Cyanobacterial biomass as carbohydrate and nutrient feedstock for bioethanol production by yeast fermentation
© Möllers et al.; licensee BioMed Central Ltd. 2014
Received: 6 November 2013
Accepted: 27 March 2014
Published: 17 April 2014
Microbial bioconversion of photosynthetic biomass is a promising approach to the generation of biofuels and other bioproducts. However, rapid, high-yield, and simple processes are essential for successful applications. Here, biomass from the rapidly growing photosynthetic marine cyanobacterium Synechococcus sp. PCC 7002 was fermented using yeast into bioethanol.
The cyanobacterium accumulated a total carbohydrate content of about 60% of cell dry weight when cultivated under nitrate limitation. The cyanobacterial cells were harvested by centrifugation and subjected to enzymatic hydrolysis using lysozyme and two alpha-glucanases. This enzymatic hydrolysate was fermented into ethanol by Saccharomyces cerevisiae without further treatment. All enzyme treatments and fermentations were carried out in the residual growth medium of the cyanobacteria with the only modification being that pH was adjusted to the optimal value. The highest ethanol yield and concentration obtained was 0.27 g ethanol per g cell dry weight and 30 g ethanol L-1, respectively. About 90% of the glucose in the biomass was converted to ethanol. The cyanobacterial hydrolysate was rapidly fermented (up to 20 g ethanol L-1 day-1) even in the absence of any other nutrient additions to the fermentation medium.
Cyanobacterial biomass was hydrolyzed using a simple enzymatic treatment and fermented into ethanol more rapidly and to higher concentrations than previously reported for similar approaches using cyanobacteria or microalgae. Importantly, as well as fermentable carbohydrates, the cyanobacterial hydrolysate contained additional nutrients that promoted fermentation. This hydrolysate is therefore a promising substitute for the relatively expensive nutrient additives (such as yeast extract) commonly used for Saccharomyces fermentations.
KeywordsCyanobacteria Bioethanol Microalgae Saccharomyces Yeast extract
Photosynthetic biomass is a promising resource for the generation of biofuels and other valuable bioproducts. However, rapid biomass production and high-yield conversion processes are essential for successful applications. Plant-derived lignocellulosic biomass is abundant but, due to the recalcitrant nature of this material, significant challenges have to be solved if this biomass is to be used for the microbial production of biofuels and bioproducts [1–3]. Photosynthetic microorganisms constitute an appealing alternative source of biomass for many reasons. Photosynthetic microorganisms grow much faster than terrestrial plants, have a higher efficiency in using the energy of light, and can be cultivated in areas and in a manner that do not compete with plant-based food and feed production [4–7]. In this context, marine photosynthetic microorganisms have a distinct advantage in large-scale cultivation as they can be cultivated in sea water, which is not suitable for human consumption and most agricultural uses.
The most abundant photosynthetic microorganisms in nature are cyanobacteria and certain eukaryotic microalgae, including green algae, red algae, and diatoms . Despite their plant-like photosynthesis, the evolutionary origins and cellular properties of these microorganisms are very diverse. Cyanobacteria produce a wealth of high-value bioproducts and have been mass-cultivated for centuries as a nutritional supplement . Currently, much effort is being put into the genetic and metabolic engineering of photosynthetic microorganisms, especially cyanobacteria, for the production of bioproducts not naturally produced by these organisms [10, 11]. However, the direct use of biomass from cyanobacteria and other microalgae as a feedstock for the generation of biofuels and other bioproducts is underexplored [12, 13].
In terms of biomass utilization, cyanobacteria have certain advantages over eukaryotic microalgae. In spite of the overall Gram-negative-like structure of the cell envelope, the cyanobacterial cell wall contains a peptidoglycan layer that more closely resembles that of Gram-positive bacteria . The cell wall in cyanobacteria is therefore degradable by lysozyme, and is less complex and less diverse than the cell walls of most microalgae, which consist of a wide range of complex polysaccharides and proteoglycans . Furthermore, the type of storage carbohydrate is of vital importance if the biomass is to be used as a fermentation substrate for fungi. Cyanobacteria have glycogen as a storage carbohydrate [16, 17]. Glycogen is not found in any eukaryotic microalgae, which typically have either starch (green algae, red algae) or β-glucans (brown algae, diatoms) as the main storage carbohydrate . Both glycogen and starch are essentially α-1,4-glucans with α-1,6-branching but glycogen particles are small (0.04–0.05 μm) and water-soluble, whereas starch particles are much larger (0.1–100 μm) and water-insoluble . Thus, glycogen may be preferred over starch as a fermentation feedstock because in vitro starch mobilization by heating and enzymatic treatment is a more energy-intensive process than glycogen mobilization .
