature and substance

location is found to be inadequate, sunk costs occur and valuable time is lost.

concentrations (e.g. CO2, O2, nutrients, salinity, contaminants, …) realistically, it must be known which aspects of

To avoid most of these problems, the third option – in-

these entities are of relevance to the cultivation of microal-

door validation – can be chosen. In indoor validation experiments, outdoor environmental conditions and experimental

gae and which can safely be omitted in the environment reproduction.

procedures must be faithfully reproduced to ensure a reliable validation. Experimental procedures can influence the phys-

Light

iology of microalgae, which might lead to differing experimental results. For example, inoculation cultures for indoor

Light can be described by a multitude of properties, but only

validation experiments should be grown and handled the

some are important for cultivation of photosynthetic organ-

same way as outdoor inoculation cultures would be grown and handled at the envisaged outdoor cultivation site. As a

isms. These are spectral energy distribution, irradiance, timing, and duration of illumination [121, 122]. For higher

complete reproduction of outdoor environmental conditions would be very complex, an appropriate level of detail should

plants (and analogously, photobioreactors designed for considerable light transmission through the side walls), direc-

be chosen. Environmental conditions which are important to microalgae cultivation must be identified and incorporated

tionality is also important [123], however this is not the case for microalgae grown in open mass cultivation systems

realistically into the reproduction.

where the main air-water interface can be approximated as a

For a photoautotrophic process, light is very important as it is the energy source for the cellular metabolism. Fur-

two-dimensional horizontal light receiving surface. In research on photosynthetic organisms, light should be meas-

thermore, temperature of the cultivation medium and concentration of substances (both beneficial and harmful ones)

ured in appropriate radiometric SI units (irradiance, W m-2; photosynthetic photon flux density, µmol photons m-2 s-1) to

in the medium are important. For example, temperatures tolerated by Scenedesmus acutus in outdoor reactors during

avoid confusion from the use of incorrectly named or photometric units [124, 125].

midday were found to be up to 8 °C higher than upper tem-

The wavelength range of ‘photosynthetically active ra-

perature limits determined in constant-temperature laboratory experiments [120]. Mixing is important for microalgae

diation’ (PAR) is usually agreed to be 400 – 700 nm [126]. Light of these wavelengths is therefore considered important

cultivation, but mainly (apart from minor wind-induced mixing) inherent to the bioreactor and therefore independent

for microalgae and should be provided in cultivation experiments. Notwithstanding the abundant use of ‘PAR’ in sci-

of environmental conditions. For best experimental results, in the indoor experimental

entific literature, other wavelengths outside the PAR range are also important for realistic cultivation. Photons of lower

setup the microalgae should hence be exposed to the same

wavelengths (ultraviolet radiation) are highly energetic and

environmental conditions regarding light, temperature and substance concentrations as they would be outdoors [53]. As

may damage microalgae [85], but UV radiation can also drive photosynthesis [127] and exert influence on species

technological capabilities expand, researchers conceive microalgae cultivation systems with increased realism in

composition [128]. Recent findings revealed that light of higher wavelengths (infrared radiation) can also be used for

selected environmental parameters. Recently, Huesemann et

photosynthesis [129, 130]. The once used wider definition of

al. [119] realistically reproduced the spectral energy distribution of sunlight in indoor experiments in 800 L raceway

‘PAR’ as 380 – 710 nm [126] therefore slightly better described reality than today’s definition.

ponds, and Lucker et al. [112] developed a 0.5 L closed


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Although all wavelengths in the PAR range can potentially be used in photosynthesis [131], currently marketed

synthetic organisms. The spectrum was calculated for direct and circumsolar radiation based on several assumptions

plant growth lights often only supply red and blue light. With these lamps, realistic growth cannot be expected. First-

regarding latitude, solar elevation, and atmospheric influences. Although variations of these parameters could influ-

ly, despite low absorption of green light by the different

ence the actual sunlight spectrum at a given location and

chlorophylls [132], in dense cultures photons of all PAR wavelengths are eventually absorbed and therefore available

despite the omission of skylight, the ASTM spectrum is currently the best choice available [144]. The spectral ener-

to drive photosynthesis in deeper layers [133, 134] as evidenced by the existence of a euphotic depth in microalgae

gy distribution of traditional plant growth lights is very distinct from the ASTM spectrum. With the progress of light

culture [8, 135]. Secondly, the spectral energy distribution of light has significant effects on cell physiology and develop-

emitting diode (LED) technology, today LED arrays with customized spectral energy distribution can be created [145].

