i An update to this article is included at the end

Bioresource Technology Reports 5 (2019) 230–237

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Bioresource Technology Reports

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Ethanol and protein production from minimally processed biomass of a T

genetically-modified cyanobacterium over-accumulating sucrose
⁎
Maria Eugenia Sanz Smachetti, Macarena Perez Cenci, Graciela L. Salerno, Leonardo Curatti
Instituto de Investigaciones en Biodiversidad y Biotecnología (INBIOTEC-CONICET), Mar del Plata 7600, Argentina
Fundación para Investigaciones Biológicas Aplicadas (FIBA), Mar del Plata 7600, Argentina

A R T I C LE I N FO A B S T R A C T

Keywords: One of the main bottlenecks of a microalgal or cyanobacterial biomass biorefinery is the separation of different
Anabaena sp. useful fractions using simple, low energy-consuming, cost-effective, and scalable separation processes. Although
Genetic engineering the carbohydrates-rich biomass of these microorganisms presents clear advantages over conventional terrestrial
Sucrose crops as feedstocks for ethanol, it still requires acid and/or enzymatic hydrolysis for efficient fermentation. Here,
Biofuels
we show the genetic modification of carbohydrates partitioning in a filamentous cyanobacterium towards the
Bioethanol
accumulation of sucrose up to 10% (w/w) as a readily fermentable feedstock. We optimized two methods for the
preparation of concentrated sucrose syrups, which were efficiently converted into ethanol by yeasts, without the
need of additional pretreatments. Biomass drying and milling, followed by aqueous extraction of sugars and
proteins, and the recovery of proteins by short pulses of heat, kept the value of sugars as a feedstock for ethanol
and protein for feed supplements within a cost-effective biomass biorefinery.

1. Introduction produces large volume of contaminating residues, enzymatic hydrolysis

can be completed under milder temperatures, although it still requires
Most techno-economic analysis tends to indicate that costs to pro- some physical or chemical pretreatments, and it is considerably more
duce only biofuels from microalgal or cyanobacterial biomass remain expensive (Hernández et al., 2015). Nevertheless, both strategies lead
too high for profitable commercialization. Similar analyses also suggest to biomass saccharification and conversion into ethanol by microbial
that co-production of biofuels and animal feed, or other higher-value fermentation with an efficiency very close to the upper theoretical
co-products within biomass biorefineries, would largely improve cost- value, as recently compiled by Sanchez Rizza and co-workers (Sanchez
effectiveness (Laurens et al., 2017). Rizza et al., 2017; Sanz Smachetti et al., 2018).
In addition to aspects directly related to the cost of producing bio- Both microalgae and cyanobacteria can also accumulate soluble
mass (Luan and Lu, 2018), the main bottleneck for a biorefinery ap- sugars, mainly in response to salt, osmotic, desiccation, cold, or heat
proach is the separation of different useful fractions using simple, low stress (Kolman et al., 2015; Salerno et al., 2004). In cyanobacteria, the
energy-consuming, cost-effective, and scalable separation processes predominant soluble carbohydrates that accumulate upon salt stress are
(Chew et al., 2017; Luan and Lu, 2018). the disaccharides sucrose and trehalose, glucosylglycerol and gluco-
Microalgal and/or cyanobacterial carbohydrates present consider- sylglycerate, and sucroglucans (Kolman et al., 2015; Pontis et al.,
able advantages over plant-based feedstocks for the production of 2007). Sucrose is the most generalized one, and in addition to its
ethanol (Sanz Smachetti et al., 2018). The lack of lignin and its simpler function as a compatible solute during environmental stress, it also
structure tend to make the pretreatment and saccharification of mi- appears to display other functions in diazotrophic growth and/or in cell
croalgae or cyanobacterial polysaccharides less energy-intensive and signaling pathways in some strains (Cumino et al., 2007; Curatti et al.,
reagent-demanding than current plant-based feedstocks (Sanz 2002; Desplats et al., 2005; Kolman et al., 2015). Sucrose metabolism in
Smachetti et al., 2018). It has been shown that microalgal and cyano- cyanobacteria is similar to that of microalgae and plants: it is synthe-
bacterial carbohydrate–rich biomass can be efficiently hydrolyzed into sized by the sequential action of sucrose-phosphate synthase (SPS, U/
monosaccharides by chemical (acid or alkaline)/physicochemical or ADP-glucose: D-fructose-6-phosphate 2-α-D-glucosyltransferase, EC
enzymatic hydrolysis (Hernández et al., 2015). While acid hydrolysis, 2.4.1.14) and sucrose-phosphate phosphatase (SPP, sucrose-6F-phos-
mostly using diluted sulfuric acid, requires high temperatures and phate-phosphohydrolase, EC 3.1.3.24); while its catabolism is initiated

