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Recovery of bacterioruberin and proteins using

Cite this: RSC Adv., 2022, 12, 30278
aqueous solutions of surface-active compounds†
Open Access Article. Published on 24 October 2022. Downloaded on 2/2/2023 6:43:15 AM.

Bárbara M. C. Vaz,‡a Mariam Kholany,‡a Diana C. G. A. Pinto, b Inês P. E. Macário,ac

Telma Veloso,ac Tânia Caetano, c Joana L. Pereira, c João A. P. Coutinho a
and Sónia P. M. Ventura *a

Haloarchaea microorganisms are little explored marine resources that can be a promising source of
valuable compounds with unique characteristics, due to their adaptation to extreme environments. In
this work, the extraction of bacterioruberin and proteins from Haloferax mediterranei ATCC 33500 was
investigated using aqueous solutions of ionic liquids and surfactants, which were further compared with
ethanol. Despite the good performance of ethanol in the extraction of bacterioruberin, the use of
aqueous solutions of surface-active compounds allowed the simultaneous release of bacterioruberin
and proteins in a multi-product process, with the non-ionic surfactants being identified as the most
promising. The optimum operational conditions allowed a maximum extraction yield of 0.37
0.01
−1 −1
mgbacterioruberin gwet biomass and 352
9 mgprotein gwet biomass with an aqueous solution of Tween® 20
Received 22nd April 2022
Accepted 2nd October 2022
(at 182.4 mM) as the extraction solvent. In addition, high purities of bacterioruberin were obtained, after
performing a simple induced precipitation using ethanol as an antisolvent to recover the proteins present
DOI: 10.1039/d2ra02581g
in the initial extract. Finally, a step for polishing the bacterioruberin was performed, to enable solvent
rsc.li/rsc-advances recycling, further closing the process to maximize its circularity.

Recently, marine resources like algae and cyanobacteria have

Introduction gained signicant interest as renewable feedstocks to produce
Our continued reliance on a petroleum-based economy to several natural bioactive compounds. Their sustainable use can
satisfy humankind’s needs cannot be sustained anymore.1 The easily t the priorities of the 2030 Agenda for Sustainable
traditional model of take, make and dispose using fossil-based Development.5 They can generate biofuels and bioenergy and
feedstocks has severe implications for human health, the integrate bioactive medicinal products, food, and materials.6 In
depletion of fossil resources, and as a driver of climate change.2 this context, halophilic microorganisms from the domain
In this sense, the development of sustainable technologies and Archaea, with high commercial potential, have emerged as
practices to ensure a gradual transition to a renewable (bio- underexplored marine resources, easily found around the globe,
based) economy should be encouraged.1,3 By considering the mostly in hypersaline environments. Additionally, they are
concepts of circular economy and integrated bioreneries, the natural producers of several relevant high-demand products,
complete valorization of all biomass fractions could be ach- such as proteins, poly(3-hydroxybutyrate), poly-
ieved. Besides maximizing the value of raw materials, waste hydroxyalkanoates, and carotenoids.7
production could be minimized or avoided, which leads to an Proteins are complex molecules with a unique three-
increase in the economic viability of downstream processes for dimensional structure composed of numerous combinations
industrial implementation.4 of the 20 different amino acids attached to form long chains.8
There is a problem in searching for new sources of proteins for
food purposes. However, due to their multiple biological roles,
their biotechnological interest is vast,9,10 and thus, depending
a
CICECO – Aveiro Institute of Materials, Department of Chemistry, University of on the nal application, their purication may be required. In
Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal. E-mail:
halophilic archaea (or haloarchaea), halophilic proteins assist
[email protected]
adaptation to extreme saline conditions by modifying their
b
LAQV – REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro,
Portugal structure. The presence of acidic amino acids on the protein
c
CESAM – Centre for Environmental and Marine Studies, Department of Biology, surface confers a more hydrophilic and exible surface, capable
University of Aveiro Campus Universitário de Santiago, 3810-193 Aveiro, Portugal of increasing the hydration of the protein. Hence, its aggrega-
† Electronic supplementary information (ESI) available. See DOI: tion is prevented, and the protein’s functionality is retained.11
https://doi.org/10.1039/d2ra02581g On the other hand, carotenoids are natural colorants well
‡ These authors contributed equally to this manuscript.