ment of microalgae, which could result in competitive ad-

In addition to spectral energy distribution, realistic re-

vantages for certain strains and hence a domination of strains in the indoor experiment which would not dominate

production of the outdoor light environment requires correct irradiance, timing, and duration. Irradiance is the main de-

in outdoor cultivation. Another possible result of the use of light sources with a spectral energy distribution differing

terminant of the potential amount of growth and influences photosynthetic efficiency, photoinhibition, and photodamage

significantly from sunlight is influence on cultivation parameters such as growth rate, leading to flawed determina-

[146]. To be able to reproduce irradiance, timing, and duration realistically, the PAR or global radiation at the consid-

tion of these parameters [122, 136–141].

ered outdoor location must be known with high temporal

For realistic conditions in microalgae cultivation, it is therefore recommended to use light sources with a spectral

resolution for the period of time to be reproduced. There are several options to determine these values.

energy distribution similar to sunlight. A comparison of the spectral energy distribution of sunlight and some commonly

Own measurements can be conducted using a PAR quantum sensor, a pyranometer or a spectroradiometer. A pyranome-

used light sources is shown in Fig. 4, further examples were presented by Barnes et al. [142]. Sunlight is represented by

ter measures global radiation, from which PAR can be calculated. The irradiance ratio of PAR to global radiation

the ASTM G173-03 spectrum [143], which was developed

depends on several conditions, e.g. location, season, and

as standard solar spectrum for testing of photovoltaic equipment and can analogously be used as reference spec-

weather, and usually is 40 – 50 % [126, 147–149] which can be used as a first approximation. Instead of conducting

trum for light source selection in experiments with photo-

lengthy measurement campaigns, publicly available data

Fig. 4 Normalized spectral energy distribution of different light sources in the wavelength range of 350 – 850 nm. Sunlight at the earth’s surface (black, solid line; ASTM G173-03 direct+circumsolar [143]), incandescent lamp (red, dot-dashed), white LED (green, dotted; Labfors 5 Lux photobioreactor, Infors, Bottmingen, Swiss), fluorescent tube (blue, dashed; Fluora, Osram, Munich, Germany). Spectra measured using BlueWave UVNb spectroradiometer (StellarNet, Tampa, FL, USA).


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from nearby meteorological stations can be used. A source for worldwide high-quality measurements of global radia-

mended to use other LED dimming methods such as constant current reduction.

tion in fine temporal resolution is the Baseline Surface Radiation Network [150]. Some weather station networks (e.g.

Temperature

SURFRAD [151, 152]) are equipped with both pyranometers and PAR quantum sensors. As an example, environmental conditions of a desert location are shown in Fig. 5. For

In outdoor cultivation algae experience day/night, weatherbased, and seasonal cycles of cultivation medium tempera-

realistic growth conditions, the outdoor irradiance graph of the location envisaged for large-scale outdoor cultivation

ture. Since temperature influences growth of microalgae, it should be reproduced correctly for realistic results. Howev-

should be faithfully reproduced in indoor experiments. With less accuracy, PAR data can also be calculated from satellite

er, heat fluxes in indoor and outdoor experiments could be different for several reasons. In glass houses, roof glazing

data [153] or using completely theoretical models of the

materials could filter solar infrared radiation [159], while

solar elevation angle. Local weather deviations such as industrial emissions [65] or regular presence of fog [104] will

artificial illumination could supply more or less infrared radiation than the sun would supply outdoors. Significant

however not be revealed by this approach. [154, 155] If the spectrum of the artificial light source to be used is

radiative cooling occurs in outdoor reactors during the night [160], but could be reduced in indoor experiments due to the

remarkably different from the solar spectrum, a conversion from irradiance to photosynthetic photon flux density is

differing radiative properties of the ceiling. Heat loss by evaporation or conduction/convection could be different due

useful to ensure realism in terms of the amount of PAR

to the absence of wind indoors or a different setup of the

photons supplied. Conversion factors for common light sources were published by Biggs [125], for other spectra

cultivation systems. Furthermore, if water is added during the cultivation process, its temperature could be different

such as custom LED arrays they can be calculated [126]. To reproduce variable irradiance as experienced in outdoor

from the temperature of the water that would be used outdoors. A heat balance as described by Béchet et al. [107] can

cultivation, LEDs can be dimmed. Pulse width modulation, a frequently used dimming method [156, 157], works by

be used to assess and optimize the comparability of the experimental setup indoors and the real outdoor situation.

rapidly switching the LEDs on and off, hence the flashing-

Weather stations can provide data to establish realistic

light effect [2, 158] could be inadvertently used. For realistic conditions in microalgae cultivation, it is therefore recom-

boundary conditions in a such heat balance calculation (Fig. 5).