⁎
Corresponding author at: Instituto de Investigaciones en Biodiversidad y Biotecnología (INBIOTEC), Vieytes 3103, Mar del Plata 7600, Argentina.
E-mail address: [email protected] (L. Curatti).

https://doi.org/10.1016/j.biteb.2019.01.019
Received 29 December 2018; Received in revised form 26 January 2019; Accepted 27 January 2019
Available online 30 January 2019
2589-014X/ © 2019 Elsevier Ltd. All rights reserved.
M.E. Sanz Smachetti et al. Bioresource Technology Reports 5 (2019) 230–237

by the activity of three different enzymes: (i) sucrose synthase (SuS, A/ inoculated into 500 mL bottles containing 300 mL of BG11 medium
UDP-glucose: D-fructose 2-α-D-glucosyltransferase, EC 2.4.1.13); (ii) supplemented with 8.4 g·L−1 NaHCO3 and sparged from the bottom
alkaline/neutral invertase (A/N-Inv, an α-glycosidase, EC 3.2.1.261); with filtered CO2-enriched air (at about 5000 ppm) at 0.3–0.5 L·min−1
and (iii) amylosucrase (AMS, EC 2.4.1.4) (Kolman et al., 2015; Salerno and illuminated with constant white light at 75 μmol photons m−2·s−1.
and Curatti, 2003). Sucrose accumulation experiments were carried out in 5 L air-lift
Soluble carbohydrates such as glucose or sucrose are very attractive column photobioreactors (PBRs) containing 4.5 L of BG11 supple-
carbon sources because they are readily up-taken by yeasts and most mented with 8.4 g·L−1 NaHCO3, sparged with air from the center of the
microorganism of industrial importance to produce ethanol and other riser tube at 6 L·min−1 (up flow circulation) and with pure CO2 from the
fermentation products without the need of any of the pretreatments bottom of the down flow circulation at 0.2 L·min−1, and illuminated
already described (Marques et al., 2016; Verstrepen et al., 2004). Thus, with constant white light at 213.8 μmol photons m−2·s−1. Temperature
during the last years, much attention has been devoted to the genetic was maintained constant at 28 ± 1 °C in all cases. As indicated in
modification of unicellular cyanobacteria to increase biosynthesis and place, cultures were supplemented with 80 mM or 120 mM NaCl.
release of glucose or sucrose into the medium as an alternative to plant Cells used to determine SPS activity were cultivated in 250 mL
crop-based carbohydrates as fermentation feedstocks (Du et al., 2013; Erlenmeyer flasks containing 50 mL of BG11 medium with constant
Duan et al., 2016; Ducat et al., 2012; Hays et al., 2017; Kirsch et al., agitation at 120 rpm and illuminated with constant white light at
2018; Löwe et al., 2017; Smith and Francis, 2017; Song et al., 2016; 30 μmol photons m−2·s−1.
Weiss et al., 2017). Some estimations suggested that sucrose pro- For fermentation analysis, the yeast Saccharomyces cerevisiae
ductivity of genetically modified Synechococcus elongatus could be (Levex®, Argentina) was maintained in YPD-agar containing 1% yeast
several-fold higher than that of sugarcane, assuming the scalability of extract, 2.5% peptone, 2% dextrose and 1% agar-agar, at 28 °C in the
laboratory results on either a volumetric or areal basis (Ducat et al., dark.
2012). These sugar-exporting strains cross-fed sugars to heterotrophic
microorganisms such as the yeast Saccharomyces cerevisiae or bacteria 2.3. Biomass processing and sugars extraction
when co-cultivated (Du et al., 2013; Duan et al., 2016; Ducat et al.,
2012; Hays et al., 2017; Löwe et al., 2017; Smith and Francis, 2017; To obtain cyanobacterial sugar-rich extracts, 4.5 L of Anabaena sp.
Weiss et al., 2017). In some cases, co-cultures allowed production of PCC 7120 wt or spsB+ cultures were promptly cooled down by adding
target compounds, such as polyhydroxybutyrate, from atmospheric CO2 crashed ice and supplemented with 240 mg·L−1 FeCl3 to promote
(Löwe et al., 2017; Smith and Francis, 2017). flocculation. The decanted-cells slurry was further dewatered by cen-
In this report we show that a filamentous cyanobacterium over- trifugation at 6000 ×g. For the sugars extraction-method based on
expressing a sucrose-phosphate synthase encoding-gene over-accumu- microwaves (MW), the cell paste was subjected to extraction by mi-
lates sucrose intracellularly up to 10% (w/w) of its dry weight after crowaves at 200 W of power, for 4 cycles of 2 min each, in a microwave
induction by a NaCl stress. We show optimization of two low energy- oven (BGH Quick Chef® 15140, Argentina) and the soluble fraction was
and reagents-demanding methods for cell collection and preparation of separated by centrifugation at 17,211 ×g for 15 min. The removed