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known for their biotechnological applications and key proper- simple and efficient at the lab scale, is not industrially
ties for human health.12,13 The haloarchaea group can produce adequate. In this sense, more efforts are needed to develop
many carotenoids, including b-carotene, salinixanthin, bacter- efficient and sustainable approaches with scale-up potential
ioruberin, and its precursors lycopene and phytoene.7,12 Among encompassing the valorization of multiple products obtained
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these, bacterioruberin represents the most abundant carot- from this biomass.
enoid produced, since all the species with sequenced genomes In this work, aqueous solutions of surfactants and ionic
encode its biosynthetic pathway.14 Bacterioruberin can inu- liquids (ILs) were investigated with the prospect of simulta-
ence the membrane uidity, acting as a water barrier and neously recovering different products from the same biomass in
allowing the permeability of oxygen and other molecules a more sustainable way. As surfactants and tensioactive ILs are
needed for the cell’s survival in hypersaline environments. amphiphilic molecules,33 they become effective solvents in the
Open Access Article. Published on 24 October 2022. Downloaded on 2/2/2023 6:43:15 AM.

Consequently, the membrane structure rigidity is extraction of both more hydrophilic compounds, e.g.
increased.7,15,16 Of great relevance, this C50 isoprenoid, proteins,10,34–37 and more hydrophobic molecules,37–41 such as
composed of 13 conjugated double bonds and 4 hydroxyl bacterioruberin. Furthermore, their use has several advantages,
groups, has a higher antioxidant activity than other molecules. namely milder process conditions while using water as the
Therefore, higher protection can be achieved against intense main solvent, which could also be attractive for cell disruption,
light, gamma irradiation, and DNA damaging agents such as as most haloarchaea lyse rapidly when suspended in water.7
radiography, UV-irradiation, and H2O2 exposure.7,15,16 These Aer the extraction of multiple compounds, a purication step
characteristics make bacterioruberin an appealing bioactive is usually needed to obtain each compound with the appro-
compound to be applied in the food, cosmetic and pharma- priate purity for the specic application. Induced protein
ceutical industries.17 Moreover, their commercial potential goes precipitation was applied in this work. The addition of
beyond the applications previously mentioned. More recently, a precipitating agent lowers the solubility of proteins in water,
some of us have investigated its optical potential to develop causes their precipitation42 and allows the separation of the
optical sensors and retinal gene therapy formulations (ongoing solvent. As well as its simplicity, it has industrial potential,43
work), with promising results. Despite the high potential of being recognized as having a low carbon footprint and
bacterioruberin and halophilic proteins, efficient methods of economic impact.44,45 In the end, a polishing step was also
extraction and purication are still at an early stage. Most applied to bacterioruberin, allowing the solvent to be recycled.
literature focuses on exploring protein structures and their
adaptations to extreme environments,18–23 and optimizing bac-
terioruberin production and its possible applications.15,17,24–26 Results and discussion
The studies found just quantify bacterioruberin extracted using Screening of surfactants and ILs as solvents
organic solvents, without considering biomass valorization. A large screening of aqueous solutions of surface-active
Regarding the purication step, some works used thin-layer compounds belonging to different classes (cationic, anionic,
chromatography.17,27–32 However, this technique, despite being non-ionic) and other non-surfactant agents was performed at

Fig. 1Bacterioruberin yield of extraction using aqueous solutions of cationic, anionic, and non-ionic compounds at 100 mM, as well as ethanol,
used as a control solvent, at room temperature (20–25  C), under a constant vertical rotation at 50 rpm, for 45 min, protected from light
exposure, and at a fixed SLR of 0.1 gwet biomass mLsolvent−1.

