DOI: 10.3303/CET2293058 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Paper Received: 9 December 2021; Revised: 9 March 2022; Accepted: 21 April 2022 
Please cite this article as: Valentini M., Mazzelli A., Iannone A., Mrotek E., Sapone V., Bravi M., 2022, Techno-economic Evaluation of 
Heterotrophic Microalgal Cultivation Approaches, Chemical Engineering Transactions, 93, 343-348  DOI:10.3303/CET2293058 
  

 CHEMICAL ENGINEERING TRANSACTIONS  
 

VOL. 93, 2022  

A publication of 

 

The Italian Association 
of Chemical Engineering 
Online at www.cetjournal.it 

Guest Editors: Marco Bravi, Alberto Brucato, Antonio Marzocchella 

Copyright © 2022, AIDIC Servizi S.r.l. 

ISBN 978-88-95608-91-4; ISSN 2283-9216 

Techno-economic Evaluation of Heterotrophic Microalgal 

Cultivation Approaches 

Marco Valentini a*, Alessio Mazzelli b, Annalaura Iannone a, Eryka Mrotek a, Vittoria 

Saponea, Marco Bravia  

a Dept. Chemical Engineering Materials Environment, University of Rome La Sapienza; via Eudossiana 18, 00184 Rome, 

Italy 
b NextChem S.p.A. via di Vannina 88/94, 00156 Rome, Italy 

ma.valentini@uniroma1.it 

This work focuses the attention on the evaluation of different heterotrophic microalgal cultivation approaches, 

finding the best process solution to achieve the accumulation of high value-added metabolites, such as 

astaxanthin and polyunsaturated fatty acids (PUFA), trying also to use a wastewater as carbon source.  

Three different approaches are evaluated: 1) Classical batch cultivation in sterilized fermenter; 2) Impulse 

nutrient feeding without sterilizing the reactor; 3) Impulse nutrient approach coupled with wastewaters’ usage in 

two different parallel reactors. This latter strategy is the one adopted in the European Project MEWLIFE 

(MicroalgaE biomass from phototrophic-heterotrophic cultivation using olive oil Wastewaters) that aims to 

produce microalgal biomass in an integrated photo-heterotrophic cultivation system using preconcentrated (in 

a membrane filtration plant) olive oil mill wastewater (OOMWW) as carbon source for growing algae, thus 

contributing to waste reuse and valorisation. Thanks to the Aspen Custom Modeler software, it is possible to 

write codes to represent the fermentation units, which are normally not present in the databases of most 

common simulators. Finally, a technical-economic analysis is conducted to evaluate the most advantageous 

process. At present, the pulse technique represents an extremely promising strategy with margins of 

improvement, deserving thus further investigation. 

1. Introduction 

Various microalgae species can be used in raw or semi-decomposed form as organic biofertilizers, for 

remediation or biosorption of environmental problems and for the production of biofuels and carbon dioxide 

sequestration (Aishvarya et al. 2015). 

In addition to proteins, pigments, biopolymers and fatty acids, microalgae can produce antioxidant substances 

for commercial and pharmaceutical purposes (Mazzelli et al. 2019a, Mazzelli et al 2019b, Mazzelli et al. 2022). 

Microalgae must be cultivated in a way that meets all their requirements for growth and maintenance. In addition, 

it is necessary to consider several factors and the most important variables that regulate algal growth, such as 

nutrient quantity and quality, light, temperature, and pH. (Mazzelli et al. 2018, Acièn 2013). Cell biomass 

productivity is not only affected by all these parameters, but they also affect the pattern, pathway, and activity 

of cellular metabolism and the resulting change in cellular composition. The cultivation methods are classified 

based on their exposure to light (phototrophy) or absence of light (heterotrophy). It is possible to reach higher 

cell densities and to use traditional fermenters for microalgae cultivation by using heterotrophic cultivation, which 

does not require CO2 (unlike photoautotrophic cultivation). (Hua et al. 2017).  