Green microalgae and cyanobacteria typically accumulate starch or glycogen to a content of 10 to 50% of their biomass, depending on the strain and growth conditions, and this polysaccharide is potentially useful as substrate for biofuel fermentation . Whole-cell material from starch-enriched green microalgae [19–22] and glycogen-enriched cyanobacteria  has recently been used as feedstock for bioethanol production by yeast fermentation. These studies employed various enzymatic, chemical, and physical treatments (including drying, heating, and acid- and base-treatment) to liberate monomeric hexoses from the biomass. In the present work, the single-celled, marine cyanobacterium Synechococcus sp. PCC 7002 (hereafter denoted Synechococcus; previously known as Agmenellum quadruplicatum PR-6; ) was used as a biomass feedstock for anaerobic fermentation by the yeast Saccharomyces cerevisiae. This Synechococcus strain accumulates glycogen and cyanophycin as carbon and nitrogen storage compounds and does not produce polyhydroxybutyrate as is observed in some cyanobacteria [24–26]. Exhaustion of nitrate in the growth medium of Synechococcus causes well-coordinated and complex physiological adaptations that allow photosynthesis and growth to continue to some extent [27–30]. This results in an increased C:N ratio of the biomass, an increased carbohydrate content (mostly glycogen), and a degradation of nitrogenous components including the light-harvesting phycobilisome (PBS) antenna proteins [24, 30].
The objectives of the present work were to investigate if whole-cell, carbohydrate-loaded Synechococcus biomass treated only with enzymes is suitable as fermentation feedstock, and to explore the ethanol productivity when using a high concentration of this biomass as fermentation feedstock. We show that Synechococcus biomass indeed could be sufficiently degraded by enzymatic treatment, and that it served both as fermentable substrate and as nutrient source for the fermenting yeast. This resulted in higher ethanol productivity than previously reported with whole-cell biomass from microalgae [19–22] or cyanobacteria . In addition, the present study also suggests that enzymatically hydrolyzed cell material from cyanobacteria could have a general use as a nutrient supplement to enhance the yeast fermentations of various biomass feedstocks low in nitrogenous compounds and other nutrients.
Results and discussion
Nitrate limitation and carbohydrate accumulation in Synechococcus
Maximum total carbohydrate content obtained in Synechococcus cell cultures with various nitrate concentrations 1
NaNO3concentration (g L-1)
Time from inoculation (hours)
Cell DW (g L-1)
Total carbohydrates per cell DW (%)
0.90 ± 0.15
52 ± 8
1.65 ± 0.05
58 ± 2
3.0 ± 0.2
59 ± 4
3.7 ± 0.3
40 ± 3
In order to produce sufficient amounts of biomass for fermentation trials, the culture volumes were scaled up to 800 mL. A comparison of growth (as OD730) and other key cellular parameters in 25 mL and 800 mL culture setups showed little difference (Additional file 1: Figure S3).
Biochemical composition of Synechococcus
The carbon content of Synechococcus cell DW was 49 ± 2% w/w (n = 2) for cultures that were nitrate replete and 47 ± 0% w/w (n = 2) for cultures that were nitrate limited, which is very similar to values previously obtained with cyanobacteria . Previously obtained C:N weight ratios with the same Synechococcus strain were 5.8 during growth with nitrate (similar to the Redfield Ratio of 5.7 generally observed in marine microalgae that are not nutrient-limited ), 3.4 during growth with ammonium, and up to 13 during nitrogen starvation (assuming a carbon content of 48% w/w of DW) . In our cultures, exponentially growing Synechococcus cells in the presence of nitrate (25 hours with 1 g NaNO3 L-1; Figure 1) had a C:N weight ratio of 4.4 ± 0.4 (n = 2). The Synechococcus cells with maximum carbohydrate accumulation obtained from cultivation with 0.24 g NaNO3 L-1 for 48 hours (60% w/w total carbohydrate; Figure 1) had a C:N weight ratio of 11 ± 1.4 (n = 2). A prolonged incubation of these Synechococcus cultures for 120 hours (0.24 g NaNO3 L-1; Figure 1A) resulted in a slightly increased C:N weight ratio of 12.2 ± 1.3 (n = 2), even though the total carbohydrate content dropped to 35% w/w. Thus, a high C:N ratio is not necessarily indicative of a high carbohydrate content. Presumably the carbohydrate was eventually eliminated by respiration and other metabolic processes.
Enzymatic hydrolysis of Synechococcus
In conclusion, to promote disintegration of Synechococcus cells and mobilization of monomeric glucose for fermentation by S. cerevisiae, the cells were treated with freezing, lysozyme, and two alpha-glucanases.