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100 mM to assess their ability to extract bacterioruberin (the non-ionic form. This allows for more stable interactions
most valuable compound present). The outcomes were bench- between the pigment and the micelle hydrophobic core.
marked against ethanol. The surfactants tested were pre- However, with such conditions (with a surfactant concentration
selected considering previous works that identied them to be of 100 mM), a high viscosity was observed in most solutions
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efficient in the extraction of biomolecules, namely caroten- obtained aer extraction (see Table S1 in the ESI†), which is
oids.37,38,46 Table S1 in the ESI† reports the ability of the surface- normally correlated with the release of chromosomal DNA
active compounds tested (30 in total) to extract bacterioruberin. during cell disruption.48,49 This phenomenon, whose origin is
Fig. 1 shows the yields of extraction (mgbacterioruberin gwet yet to be proved, imposed some experimental constraints. To
−1
biomass ) of the systems with extraction capability, in terms of overcome this issue and better assess the extraction capacity of
the ability of the surfactants/ILs to induce the release of the the most efficient solvents (here, the non-ionic surfactants),
Open Access Article. Published on 24 October 2022. Downloaded on 2/2/2023 6:43:15 AM.

target compound. Photographs of all systems can be found in a second screening at 250 mM surfactant concentration fol-
Fig. S1 in the ESI.† lowed, with the respective results shown in Fig. 2. The UV-Vis
Ethanol displayed a better bacterioruberin yield of extraction spectra of the resulting extracts can be found in Fig. S2 in the
than aqueous solutions of surface-active compounds (Fig. 1). ESI.†
Ethanol is recognized for its excellent performance in pigment Since the non-ionic surfactants tested are viscous liquids at
extraction and membrane solubilization.40,47 Thus, and also room temperature, an increase in their concentration results in
owing to the presence of bacterioruberin in the cell membrane, a more viscous aqueous solution. However, the viscosity of the
which endows it with a low water permeability,7 the perfor- nal solutions (aer bacterioruberin extraction) at 250 mM was
mance of ethanol in bacterioruberin extraction was expected. revealed to be lower and more manageable than that of the
Regarding the performance of the aqueous solutions of same solutions at 100 mM. On the other hand, a very high
surface-active compounds as depicted in Fig. 1 and Table S1 in surfactant concentration in the initial aqueous solution can
the ESI,† almost none of the anionic or the non-tensioactive also lead to viscosity issues. Therefore, the choice of an
compounds were able to efficiently extract bacterioruberin. adequate concentration of surfactant seems to be a key factor in
The non-tensioactive ILs have a low capability to disrupt the this work’s success. Fig. 2 shows that the highest performances
phospholipidic cell membranes due to their hydrophilic at these higher concentrations were once again accomplished
nature.38 The low performance of the anionic surfactants can be by Tween® 20 and ethanol. Nevertheless, ethanol lacks the
attributed to the fact that both types of tensioactive compounds desired capacity to simultaneously extract bacterioruberin and
and the phospholipids (components of the cell membrane) are the proteins present in the biomass, thus failing to achieve the
negatively charged, creating repulsive forces.37 In contrast, envisioned multi-product exploitation scenario. Aqueous solu-
almost all cationic and non-ionic compounds were able to tions of surfactants provide a milder environment, thus allow-
release the compound of interest. This can be explained by their ing the extraction of different fractions of interest (proteins and
ability to establish attractive electrostatic interactions between pigments), both with applications related to the food industry.
the IL cation and the negatively charged head of the phospho- Ethanol leads to a protein extraction yield of approximately 24
lipids.38,39 Bacterioruberin is almost exclusively present in its mgprotein gwet biomass−1, while Tween® 20 leads to a protein

Fig. 2 Bacterioruberin yield of extraction using aqueous solutions of non-ionic compounds at 250 mM, as well as using ethanol as the control
solvent, at room temperature (20–25  C), under a constant vertical rotation at 50 rpm, for 45 min, protected from light exposure, and at a fixed
SLR of 0.1 gwet biomass mLsolvent−1.