sugar-enriched fractions as suitable feedstocks for ethanol fermenta- volume was replaced with distilled water and centrifuged once again.
tion. Additionally, a large fraction of protein could be recovered as a Both soluble fractions were combined.
potential animal feed supplement. For the sugars extraction-method based on dry milling of the bio-
mass (D&M), the dewatered biomass was air-dried, milled with 15%
2. Materials and methods sand (w/w) and rehydrated with water at a 1:3.5 ratio (w/v). Glass
beads were added and then, vigorously vortexed. After centrifugation at
2.1. Plasmid construction and strain transformation 11,952 ×g for 15 min, the soluble fraction was separated, and the re-
moved volume was replaced with distilled water. This step was re-
A mutant strain of Anabaena sp. PCC 7120 was obtained by cloning peated 3 times. All soluble fractions were combined and incubated at
the native spsB gene (Cumino et al., 2002) in the plasmid pRL1404 in 100 °C for 5 min to partially remove the soluble proteins. Incubation
front of its own promoter. The resulting construct was transferred into conditions that ensure maximum protein precipitation and minimal
the cyanobacterium by conjugation. Methods for DNA manipulation sucrose lost were chosen by incubating the extracts at 60 or 100 °C for
and transgenic strains isolation were described before (Curatti et al., 5, 10, 20 and 40 min, and protein and sucrose content in the extract was
2002). determined (Supplementary Materials). Regardless the method of ex-
traction, soluble fractions were stored at −20 °C until further analysis.
2.2. Strains' culture conditions
2.4. Fermentation
Anabaena sp. strain PCC 7120 and the derivative transgenic strain
were routinely cultivated in BG11 medium (0.04 g·L−1 K2HPO4; Sucrose-rich extracts from Anabaena sp. PCC 7120 wt or spsB+
0.075 g·L−1 MgSO4·7H2O; 0.036 g·L−1 CaCl2·2H2O; 0.006 g·L−1 citric biomass were taken to pH 6.5 with H2SO4 and supplemented with
acid; 0.006 g·L−1 ferric ammonium citrate; 0.001 g·L−1 EDTA (dis- 19 g·L−1 MgSO4. Micro-fermentations (1 mL) were conducted as pre-
odium salt); 0.02 g·L−1 Na2CO3, and trace metal mix A5 (2.86 mg·L−1 viously described (Sanchez Rizza et al., 2017) by inoculating the su-
H3BO3; 1.81 mg·L−1 MnCl2·4H2O; 0.222 mg·L−1 ZnSO4·7H2O; crose-rich preparation with S. cerevisiae cells at an initial OD600 of 0.25
0.39 mg·L−1 NaMoO4·2H2O; 0.079 mg·L−1 CuSO4·5H2O and for 24 or 48 h at 28 °C and agitation at 120 rpm. Samples were taken
0.049 mg·L−1 Co(NO3)2·6H2O)), containing 1.5 g·L−1 NaNO3 as a ni- periodically, centrifuged at 16,300 ×g for 5 min, and supernatants were
trogen source. For isolation and maintenance of the transgenic strain, stored at −20 °C until further analysis. As a positive control, fermen-
streptomycin and neomycin were used at 5 mg·L−1 and 150 mg·L−1, tation of YPD medium, at a dextrose concentration in the range of su-
respectively. crose content in the samples, was routinely conducted.
Strains were maintained in BG11-agar Petri dishes illuminated with
constant white light at 6 μmol photons m−2·s−1. For inoculum pre- 2.5. Analytical methods
paration, strains were first cultivated in 100 mL Erlenmeyer flasks
containing 20 mL of BG11 medium supplemented with 2.4 g·L−1 Hepes- For dry weight determinations, 50 mL of resuspended cells in cul-
NaOH, pH 7.5; with constant agitation at 120 rpm and illuminated with ture medium were first centrifuged at 3900 ×g for 10 min, transferred
constant white light at 30 μmol photons m−2·s−1. Then, cultures were into a 1.5 mL centrifuge tube and then, centrifuged at 16,300 ×g for

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Fig. 1. Effect of spsB gene overexpression on growth

and sucrose accumulation. A) Cyanobacterial strains
doubling time in the presence of different con-
centrations of NaCl. Data at 0 and 80 mM NaCl re-
present the mean and SD of five to eight independent
experiments; and at 120 mM, the mean and SD of
two independent experiments. B) Sucrose content, as
percentage of dry biomass, of cells cultured in the
presence of different concentrations of NaCl. Data at
0 and 80 mM NaCl represent the mean and SD of four
to ten independent experiments; and at 120 mM, the
mean and SD of two to four independent experi-
ments. C) Sucrose productivity of Anabaena sp.
strains cultured at different concentrations of NaCl.
Data at 80 mM NaCl represent the mean and SD of
five or six independent experiments; and at 120 mM,
the mean and SD of two to four independent ex-
periments.