The purpose of this study is to examine three different microalgal heterotrophic cultivation techniques in terms 

of productivity and economics. The first one involves classical batch fermentation followed by sterilization, in 

which the nutrients are supplied at the beginning, and the microalgae are added after having been sterilized. To 

grow the microorganism, the fermenter is closed, and the temperature and pH are kept constant. Using this 

technique without sterilization causes problems with bacterial contamination since bacteria and fungi compete 

for growth with microalgae (Sandani et al. 2020).  

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This study analyzes the impulse nutrient feeding without sterilizing the reactor; an innovative solution patented 

by NextChem (EP3498855) as a second cultivation technique. By using this approach, it is possible to avoid the 

use of sterilization and aseptic conditions, since the carbon and nitrogen sources are injected respectively into 

the growth medium in alternate pulses. The principle behind the implemented cultivation strategy is that 

microalgae can grow in the absence of nitrogen by consuming nitrogen stored in the cells, while most bacteria 

can grow only if all nutrients are simultaneously present in the culture medium. Using the pulse technique with 

the OOMWW-based feeding (as organic carbon source), the third and last technique studied aims to both 

accumulate metabolites and treat wastewater. This method was used in the MEWLIFE (https://www.mewlife.eu) 

project. MEWLIFE was a LIFE project aiming to demonstrate the environmental benefit and economic feasibility 

of an innovative approach to produce microalgal biomass in an integrated phototrophic – heterotrophic 

cultivation system using preconcentrated (in a membrane filtration plant) olive oil wastewaters as carbon source 

for growing algae, thus contributing to waste reuse and valorization. 

According to estimates, the annual world production of OOMWW is between 7 and over 30 million m3. In this 

context, there is an urgent need to find ways to treat this liquid residue and other by-products of the olive oil 

industry. Considering these difficulties in the disposal of vegetation water, its use as a growth medium for 

microalgae can be a very promising alternative.  

Lastly, this work discusses a techno-economic evaluation comparing a classical cultivation technique, the pulse 

technique and OOWMM + pulses. A techno-economic evaluation of the innovative pulse cultivation technique 

and OOWMM + pulses has not been done so far. 

2. Materials and methods 

2.1 Experimental conditions and modeling 

In the present work the experimental data, for the pulsed technique, are taken from NextChem database related 

to MEWLIFE project. In this project indeed two fermenters working in parallel, with an operative volume of 3 m3 

each, have been used. One is F-101, always operating according to the pulse technique to accumulate nitrogen 

inside the microalgal cells, using pure glucose as carbon source. Once the biomass in F-101 reaches the desired 

concentration, ending its cultivation cycle only when the nitrogen in the broth is consumed, a part of it is 

transferred to F-102. This biomass will then have the maximum possible intracellular nitrogen content that will 

be exploited in the second fermenter, since only organic carbon will be present in the reactor as a nutrient in 

form of OOMWW, to induce the accumulation of starch and lipids in the microalgae.  

To create a tool for the simulation of these coltivation approaches, the following model is used, relating this 

model to the existing fermenters with an operating volume of 3 m3. The specific growth rate is defined as a 

function of the maximum specific growth rate (𝜇𝑚) and several growth-limiting factors according to Eq. 1 

(Murwanashyaka et al. 2020). 

𝜇 = 𝜇𝑚 ∗ ∏ 𝑓(𝑆𝑖)
𝑛
𝑖=1      (1) 

n is the total number of limiting factors and f(Si) is the function that defines the extent to which the Si factor limits 

growth. The function describing how much a limiting factor affects the growth rate can take many different forms 

including the Monod function which is widely used. 

𝑓(𝑆𝑖) = 
𝑆𝑖

𝐾𝑆+𝑆𝑖
    (2) 

Ks is the semi-saturation constant.  

Consequently, the specific growth rate of algal biomass, assuming glucose G, nitrate N, and phosphate P as 

the limiting substrates, can be expressed as follows. 