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extraction yield of approximately 352 mgprotein gwet biomass−1, the process. The concentration of Tween® 20 has a great inuence
increasing by over 14 times the amount of protein obtained. As in the extraction process, where an optimum value was reached at
a result, the following work used Tween® 20 as the best solvent. 182.4 mM, the minimum level tested for this variable. These
results suggest that a higher extraction yield could be achieved at
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concentrations of Tween® 20 below 182.4 mM, although an

Optimization of the solid–liquid extraction increase in the tested range would not be suitable due to the
An optimization of the operational conditions was performed viscosity issues noticed (see above). SLR has a positive inuence on
based on a central composite rotatable design (CCRD-23) the yield of extraction as predicted by the model, with the best
comprising three independent variables, namely SLR (in gwet response being obtained at 0.06224 gwet biomass mLsolvent−1. These
−1 results can be conrmed by the data present in Table S3† in the
biomass mLsolvent , X1), time of extraction (t in min, X2) and
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concentration of surfactant in water (Csurf in mM, X3). Eighteen ESI,† the Pareto chart (Fig. S3 in the ESI†), and the predicted vs.
assays with four central (level 0) and axial points (−1.68 observed values present in Fig. S4 in the ESI.† Here, it becomes
and +1.68 levels) were studied in terms of bacterioruberin yield evident that the solid–liquid ratio and the concentration of
of extraction (in mgbacterioruberin gwet biomass−1) ranging from Tween® 20 in water were the variables that most constrained the
0.111 mgbacterioruberin gwet biomass−1 in assay 5 to 0.331 model regarding the bacterioruberin yield of extraction. Therefore,
mgbacterioruberin gwet biomass−1 in assay 9 (Table S2 in the ESI†). aer nding the optimal operational conditions (8.04 min,
The tted model expressed in eqn (1), obtained using the SS 182.4 mM, and 0.06224 gwet biomass mLsolvent−1), a model validation
residual from Analysis of Variance (ANOVA), revealed good was developed. A bacterioruberin yield of extraction of 0.37
0.01
predictability at a condence level of 95% with R2 ¼ 0.80912 and mgbacterioruberin gwet biomass−1 was obtained experimentally (Table
Fcalculated > Ftabulated. S4 in the ESI†), encompassing a mean relative deviation of 11.6%.
Under these conditions, a protein yield of extraction of 352
9
Yield of extraction (mgbacterioruberin gwet biomass−1) mgprotein gwet biomass−1 was achieved.
¼ 0.147055 − 0.270072(X1) + 0.002002(X3)2 (1)

The response surfaces plotted in Fig. 3 show low effects on yield Protein induced precipitation
imposed by the extraction time. Therefore, the minimum time Ethanol is a common solvent used as a precipitating agent since
tested is the obvious choice to reduce the energy consumption of it possesses a lower dielectric constant than water,50 which leads

Fig. 3 Response surface plots obtained for the CCRD (23) using an aqueous solution of Tween® 20 regarding SLR (gwet biomass mLsolvent−1),
concentration of surfactant in water (Csurf, mM), and time of extraction (t, min) in terms of bacterioruberin yield of extraction (yield of extraction,
mgbacterioruberin gwet biomass−1).

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to an increase in the attraction forces between proteins, Finally, the volume of ethanol was considered (Fig. 4C) under
specically at low temperatures.51–53 To achieve the minimum xed conditions of 25  C and 2 min, and it was shown that the
amount of protein in the supernatant fraction (SF), an evalua- volume of ethanol is the variable that most inuences the
tion of the process conditions, regarding temperature, volume precipitation process: protein content decreases with an
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of ethanol added to the initial extract, and time of the precipi- increase in the ethanol volume added to the initial extract. A
tation, was performed between −20 and 30  C, 0.5 and 4 mL and volume of 3 mL of ethanol was selected as the difference
2 and 30 min, respectively (Fig. 4). between 3 mL and 4 mL corresponds to only 1.5% protein
The inuence of temperature on the precipitation was content. Thus, with a set of optimum conditions of 25  C, 2 min,
assessed at a xed time of 20 min and a volume of ethanol of 3 and 3 mL of ethanol, a yield of 234.5
0.9 mgprotein gwet
−1
mL. Lower temperatures, particularly 4  C, slightly reduce the biomass (quantied and calculated aer protein redissolution
Open Access Article. Published on 24 October 2022. Downloaded on 2/2/2023 6:43:15 AM.