5 min, at 4 °C. Pellets were dried out in an oven at 90 °C until constant EDTA; 20 mM MgCl2; 2% (v/v) ethylene glycol; 20 mM 2-mercap-
weight (2–3 days). toethanol; and 20% glycerol, frozen under liquid N2 and milled in a drill
For growth curves analysis, cell density was estimated by recording in the presence of acid-washed glass dust. After clarification by cen-
periodically OD at 750 nm in a UV-1800 spectrophotometer (Shimadzu, trifugation at 9600 ×g for 20 min at 4 °C, protein extracts were desalted
Japan). Data were plotted using the GraphPad PRISM software through BioGel P10 resin, previously equilibrated in the same buffer.
(Intuitive Software for Science, US) and doubling times were obtained SPS activity assays, containing 100 mM Hepes-NaOH, pH 7.5; 10 mM
by fitting the experimental data to theoretical curves of exponential Fru-6-P; 10 mM UDP-Glc; 20 mM MgCl2; 50 mM NaF; and 5 mM ar-
growth with R2 above 0.96 (Supplementary Materials). Doubling time butin, were incubated at 30 °C. The production of sucrose-6-P was de-
was also calculated from dry weight data for an observed correspon- termined by the thiobarbituric acid method, using fructose as a stan-
dence below 10% difference between both methods. dard (Pontis, 2017).
For protein determination, samples of the sucrose-rich extracts were
subjected to the Lowry's method (Lowry et al., 1951), using bovine
serum albumin as a standard. Alternatively, crude protein was calcu- 3. Results and discussion
lated after the combustion of the samples in an atmosphere of ultrapure
O2 and helium at 850 °C, total N was determined in a LECO FP 528 3.1. Genetic modification of sucrose accumulation in Anabaena sp. PCC
system, using EDTA as a calibration standard, and the standard N-to- 7120
protein conversion factor 4.44, characteristic of cyanobacterial protein,
was applied (González López et al., 2010). We constructed an Anabaena strain over-expressing the spsB gene as
Total lipids were determined gravimetrically after lipids extraction, an alternative platform for producing readily fermentable sugars. Thus,
basically according to Bligh and Dyer (1959) with modifications (Do we placed the native Anabaena sp. PCC 7120 spsB gene, encoding a SPS
Nascimento et al., 2012). enzyme (Cumino et al., 2002) and its putative promoter, downstream of
For total carbohydrates determination, samples were reacted with the constitutive promoter of pDU1 of the shuttle vector pRL1404.
the anthrone reagent (Dreywood, 1946). Standard sucrose extraction The transgenic strain showed an average increase of SPS activity of
and determination were conducted essentially as reported (Pontis, 2-fold, from 1 nmol Fru. mg prot.−1·min−1 to 2 nmol Fru. mg
2017). Briefly, 50 mL of Anabaena sp. PCC 7120 wt or spsB+ culture prot.−1·min−1, when cells were cultivated under standard growth
were centrifuged at 3600 ×g for 15 min, at 4 °C. Cells were resuspended conditions. Under this culture condition, the transgenic strain showed
in 2 volumes of boiling alkaline water (pH 8), incubated at 100 °C for no growth difference when compared to the parental strain, with
5 min and then, centrifuged at 9600 ×g for 5 min, at 4 °C. Extraction doubling times of 25.3 ± 8.7 h−1 and 24.1 ± 8.6 h−1, respectively.
was repeated two more times and the fractions were combined and However, the transgenic line was more tolerant to NaCl loading in the
stored at −20 °C until further analysis. For sucrose determination, culture medium, especially at 80 mM NaCl, displaying a doubling time
samples in 100 mM NaOAc, pH 4.5 buffer were incubated at 55 °C in the of 36.2 ± 11.1 h−1, in comparison to 55.1 ± 11.4 h−1 of the wild
presence of 80 μg·mL−1 acid invertase (Sigma-Aldridge) for 30 min for type strain (Fig. 1A and Supplementary Materials).
its conversion into glucose and fructose, which were later determined As expected, under standard growth conditions, the mutant strain
by the Somogyi-Nelson's method with a standard curve using sucrose accumulated 10-fold more sucrose in its biomass than the parental
(Pontis, 2017). strain. Under saline stress at 80 mM or 120 mM NaCl for 48 h, sucrose
For ethanol determination, yeast spent medium after fermentation content of the transgenic line was 2- or 4-fold higher than the wild type,
was subjected to a method previously reported (Sanchez Rizza et al., reaching values of 6.3 ± 1.3 (w/w) or 8.7 ± 2.7% (w/w) of their dry
2017). Briefly, the standard ethanol assays contained 50 mM Tris-HCl, biomass, respectively (Fig. 1B). However, the overall sucrose pro-
pH 8.4; 2.5 mM NAD+ and 3 μg protein preparations containing alcohol ductivity was offset by slower growth under more stringent stressing
dehydrogenase activity and the samples in a total volume of 100 μL and conditions for a similar productivity at around 0.8 mg Suc. L−1·h−1
were incubated at 30 °C for 25 min. Ethanol was determined as the (Fig. 1C).
ethanol-dependent reduction of NAD+ at 340 nm and compared with a Fig. 2 shows that biomass of both Anabaena strains presented similar
standard curve made with 99% (v/v) analytical grade ethanol. levels of total carbohydrates at about 27% (w/w) (Fig. 2A). However,
SPS activity was assayed essentially as reported before (Porchia and while sucrose represented a minor fraction of total carbohydrates of
Salerno, 1996). Protein extracts were prepared from cells collected from non-stressed cells of both strains, its level increased up to 43.6 ± 9.7%
50 mL of culture. Cells were resuspended in 3 volumes (fw/v) of a (w/w) of total biomass carbohydrates in the transgenic strain (Fig. 2D).
buffer containing 100 mM Hepes-NaOH, pH 7.5; 50 μM PMSF; 2 mM Thus, NaCl stress appears to exert a major shift in carbohydrates
partitioning, which can be exacerbated by over-expression of a SPS