Fig. 5 Environmental conditions in a desert. Temperature (red, solid line), Relative Humidity (blue, dotted), Global Radiation (black, thick dashed), PAR (green, thin dashed). On the second day, minor cloud cover can be observed in the irradiance. Data from SURFRAD station in Desert Rock, NV, USA on Sep 2/3, 2014 [154]. To convert PAR irradiance (W m-2) to photosynthetic photon flux density (µmol photons m-2 s-1), multiply by 4.6 [155].


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Substance concentrations

Conclusion

Realistic reproduction of substance concentrations appears feasible because the devices for nutrient feeding and CO2

Major engineering challenges in the implementation of commercial large-scale open photobioreactors for the culti-

supply as well as the temporal profiles of these substance

vation of microalgae are poor and uneven mixing, low bio-

additions can easily be implemented in a scaled-down version in the indoor experiment. However, the indoor envi-

mass concentration, low CO2 utilization efficiency, contamination, and the strong influence of weather on the cultiva-

ronment protects from rainfall and modifies evaporation, resulting in reduced fluctuation of the cultivation volume

tion. In the history of microalgae mass cultivation, different solutions for these problems using bioprocess and bioreactor

and deceptively undisturbed process operation. Realism in evaporation in indoor experiments can be increased by cor-

engineering considerations have been conceived. Further research and development is necessary as up until now, only

rect reproduction of relative air humidity, wind speed, culti-

few microalgae cultivation processes have reached commer-

vation medium temperature, and irradiance prevalent at the location envisaged for large-scale outdoor cultivation; the

cial viability. Using modeling and simulation tools, development can

effects of rainfall can be reproduced by pulsed addition of water.

be conducted using a model-based approach for costeffective design of novel bioreactors and bioprocesses that

During scale-up from laboratory to industrial scale, a transition from reagent grade to technical grade nutrients

could outperform currently existing mass cultivation systems. Validation is required to ensure accurate representa-

must be made to reduce cost in industrial-scale cultivation. It

tion of reality by the model. It was shown that indoor valida-

is advisable to test the effects of less pure technical grade chemicals in indoor experiments before a cultivation system

tion is advantageous compared to outdoor validation if relevant elements of the outdoor environment can be faithfully

is implemented in large scale. Additional variation of cultivation medium components

reproduced indoors. Considerations for indoor reproduction of the most important conditions in photoautotrophic culti-

can stem from the use of different water sources. In indoor experiments, cultivation medium is usually created from

vation of microalgae – light, temperature, and substance concentrations – were elaborated, and the current state of the

purified water (in laboratory scale) or tap water (in larger

art was discussed.

experiments). Tap water is less pure and can have minor variations in its composition due to external effects (e.g.

The impact of more or less realistic reproduction of environmental elements on a cultivation experiment varies

increased nutrient concentration due to manure spread on fields in the drainage basin of the tap water source). Contra-

depending on the degree of the deviation and on the element itself. Hence, further evaluation of this subject is necessary.

ry to that, due to the large volume required, water in largescale outdoor cultivation would be sourced with basic saniti-

As a general conclusion, increased effort in realistic environment reproduction will lead to more realistic cultivation

zation equipment from a nearby natural water body (e.g.

results, more accurate process models, and ultimately to

ground, lake, sea or river water) influenced by environmental conditions and would therefore contain significantly

more relevant improvements in design and operational strategies of open photobioreactors for mass cultivation of mi-

more components at varying concentrations than tap water. Knowledge of the different water compositions is important

croalgae.

for indoor development of nutrient feeding strategies adapted to outdoor water quality. Finally, most contamination sources are not present in an indoor environment. For realistic development of contamination control strategies, artificial contamination experiments can be conducted indoors by introducing contaminating substances such as other algae strains or sand into the open photobioreactor.

Acknowledgments Funding for this work was provided by the Bavarian State Ministry for Economic Affairs and the Media, Energy and Technology (Munich, Germany), the Bavarian State Ministry of Education, Science and the Arts (Munich, Germany) and Airbus Group (Leiden, The Netherlands). NOAA's Earth System Research Laboratory/Global Monitoring Division - Radiation (G-RAD, Boulder, CO, USA) is acknowledged for providing SURFRAD data. The authors thank Skye Thomas-Hall, Peer Schenk (The University of Queensland, Brisbane, Australia), and Thomas Brück (Industrial Biocatalysis, Technical University of Munich, Garching, Germany) for fruitful discussions on microalgae research.


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