protein losses of the process (Fig. 4A). However, as the differ- in PBS), which corresponds to 91% of the proteins present in
ence in the protein content between 4  C and 25  C corresponds the initial extract, was obtained.
to only 3.9%, a temperature of 25  C was seen as preferable, as it Moreover, further studies were conducted to understand the
lowers the energetic burden of the overall process. Taking this effect of consecutive precipitations (Fig. S5 in the ESI†). Aer
into account, the precipitation time was evaluated at a xed the rst precipitation, and consequent recovery of the pellet
temperature of 25  C and 3 mL of ethanol. The data obtained fraction, a second precipitation was carried out by adding
(Fig. 4B) show an almost constant protein content with another 3 mL to the supernatant fraction and repeating the
a minimum at 2 min, and this time was chosen to proceed. procedure. Only a very small fraction of approximately 1.0% of
the total proteins present in the initial extract was obtained in
the second precipitation. This corresponds to a yield of 2.7
0.3
mgprotein gwet biomass−1 (quantied and calculated aer protein
redissolution in PBS), which is markedly lower when compared
to the rst, and thus the application of a second step of
precipitation was disregarded. This further conrms that over
90% of the protein fraction was recovered in a single step of
precipitation, as also visible by the abundance of proteins
separated through SDS-PAGE electrophoresis of the precipi-
tated proteins aer dissolution in PBS (1, pH 7.4), Fig. S6 in
the ESI.† Finally, the bacterioruberin content was measured,
aer ethanol evaporation, to investigate bacterioruberin losses
during induced precipitation, with a loss xed at 12%.

Bacterioruberin polishing and conceptual process design

A bacterioruberin polishing step was accomplished to achieve
the recycling of Tween® 20. Here, the objective was the sepa-
ration of the bacterioruberin and the solvent, thus facilitating
its further application. Briey, to perform this step, the water
content was rst removed from the bacterioruberin extract
(extract obtained aer induced precipitation and ethanol
evaporation) by freeze-drying. Then, the formation of two pha-
ses was achieved by adding ethanol to the sample [1 : 20 (Vbac-
terioruberin extract : Vethanol)]. Aerwards, the solution obtained was
stored in the freezer at −80  C for three days. Thus, bacterior-
uberin migrated to the ethanol-rich (top) phase, while Tween®
20 remained in the bottom phase. At the end, the ethanol-rich
phase was analyzed by 1H NMR spectroscopy (Fig. S7 in the
ESI†) to evaluate if Tween® 20 was fully separated, and no
surfactant was detected. The presence of bacterioruberin in the
top phase of the collected sample was conrmed by UHPLC-MS
analysis. It was possible to identify a mixture of bacterioruberin
(m/z ¼ 741) and monoanhydrobacterioruberin (m/z ¼ 724) at
around 12.59 min, with bacterioruberin being the major
compound in the mixture (85%). However, under the conditions
Fig. 4 Protein content in the supernatant fraction (SF) after protein used (positive mode), these two compounds’ pseudomolecular
precipitation considering different temperatures (A), times of precipi- ions appear as [M + H]+ and easily produce more stable frag-
tation (B), and volumes of ethanol added to the initial extract (C). ment ions with m/z ¼ 635 and 591, respectively (see Fig. S8 in

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the ESI†). Finally, the bottom phase was analyzed by UV-Vis and incubated under the same conditions (i.e. 37  C, 180 rpm,
spectroscopy, with only 2.8% of bacterioruberin detected, 4000 lux) for 96 h. Archaea growth was monitored by optical
indicating that around 97.2% of bacterioruberin was success- density determined at 600 nm. Aer the batch culturing period,
fully concentrated in the top phase. the biomass was centrifuged in a Thermo Scientic Heraeus
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A nal diagram of the process developed in this work was Megafuge 16R centrifuge at 18 894g for 15 min at room
proposed (Fig. 5), in which all steps were considered. In the end, temperature (20–25  C). The supernatant was discarded, and
the colorant can be provided in ethanolic solution or in its dry the pellet was stored in the dark at −20  C until further use.
form, depending on the desired end application. Drying should Some natural variability was found in the data due to the use of
be performed using a vacuum dryer at low temperature and different cultivation batches.
pressure. This not only prevents pigment degradation, thus Chemicals. Ethanol absolute (analytical reagent grade, CAS
Open Access Article. Published on 24 October 2022. Downloaded on 2/2/2023 6:43:15 AM.