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Fig. 2. Effect of spsB gene overexpression on biomass composition and carbohydrates partitioning. A) Total carbohydrates, total proteins and total lipids content after
48 h of induction with 80 mM NaCl. For carbohydrates, proteins and lipids, data represent the mean and SD of four, independent experiments; a single determination
obtained from the pool of four independently obtained samples; and two independent experiments, respectively. B–D) Soluble carbohydrates partitioning between
sucrose and other carbohydrates at 0 (B), 24 (C) and 48 (D) h of induction with 120 mM NaCl. Data represent the mean and standard deviation of two independent
experiments (B and C) or four independent experiments (D).

encoding-gene. Interconnection between the glycogen and sucrose cells were efficiently collected by flocculation in the presence of FeCl3
pools has been demonstrated in some unicellular cyanobacteria, (Ducat and further dewatered by mild centrifugation. Then, we optimized two
et al., 2012; Miao et al., 2003; Qiao et al., 2018; Suzuki et al., 2010; Xu methods for sugar extraction: one consisted in drying and milling (D&
et al., 2013) and also in Anabaena spp. (Curatti et al., 2008). Although it M) the biomass, followed by an aqueous extraction at room tempera-
has been generally interpreted that sucrose and glycogen synthesis were ture (about 22 °C) and the other one was based on microwaves (MW)
competing pathways at the levels of the common substrate glucose-1- treatment of the wet biomass. Both procedures required further clar-
phosphate, a recent study in S. elongatus PCC 7942 showed that gly- ification by centrifugation. The D&M or MW methods allowed re-
cogen synthesis was required for NaCl induction of sucrose accumula- coveries of 54.5 ± 11.3% or 84.3 ± 13.7% of the sucrose content of
tion (Qiao et al., 2018). This result suggested that, in addition to mu- the transgenic strain's biomass, respectively. However, the opposite
tation of genes for sucrose breakdown (Ducat et al., 2012; Kirsch et al., trend was consistently observed for biomass of the wt strain (Fig. 3).
2018), metabolic engineering of glycogen synthesis and mobilization Although the reason for this difference is currently not understood, we
could serve as an additional source of glucose-1-phosphate to further speculate it can be related to some other pleiotropic effect of the mu-
boost sucrose accumulation in sps genes over-expressing strains. tation on the structure and/or biochemical composition of the biomass.
It has been pointed out that in order for cyanobacteria to be utilized The maximum soluble-carbohydrates and sucrose content of the
successfully as biofactories, especially for commodities, tolerance to transgenic strain biomass, induced at 120 mM NaCl and extracted by D
environmental stress must be increased (Kitchener and Grunden, 2018). &M method was 21.5 ± 0.3 g·L−1 and 12.9 ± 1.1 g·L−1, respectively
Moreover, identifying or creating hyper-tolerant strains either to nat- (Fig. 4).
ural (or even artificially enhanced) stressing conditions, might re- As a platform for producing fermentable sugars, this alternative
present a crop protection strategy by discouraging competitors and/or strategy brings some advantages over unicellular cyanobacteria over-
predators. Sucrose accumulation upon salt stress has long been de- producing and excreting sucrose. First, larger and filamentous strains,
monstrated in Anabaena spp. as well as in other cyanobacteria. This has aided by flocculation, are easier to separate from the culture medium.
led to the general view that it serves the function of an osmolite en- Additionally, sugar retention in the biomass and simplified biomass
abling cells to stand salt stressing conditions (Kolman et al., 2015;
Kirsch et al., 2018). Both sucrose accumulation under osmotic stress
and/or high temperatures have led to identical interpretations (Warr
et al., 1985). Kitchener and Grunden (2018), discussed the challenge of
diverting cellular resources towards an efficient stress tolerance re-
sponse alongside maximizing accumulation of the target product. The
coincidental nature of sucrose as a key player of the stress tolerance
response and the target product would represent a very interesting
advantage of this platform.
Until this study and regardless of the vast cumulative evidence
concerning sucrose accumulation upon salt stress, it has been difficult
to accurately estimate up to what extent sucrose accumulation alone
could promote salt stress tolerance. Salt stress tolerance is a complex
process that, in addition to accumulation of osmolites, also involves
active efflux of ions and change in expression of many genes
(Hagemann, 2016). Our results suggest the feasibility of increasing
tolerance to stress by the same kind of mutations that promote the
accumulation of the target product.
Fig. 3. Biomass sucrose extraction by two alternative methods. D&M and MW
3.2. Sucrose extraction from Anabaena biomass methods. Sucrose extracted by the different methods was expressed as a per-
centage of total biomass sucrose. Data represent the mean and range of two
Cyanobacterial strains were cultivated in 5 L photobioreactors and independent experiments.