𝜇 =
𝜇𝑚∗ 𝐺∗𝑁∗𝑃

(𝐾𝑛+𝐺 )+(𝐾𝑛+𝑁)+(𝐾𝑝+𝑃) 
    (3) 

Kg, Kn, and Kp are the semi-saturation constants for glucose, nitrate, and phosphate consumption, essential 

substrates for cell growth. Expressions of their consumption rates are the critical elements of the model since 

their uptake regulates cell growth and metabolite accumulation. Typically, in a simplified manner, these rates 

are linearly related to the biomass growth rate according to the Luedeking-Piret expression as follows.  

𝜌𝑔 = −(
1

𝑌𝑔
) ∗ 𝜇 − 𝑚𝑔     (4) 

𝜌𝑛 = −(
1

𝑌𝑛
) ∗ 𝜇 − 𝑚𝑛     (5) 

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𝜌𝑝 = −(
1

𝑌𝑝
) ∗ 𝜇 − 𝑚𝑝     (6) 

Yg, Yn, and Yp are the biomass yield coefficients, and mg, mn, and mp are the maintenance coefficients on carbon, 

nitrogen, and phosphorus consumption, respectively. In microalgae culture, once growth has been limited due 

to nutrient depletion, assimilated carbon can be accumulated as storage products in the form of starch and/or 

lipids (Breuer et al. 2015). Based on the above formulations, the dynamics of the algal culture in this batch work 

are therefore described by a system (Eq. 7) set of six differential equations. 

{
 
 
 
 

 
 
 
 
𝑑𝑋

𝑑𝑡
= 𝜇 ∗ 𝑋 

𝑑𝐺

𝑑𝑡
= 𝜌𝑔 ∗ 𝑋

𝑑𝑁

𝑑𝑡
= 𝜌𝑛 ∗ 𝑋

𝑑𝑃

𝑑𝑡
= 𝜌𝑝 ∗ 𝑋

𝑑𝑆

𝑑𝑡
= 𝜋𝑠 ∗ 𝑋

𝑑𝐿

𝑑𝑡
= 𝜋𝑙 ∗ 𝑋

     (7) 

The mathematical model describing the dynamic behavior of the system is a system of ordinary differential 

equations of the following general form: 𝑑𝑥(𝑡) 𝑑𝑡 = 𝑓(𝑥(𝑡), 𝜃) where 𝑥 is the vector of differential variables 

(X,G,N,P,S,L) and θ is the vector of model parameters to be determined. Solved by using the Simulated 

Annealing Constrained method to find the best set of parameters. Table 1 shows the parameters used in "run 

9" found in the Murwanashyaka et al. 2020 model. 

Table 1: Reported by Murwanashyaka et al. 2020  

Kg [gC L-1] Kn [gC L-1]  Kp [gP L-1] μm [h-1] αs [g gDW-1] βs [g gDW-1 h-1] αl [g gDW-1] βl [g gDW-1 h-1] 

1 1.5*10-2 2*10-3 1.1*10-1 3.1*10-1 4.4*10-4 3.6*10-2 3.4*10-4 

 

Yg [gDW gC-1] mg [gC gDW-1h-1]  Yn [gDW gN-1] mn [gN gDW-1h-1] Yp [gDW gP-1] mp [gP gDW-1h-1] R2 

1.2 4.3*10-4 16 1*10-7 68 3.7*10-5 0.97 

2.2 Batch and Pulsed technique simulation 

For the batch simulation performed in this work, the parameters of Murwanashyaka et al. 2020 are used, since 

the concentration of substrates fed to the fermenter reflected the relative ratio of nutrients used in this 

experimental trial. ASPEN CUSTOM MODELER® (ACM), integrated into Aspen Plus® is used for fermentation 

modeling (Atikah et al. 2019).  

The heterotrophic cultivation of microalgae poses a severe problem of managing the contamination of bacteria 

that have growth rates an order of magnitude higher than those of microalgae (Sandani et al. 2020). To solve 

this problem the High-Pressure, High-Temperature (HTHP) treatment is considered in this work and applied in 

the process simulation to the inoculum before it enters the fermenter, using for sterilization phase T=121°C, 

P=0.2 MPa with a treatment time of 15 min (Ashidate et al. 2018).  