allowing the recycling of the solvents, Tween® 20 and ethanol, 64-17-5) used in solid–liquid extraction and in induced protein
but also allows easy scale-up. precipitation was acquired from Fisher Scientic. Sodium
chloride (NaCl, 99.5 wt%, CAS 7647-14-5), potassium chloride
(KCl, 99.5 wt%, CAS 7447-40-7), anhydrous di-sodium hydrogen
Experimental phosphate (Na2HPO4, 99 wt%, CAS 7558-79-4) and monobasic
Materials and methods potassium phosphate (KH2PO4, 99.5–100.5 wt%, CAS 7778-77-
0), used to prepare phosphate-buffered saline (PBS), were
Archaea cultivation. Haloferax mediterranei ATCC 33500 was purchased from Fisher Scientic, Chem-Lab, Panreac, and
produced under controlled conditions targeting maximum Honeywell, respectively. To prepare the loading buffer for SDS-
bacterioruberin yield, in YPC-Hv (yeast, peptone and casamino PAGE analysis, glycerol (99.88 wt%, CAS 56-81-5) acquired from
acids media) culture medium: peptone of meat 1.0 (g L−1); Fisher Chemical, bromophenol blue sodium salt (CAS 34725-61-
casamino acids 1.0 (g L−1); yeast extract 5.0 (g L−1); NaCl 144 (g 6) from Merck, dithiothreitol (DTT, 98 wt%, CAS 3483-12-3)
L−1); MgSO4$7H2O 21.0 (g L−1); MgCl2$6H2O 18.0 (g L−1); KCl from NZYtech, and tris(hydroxymethyl)-aminomethane
4.2 (g L−1); Tris–HCl 12 mM (pH 7.5); KOH 1 M, and 0.5 M ((HOCH2)3CNH2, 99 wt%, CAS 77-86-1) from Alfa Aesar, were
CaCl2.54 The medium was sterilized at 121  C prior to Archaea used. RunBlue Teo 20 Teo Tricine SDS and RunBlue SDS gel 4–
culturing. A single colony was selected from the agar plate (YPC- 12% 10 cm  10 cm were supplied by Expedeon. BlueSafe used
Hv broth with 1.5% agar) and inoculated into 25 mL of YPC-Hv to stain the proteins and Amersham™ ECL™ Rainbow™
broth in a 100 mL Erlenmeyer ask and cultured at 37  C, Marker – full range were acquired from NZYtech and Cytiva,
180 rpm, under continuous light (4000 lux) for 72 h. To increase respectively. Information regarding the ILs and surfactants
the cell mass concentration, 20 mL of this pre-inoculum were screened in this work such as purity, CAS number and supplier
resuspended in 400 mL of fresh broth in a 1 L Erlenmeyer ask

Fig. 5 Schematic diagram of the downstream process developed in this work, consisting of (i) cell disruption/solid–liquid extraction of bac-
terioruberin and proteins using Tween® 20 (aq) as the solvent, (ii) protein induced precipitation with ethanol, and redissolution of proteins in PBS,
and (iii) bacterioruberin polishing and recycling of the solvents. Dashed lines are only a suggestion of how the process could be industrially
implemented, and these steps were not experimentally performed in this work.