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Fig. 4. Time course of the fermentation of the sucrose-rich syrups. Sucrose accumulation was induced at 120 mM (A–D) or 80 mM NaCl (E–L). Sucrose was extracted
from the biomass using the D&M (A–H) or the MW (I–L) procedures. A, E and I) Depletion of total soluble carbohydrates. B, F and J) Sucrose consumption. C, G and K)
Growth curve of S. cerevisiae (OD600). D, H and L) Ethanol production. The extract obtained via the MW method was supplemented with yeast extract and peptone.
The data represent the mean and range of two independent experiments.

recovery allowed us to produce more concentrated preparations of up be needed to reduce distillation costs (Möllers et al., 2014). In such a
to 22 g·L−1 of sugars and up to 13 g sucrose·L−1. This result improves case, and according to the stoichiometry of ethanol fermentation, and
most frequent concentrations around 1.5 g sucrose·L−1 from sucrose assuming a 100% conversion, this would demand a fermentation broth
exporting Synecchococcus sp. PCC 7942 strains (Du et al., 2013; Duan containing at least 80 g sugar·L−1. A recent study showed the produc-
et al., 2016; Ducat et al., 2012; Hays et al., 2017; Kirsch et al., 2018; tion of up to 72.9 g sugar·L−1 after saccharification of a microalgal
Löwe et al., 2017; Niederholtmeyer et al., 2010; Smith and Francis, biomass with H2SO4 at high temperature and quantitative conversion
2017; Song et al., 2016; Weiss et al., 2017) and also the most recent into ethanol by S. cerevisiae (Sanchez Rizza et al., 2017).
improvement after genetic manipulation of sucrose synthesis and ex- The procedures optimized in this study largely improved the state-
port, and cell proliferation at 6 g sucrose·L−1 (Abramson et al., 2018). of-the-art of the production of concentrated preparations of sugars as
This aspect may have technological implications if this kind of cyano- fermentation feedstocks without the need of biomass hydrolysis with
bacteria-based platforms are to be envisioned as a source of sugars for acids and/or enzymes. However, considerable improvements are still
the production of ethanol, since a minimum of 40 g ethanol·L−1 would required to bust sugars concentration. These improvements may result