Similar approach is used for the pulsed feeding (both for OOMWW and glucose usage), modeling it in an ACM 

script to obtain a simulation of heterotrophic growth. Indeed, the same formula described above for the metabolic 

and growth modeling are used but the typical sawtooth trend is obtained considering the correct timing, between 

the start/end of glucose/nitrate phase, taken from the experimental results database of NextChem. The pulse 

technique allows to have a reactor management less critical from the point of view of bacterial contamination. 

This is reflected in lower investment and management costs.  

3. Results 

3.1 Process simulation  

The results of the simulation of the batch culture technique on ACM are shown in Fig. 1a. Using a 3 m3 fermenter, 

a final biomass concentration of approximately 15.5 g/l is achieved, with initial glucose and nitrate amounts of 

10 and 0.6 g/l, respectively. Interestingly, despite the depletion of the nitrogen source from the growth medium, 

the accumulation of carbohydrates and lipids by the biomass continues. Indeed, under nitrogen deficiency 

conditions, the biomass undergoes to a stress condition that leads it to accumulate starch and lipids as storage 

products necessary for its survival. Considering a process time of about 5.5 days, including the phases of filling, 

345



cleaning and sterilization, the following results are obtained: Biomass productivity 5.9 (kg/day), Glucose 

consumption 5.4 (kg/day) and Nitrate consumption 0.32 (kg/day). 

Regarding the simulation of the pulse technique the results are shown in Fig 1b. The profile obtained shows the 

trend of saw-tooth from the biomass (red line), with growing trends during the glucose phases (blue line) and 

consumption trends during nitrate phases (green line). The plot also shows the profile of the internal nitrogen 

quota (light-blue line), which shows an oscillating trend between the maximum and minimum values that were 

determined previously. Considering a process time of about 8 days, also considering filling and cleaning of the 

fermenter, the following values of productivity and consumption are obtained: Biomass productivity 5.91 

(kg/day), Glucose consumption 16.03 (kg/day) and Nitrate consumption 1.7 (kg/day). 

 

 

Figure 1: Profile concentration of a) Batch simulation, b) Pulsed technique simulation 

With respect to the third option applied in the MEWLIFE project, the operating phase is divided in two fermenters 

working in parallel as described in previous paragraph. The cycle in F-101 is interrupted at the end of the second 

nitrate phase (after 6.5 days), when a biomass concentration of about 10 g/l is reached and a part of this 

suspension is sent to F-102 cultivation with only OOMWW. 

Specifically, the microalgae suspension of 3 m3 of the first fermenter is in part (1.33 m3) recirculated to the F-

101 to obtain, through dilution, the initial concentration of 4.5 g/l of biomass with which to start the cycle again, 

while the remaining part (1.67 m3), will be inoculated into the second fermenter F-102, on which only the first 

glucose phase (substituting glucose with OOMWW) of the pulsed strategy is carried out, thus starting from 5.63 

g/l and arriving at 11.3 g/l of microalgal biomass. The two fermenters run in parallel; the F-102 can run two 

consecutive cycles of about 3 days each before the F-101 ends.  

  

Figure 2: a) Biomass and Nutrient Profile in F-101 b) Biomass and Nutrient in F-102 

The second cycle of F-102 is inoculated with the microalgal suspension derived from the previous phototrophic 

section since it has not yet completed its cycle and therefore cannot be sent to F-102. After reaching 4.5 g/l of 

biomass concentration in the second cycle, F-102 will reach 9 g/l of biomass concentration. To obtain a saving 

on the glucose to be supplied, a concentrate of vegetation waters is also sent to the second fermenter. Fig. 2 

shows the biomass and nutrient profiles of the two fermenters. Overall, the following data are obtained: Biomass 

productivity 7.27 (kg/day), Glucose consumption 15.0 (kg/day) and Nitrate consumption 2.07 (kg/day). 