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can be found in Table S5 in the ESI.† The molecular structures Yield of extraction mgbacterioruberin gwet biomass 1
are shown in Fig. S9 in the ESI.†
½bacterioruberin  volume
Cell disruption and solid–liquid extraction. Cell disruption ¼ (2)
weight
and solid–liquid extraction steps were performed simulta-
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neously following an adapted protocol previously developed by where “[bacterioruberin]” corresponds to the concentration of
us.38 Briey, this step was carried out in a shaker (IKA Trayster bacterioruberin in the extract (mg mL−1), “volume” is the
Digital) at room temperature (20–25  C) under a constant volume of solvent (mL) and “weight” is the weight of the wet
vertical rotation at 50 rpm, for 45 min, protected from light cells tested (g).
exposure. Several solvents, including aqueous solutions of ILs Protein induced precipitation. Protein precipitation was
and surfactants (cationic, anionic, non-ionic, and non- performed using ethanol as the precipitating agent which was
added to the initial extract (extract obtained from the solid–
Open Access Article. Published on 24 October 2022. Downloaded on 2/2/2023 6:43:15 AM.

tensioactive), were screened at 100 mM to assess their

capacity to release rst bacterioruberin (the most valuable liquid extraction step). Different operational conditions, namely
compound) from the biomass. The critical micelle concentra- temperature ( C), volume of ethanol added (mL), and time for
tion (CMC) of all tested surface-active compounds is reported in precipitation (min), were evaluated in a range of −20 to 30  C,
Table S5 in the ESI.† Finally, a control extraction using pure 0.5 to 4 mL, and 2 to 30 min, respectively. The samples were
ethanol was also performed. centrifuged, and two different fractions were obtained, a pellet
An additional screening of aqueous solutions of non-ionic and a supernatant, using a Thermo Scientic Heraeus Megafuge
surfactants at 250 mM was also carried out. All tests were con- 16R centrifuge at 4700g, for 15 min. The protein recovery results
ducted at a xed solid–liquid ratio (SLR) of 0.1 gwet biomass were analyzed in terms of the remaining protein content
mLsolvent−1, i.e. 0.1 g of wet biomass and 1 mL of the respective present in the supernatant fraction relative to the protein
solvent. Finally, a centrifugation step was performed at 16 200g content in the initial extract (in percent, %). The pellet fraction
for 15 min in a VWR Microstar 17 centrifuge, at room temper- from the optimized precipitation was resuspended in 2 mL of
ature (20–25  C). The resulting supernatant was recovered, and PBS (1, pH 7.4) for further analysis. The yield of precipitation
the biomass debris was discarded. All the assays were done in (mgprotein gwet biomass−1) was quantied using a calibration curve
duplicate. previously obtained with bovine serum albumin (BSA) as
Optimization of the cell disruption/solid–liquid extraction a standard protein at 280 nm. Finally, the ethanol content of the
step. A response surface methodology (RSM) was applied to supernatant fraction was evaporated to evaluate bacterioruberin
simultaneously study different variables and to identify the most content aer precipitation and to further pursue its polishing.
signicant parameters and their interaction, aiming at dening Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
the optimum conditions that maximize the yield of bacterior- (SDS-PAGE). The resulting protein extract (pellet fraction
uberin extraction. A central composite rotatable design (CCRD- resuspended in PBS) was analyzed by SDS-PAGE following the
23) was applied to the best cell disruption/solid–liquid extraction procedure of Laemmli (1970).57 First, the sample was diluted
solvent in a total of 18 assays with 4 central points and axial (1 : 1) in loading buffer (4% (w/v) SDS, 20% (w/v) glycerol,
points. The independent variables studied were the SLR (1 gwet 0.5 mM Tris–HCl pH 6.8, 0.02% (w/v) bromophenol blue, 3.1%
−1
biomass mLsolvent ), the concentration of surfactant in water (Csurf,
(w/v) DTT) and incubated for 5 min at 95  C for denaturation to
mM), and time of extraction (t, min), with the dependent variable occur. Then, it was loaded into a polyacrylamide gel along with
being bacterioruberin yield of extraction (in mgbacterioruberin gwet a molecular weight marker (Amersham™ ECL™ Rainbow™
−1
biomass ). The results were statistically analyzed using Statistica® Marker – Full range) and le to run for 2 h at 110 V. Finally, to
7 soware and with a 95% condence level, following the theory stain the proteins, incubation with BlueSafe was carried out
proposed by Dean et al.55 and Rodrigues and Lemma.56 The real under mild agitation for 2 h.
values are presented in Table S2 in the ESI.† Lastly, using the Bacterioruberin polishing. The bacterioruberin polishing
means of relative deviation (%) the optimum conditions deter- procedure used was adapted from the literature.58,59 First, the
mined were validated in triplicate. water content present in the bacterioruberin extract was
Bacterioruberin quantication. The quantication of bac- removed by freeze-drying the sample. Then, ethanol was added
terioruberin was determined using a UV-Vis microplate reader in the proportion of 1 : 20 (Vbacterioruberin extract : Vethanol) and the
(Synergy HT microplate reader – BioTek). The absorption solution was kept for three days at −80  C, to induce bacter-
spectra were measured between 350 and 700 nm and bacter- ioruberin release from the micellar complex present in Tween®
ioruberin content was determined by eqn (2), using calibration 20. Aer this period, two phases were formed, a bottom viscous
curves previously obtained at 494 nm for ethanolic solutions surfactant-rich phase and a top ethanol-rich phase, to which the
and 504 nm for aqueous solutions. The bacterioruberin stan- pigment migrated. The bacterioruberin presence in the top
dard, used to determine the calibration curves, was obtained by phase was further evaluated by collecting a sample, removing
preparative thin-layer chromatography (TLC) in silica gel 60. A the ethanol, and redissolving it in a controlled volume of 0.5 mL
mobile phase of acetone : dichloromethane (1 : 1) was used, of ethanol. In addition, a 1H NMR analysis was also performed
aer the solid–liquid extraction of bacterioruberin with ethanol to assess Tween® 20 content in the top phase using a Bruker AC
from Haloferax mediterranei ATCC 33500, and subsequent 30 spectrometer (250 MHz) at room temperature, where deute-
redissolution of the resulting extract in dichloromethane. rium oxide was used as the solvent.