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from a combination of additional genetic modifications and/or host engineering of Anabaena sp. PCC 7120 to express Zymomonas mobilis
strain selection, and further optimization of biomass processing. pyruvate decarboxylase and Synechocystis sp. PCC 6803 alcohol dehy-
drogenase genes exclusively in heterocysts. In contrast to the O2-evol-
3.3. Ethanol production from Anabaena sugar preparations ving photosynthetic vegetative cells, heterocysts differentiate from ve-
getative cells upon combined N shortage and execute a complex shift in
Ethanol production represents one key example of several alter- genes expression which down-regulates O2-evolving photosynthesis and
native fermentation end-products using sugars syrups from cyano- induces N2-fixation, which is a strict anaerobic pathway. A mutant
bacterial or microalgae. Preliminary attempts of fermenting sugar strain additionally overexpressing an invertase gene in heterocysts, and
preparations obtained by the D&M procedure with S. cerevisiae pro- assisted by an optimized gas-stripping strategy, accumulated ethanol at
duced low levels of ethanol (data not shown). We observed that this 1.7 g in 23 days. This yield represented a three-fold improvement in
extraction procedure also recovered a large proportion of soluble pro- comparison to previous similar studies conducted in unicellular cya-
tein at 23–29 g·L−1, which increased the viscosity of the extracts (not nobacteria in which, as expected, fermentation appeared to be atte-
shown). Thus, we analyzed the effect of temperature on sugar and nuated by the O2 produced by photosynthesis (Ehira et al., 2018). The
protein separation and the stability of sucrose. We observed that in- platform described in our study produced considerably higher levels of
cubations as short as 5 min at 100 °C were enough to recover about 90% ethanol from Anabaena sp. PCC 7120 cells at about 8 g in three days
of the proteins as insoluble material while sucrose content remained (two for biomass production and one for fermentation). Regardless of
unchanged (Supplementary Materials). this progress, it becomes quite evident that achievable yields are still far
Deproteinized sugar preparations were quantitatively converted away in comparison to plant feedstocks (Sanz Smachetti et al., 2018) to
into ethanol by fermentation with S. cerevisiae. Fig. 4 shows the time envision commercial competitiveness of a platform dedicated to the
course of total sugars (Fig. 4A, E and I) or sucrose (Fig. 4B, F and J) production of only ethanol. However, ethanol production might be
depletion, the concomitant growth of the yeast (Fig. 4C, G and K), and considered if it occurs within a biomass biorefinery for additional
increase in ethanol production (Fig. 4D, H and L). Sucrose-rich extracts products (Laurens et al., 2017), and especially, if it minimizes the use of
from biomass induced at 120 mM NaCl and processed according to D& chemicals and enzymes.
M method (Fig. 4A–D) contained total soluble carbohydrates at
7.7 ± 0.8 g·L−1 or 21.6 ± 0.5 g·L−1 for the wt type or transgenic 3.4. Additional cyanobacterial biomass biorefinery for recovery of protein
biomass, respectively. These preparations also contained sucrose at and other cellular fractions
4.7 ± 0.4 g·L−1 or 12.9 ± 0.5 g·L−1 for the wt type or transgenic
biomass, respectively. Fermentation of these preparations produced Both methods allowed the separation of a soluble fraction rich in
ethanol up to 3.4 ± 0.2 g·L−1 or 7.7 ± 0.6 g·L−1 for the wt type or sugars as a direct feedstock for ethanol. Since in the MW method pro-
transgenic biomass, respectively (Fig. 4D). Consumed carbohydrates tein precipitation occurred inside the cells, protein can be recovered
were converted into ethanol at least up to 89.4% or 70.2% of the along other water insoluble cell materials. Conversely, if extraction
maximum theoretical value of 0.51 g ethanol·g glucose−1, from the proceeded through the D&M method, about 50% of the total biomass
biomass of the wt or the transgenic strain, respectively. crude-protein could be isolated in a water-soluble form from either
While sucrose was completely consumed by the yeast (Fig. 4B, F and strain (Fig. 6). After mild heating of these preparations, sugars re-
J), a soluble carbohydrates fraction remained non-metabolized by S. mained soluble and were separated from the insoluble protein fraction
cerevisiae under the used fermentation conditions (Fig. 4A, E and I). (Supplementary Materials). About 20% of the dry biomass could be
Although not experimentally demonstrated in this work, it is presumed recovered in these fractions (Fig. 6).
that the carbohydrates fraction that remained non-metabolized by S. There is an increasing interest to partially replace conventional
cerevisiae could correspond to sucroglucans. These oligosaccharides sources of protein, such as soybean meal, fish meal, rice bran, etc., by
represent, after sucrose, the second most abundant fraction of the so- microalgal and/or cyanobacterial protein for animal feeding, especially
luble carbohydrates that accumulate in Anabaena and Nostoc strains poultry and in aquaculture (Becker, 2007). Both, nutritional and tox-
exposed to salt stress (Salerno et al., 2004). Sucroglucans share the icological determinations supported the suitability of algal biomass as a
general structure [α-D-Glcp-(1 → 2)]n-α-D-Glcp-(1 → 2)-β-D-Fruf, and valuable feed supplement or substitute. Comparative data on biological
are characterized by a [α-D-Glcp-(1 → 2)] linkage that is quite non value, digestibility coefficient, net protein utilization, and protein ef-
frequent in nature (Pontis et al., 2007). Although accumulation in cy- ficiency ratio of algal proteins suggested a quality slightly lower than
anobacteria can be reverted upon reversal of the salt stress (Salerno those of casein or egg protein (Becker, 2007). However, poor digest-
et al., 2004), the catabolism of sucroglucans is largely unknown, and ibility of whole algal biomass due to the cellulosic cell wall, especially
appeared not to involve [α-D-(1 → 4)] glucosidase, invertase or sucrase of some microalgae, is frequently one of the major drawbacks towards
activities (Pontis et al., 2007). Thus, to further improve this platform algal biomass used as feed (Becker, 2007). Thus, efficient (Lupatini
for the production of fermentable sugars, the nature of these carbohy- et al., 2017; Safi et al., 2014) and especially cost-effective treatments, as
drates must be confirmed, and its metabolic pathways elucidated in those proposed in this study, are necessary to increase the feasibility of
order to design engineering strategies to further modify the partitioning algal biomass as a partial replacement of protein- for- feed production.
of carbon in the cyanobacterium and/or to transfer appropriate genes to It has been reported that Scenedesmus obliquus biomass drying and
microorganisms to allow fermentation into products. cooking improved the digestibility coefficient and net protein utiliza-
Similar results were obtained from cyanobacteria induced with tion (Becker, 2007).
80 mM NaCl using the D&M (Fig. 4E–H) or the MW (Fig. 4I–L) methods, Cyanobacterial biomass is characterized by a low content of lipids
respectively. However, unlike with the D&M method, sugars prepared (Hu et al., 2008). However, although not demonstrated here, the re-
by the MW method, required supplementation of an additional source sidual biomass after applying the D&M method followed by aqueous
of nutrients such as yeast extract, for a more efficient conversion into extraction of carbohydrates and protein would become enriched in li-
ethanol (Figs. 4 and 5, and Supplementary Materials). pids, which could be further biorefined for feed supplements and/or a
When integrating results from sucrose productivity by the cyano- biodiesel feedstock, as needed.
bacteria, efficiency of sugars extraction, and conversion into ethanol, it
was observed that the transgenic strain produced about twice the 4. Conclusion
ethanol per unit of biomass culture volume, regardless of the biomass
processing method (Fig. 5). This study shows the genetic modification of carbohydrates parti-
Recently, an elegant study showed cell type-specific metabolic tioning in a filamentous cyanobacteria towards accumulation of sucrose