346



3.2 Techno-economic analysis 

Economic analyses of the studied fermenters are performed to evaluate the most cost-effective solution starting 

with the AACE Association for the Advancement of Cost Engineering guidelines for making cost estimates of 

an engineering project. The present work can be classified as an estimation class number 3, with a cost 

estimation error ranging from 20% under to 30% over. Once both the CAPEX (Fig. 3) and OPEX needed to build 

and operate the plant are estimated, the Cost Of Production (COP) must be calculated, which is the cost that 

must be incurred per unit of product. 

 

 

Figure 3: Economic results of each cultivation approach - a) Batch with sterilization b) Pulsed without sterilization 

c) Pulsed + OOMWW usage in parallel reactors 

Although the amount of biomass produced is the same, with the pulsed strategy it is possible to store inside the 

microalgal cells a higher amount of starch and lipids. This happens because with pulsed fermenter feeding, the 

biomass is repeatedly subjected to a stress condition, due to the absence of the nitrogen source in the growth 

medium. As a result, the biomass responds to this stress by accumulating more products such as starch and 

lipids, resulting in a productivity of these metabolites that is almost double that of the batch strategy, where 

nitrogen and glucose are fed simultaneously. Fig. 3 shows the economic results in terms of CAPEX of each 

cultivation technique. Overall, for the same biomass produced, the operating costs are similar for the first two 

cultivation techniques (batch 130 €/kg, pulsed 128 €/kg), while for pulsed + OOMWW, it is 187 €/kg. Obviously, 

since the goal of the process is the extraction of these metabolites, the pulse strategy proved to be more 

promising and convenient than the batch case with sterilization of the incoming current. Despite the use of two 

fermenters and a whole section of wastewater pre-treatment, the pulse with vegetation water system, with the 

related investments, can reach production cost values very close to the single fermenter pulse strategy, by 

reason of a significant saving on exogenous glucose, with still wide possibilities of development. 

Consequently, the goal is to aim at optimizing growth in the presence of vegetation water, to be able to mitigate 

as much as possible the consumption of glucose, which is one of the main cost items that characterizes this 

cultivation strategy. Furthermore, the usage of OOMWW could further reduce the investment cost because an 

income due to the wastewaters’ treatment, paid from mill sites, should be considered for a more complete 

business plan of this technology. 

4. Conclusions 

This work shows how heterotrophic microalgae cultivation can be effectively conducted even without sterilization 

and aseptic procedures due to decoupled nutrient feeding. This also allows the production of secondary 

metabolites to be optimized based on the nitrogen starvation condition, to which the microalgae are alternately 

subjected. The problem with this cultivation technique is related to the low productivity and high glucose 

consumption. This is solved by running two fermenters in parallel, with one fed by wastewater from the olive 

mills, which reduces the extra supply of carbonaceous substrate.  

The final aim is to achieve complete remediation of the wastewater, which at present still cannot be discharged 

to the sewer after being used in the fermenter. Further upgrades of the work could be the possibility to include 

other wastes, such as those from the dairy industry, to further limit the demand for glucose.  

From an economic point of view, the pulse strategy conducted in parallel with a cultivation phase in the presence 

of vegetation water has shown extremely encouraging results. However, further evaluations should be done in 

347



case it is decided to increase productivity. Batch production, in fact, can be increased simply by providing more 

nutrients to the microalgae. The pulse technique, on the other hand, is more rigid from this point of view, as the 

concentration of substrates to be loaded in each phase must not exceed the value predicted by the internal 

nitrogen quota trend. Therefore, for this technique it would be necessary to intervene with additional investments 

related to the use of a larger fermenter or several bioreactors placed in parallel. Currently, the pulse technique 

represents an extremely promising strategy with ample room for improvement and therefore deserves further 

investigation to also optimize the usage of wastewaters as organic source, coupling thus biomass production 

and wastewater treatment.  

Acknowledgements 

This work was supported by the European Union in the framework of the LIFE program (MEWLIFE project - 

LIFE17 ENV IT 000180). The authors wish to tank Dr. Vittorio Galanti for efforts made on the Aspen Model. 

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