30284 | RSC Adv., 2022, 12, 30278–30286 © 2022 The Author(s). Published by the Royal Society of Chemistry
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Paper RSC Advances

Ultra-high performance liquid chromatography coupled FTC/MCTES for the nancial support to CESAM, UIDB/50017/
mass spectrometry (UHPLC-MS) analysis. Aer the polishing 2020 + UIDP/50017/2020 + LA/P/0094/2020, nanced by national
step, the bacterioruberin extract was analyzed by UHPLC-MS funds. The authors are also grateful to the FCT for the doctoral
using a Thermo Scientic Ultimate 3000RSLC (Dionex) equip- grants of M. Kholany (SFRH/BD/138413/2018), I. P. E. Macário
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.

ped with a Dionex UltiMate 3000 RS diode array detector and (SFRH/BD/123850/2016), and T. Veloso (SFRH/BD/147346/
coupled to a mass spectrometer. The analysis was performed in 2019). T. Caetano was funded by national funds (OE), through
positive mode. The separation of the compounds was carried FCT, in the scope of the framework contract foreseen in the
out with a gradient elution program at a ow rate of 2 numbers 4, 5, and 6 of article 23, of the Decree-Law 57/2016, of
mL min−1, at 30  C, by using a Hypersil Gold C18 column (100  August 29, changed by Law 57/2017, of July (CEECIND/01463/
2.1 mm i.d.; 1.9 mm particle diameter, Thermo Fisher). The 2017). The National NMR Network, funded within the frame-
injection volume in the UHPLC system was 5 mL and the mobile
Open Access Article. Published on 24 October 2022. Downloaded on 2/2/2023 6:43:15 AM.

work of the National Program for Scientic Re-equipment,

phase consisted of 0.1% formic acid in water (A) and acetoni- contract REDE/1517/RMN/2005 with funds from POCI 2010
trile (30) : methanol (70) (B). (FEDER) and FCT, is also acknowledged.

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30286 | RSC Adv., 2022, 12, 30278–30286 © 2022 The Author(s). Published by the Royal Society of Chemistry