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M.E. Sanz Smachetti et al. Bioresource Technology Reports 5 (2019) 230–237

Fig. 5. Effect of spsB gene overexpression on biomass

and sucrose production and conversion into ethanol.
A–C) Ethanol production on a culture volumetric
basis. Sucrose accumulation was induced at 120 mM
(A) or 80 mM (B–C) NaCl. Sucrose was extracted
according to the D&M (A–B) or the MW (C) methods.
In (C), syrups were supplemented with yeast extract
and peptone. The data represent the mean and
standard deviation of four (120 mM) or two (80 mM)
independent experiments.

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Owing to a Publisher error Declaration/Conflict of Interest state­ known conflicts of interest associated with this publication and
ments were not included in the published versions of the following there has been no significant financial support for this work that
articles, that appeared in previous issues of Bioresource Technology could have influenced its outcome.
Reports. 5. Ethanol and protein production from minimally processed biomass
The appropriate Declaration/Conflict of Interest statements, pro­ of a genetically-modified cyanobacterium over-accumulating su­
vided by the Authors, are included below. crose (Bioresource Technology Reports, 2019; 5: 220–229)
https://doi.org/10.1016/j.biteb.2019.01.019
1. Fermentable sugar production from a coffee processing by-product Conflict of Interest: The authors declare no conflict of interest re­
after deep eutectic solvent pretreatment (Bioresource Technology garding publication of the work.
Reports, 2018; 4: 174–180) 6. Operation of sediment microbial fuel cells in Tokyo Bay, an ex­
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Conflict of Interest: The Authors wish to confirm that there are no 2019; 6: 39–45)
known conflicts of interest associated with this publication and https://doi.org/10.1016/j.biteb.2019.02.001
there has been no significant financial support for this work that Conflict of Interest: The Authors wish to confirm that there are no
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2. Effective and reusable T. reesei immobilized on SBA-15 for mono­ there has been no significant financial support for this work that
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Technology Reports, 2019; 5: 199–205) 7. Two-dimensional modelling of syntrophic glucose conversion in
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clare. 8. The effect of different pre-treatments to improve delignification of
4. Fast screening of optimal acid-pretreatment conditions in the con­ eucalypt stumps in a biorefinery context (Bioresource Technology
version of wood to lignocellulosic sugars (Bioresource Technology Reports, 2019; 6: 89–95)
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