Fermentation of Synechococcushydrolysate
To this day, very few studies have investigated the fermentation of whole-cell material from cyanobacteria and microalgae. In a recent study intact cells of the cyanobacterium A. platensis were added to a concentration of about 20 g cell DW L-1 to a fermentation medium containing lysozyme (1 g L-1), yeast extract (10 g L-1), and peptone (20 g L-1) . The fermenting organism was a recombinant amylase-producing strain of S. cerevisiae, which eliminated the need for externally added alpha-glucanases. A final ethanol concentration of 6.5 g L-1 was obtained. The fermentation rate was low (1.08 g ethanol L-1 day-1), but a high ethanol yield was obtained (0.325 g ethanol per g cell DW). Similar to our study, the authors observed that lysozyme greatly enhanced the ethanol yield and that the available glucose was efficiently converted to ethanol (86% of theoretical maximum). The higher yield and rates obtained in our work are probably due to the fact that the cyanobacterial cells were partially degraded by enzymatic pretreatment prior to inoculation of yeast, thus making the glucose readily available for the yeast. An important observation from our work is that additional nutrients to support the fermenting yeast such as yeast extract and/or peptone were not necessary in our approach.
From an industrial point of view, a high final ethanol concentration is critical as it lowers energy consumption and thereby the costs of distillation. Typically 40 to 50 g ethanol L-1 is regarded as the lower threshold for economic ethanol production , but final ethanol concentrations exceeding 100 g ethanol L-1 are common in the fuel ethanol industry . Two important factors limit high final ethanol concentrations from the fermentation of biomass obtained from cyanobacteria and microalgae: the DW content in the fermentation and the carbohydrate content of the DW. If the biomass of cyanobacteria and microalgae obtained from liquid cultures is not dried, the DW content of the wet biomass obtained by centrifugation or filtration is typically not more than 10 to 20% w/w . In our case we managed to obtain a maximum carbohydrate accumulation of 60% of DW. Given these limitations, an expected maximum yield is approximately 60 g ethanol L-1, which is well above the critical ethanol concentration for distillation. However, efficient and cost effective harvest and dewatering of the biomass remain to be the most critical process steps.
In this work, carbohydrate-enriched biomass from cyanobacteria was obtained by photoautotrophic cultivation under nitrate limitation. This biomass was converted to a substrate suitable for fermentation by S. cerevisiae by simple enzymatic pretreatment. Harsh chemical treatments or separation of carbohydrate-containing fractions of the biomass were not necessary to obtain a high final ethanol concentration (30 g L-1). The maximum carbohydrate accumulation in the cyanobacterial biomass coincided with the degradation of PBS, which implies that PBS may be used as a proxy for carbohydrate accumulation in cyanobacteria in general. Although the cyanobacterial biomass was successfully used for bioethanol production alone, this biomass may be more valuable as a carbohydrate and/or nutrient source for production of products more valuable than ethanol in a biorefinery concept. This may be to supply nutrients and additional sugar to the fermentation of lignocellulosic materials low in nitrogen and other nutrients.
Cultivation of Synechococcus and Saccharomyces
Synechococcus sp. PCC 7002 (referred to as Synechococcus) was a kind gift from Donald A. Bryant (The Pennsylvania State University, PA, USA). It was grown in medium A supplemented with 1 g NaNO3 L-1  unless otherwise stated. Liquid cultures were bubbled with a constant flow of air supplemented with 1% v/v CO2 provided by a gas mixer (GMS150, Photon Systems Instruments, Drasov, Czech Republic). Small-scale cultures (25 mL) were maintained in tubes with an inner diameter of 2.2 cm and large-scale cultures (800 mL) were maintained in bottles with an inner diameter of 9.5 cm. All culture vessels were placed in the middle of a 30-liter transparent tank with thermostatically controlled water at 38°C. Constant illumination was provided by fluorescent tubes (cool white light; Philips Master TL-D, 18 W/840; Philips Electronics, Amsterdam, The Netherlands) placed against two opposite sides of the tank. Small-scale cultures were illuminated by 250 μmol photons s-1 m-2. Large-scale cultures were illuminated by 250 μmol photons s-1 m-2 at the time of inoculation. After 12 hours of growth (OD730 ≈ 1) the irradiance of large-scale cultures was increased to 400 μmol photons s-1 m-2. Small-scale cultures were inoculated to an OD730 of 0.2 to 0.3 and large-scale cultures were inoculated to an OD730 of approximately 0.1. In growth experiments where the effect of nitrate was studied, Synechococcus cells were harvested by centrifugation at ambient temperature (5000 g for 2 minutes), re-suspended in medium A, and used as inoculum. Reported measurements on Synechococcus growth experiments are the average and standard deviation of biological duplicates (Figures 1 and 3, Table 1, and in Additional file 1: Figures S1, S2 and S3). The S. cerevisiae strain Thermosacc® Dry (Lallemand Inc., Montreal, Canada) was used to ferment Synechococcus hydrolysates. The yeast was aerobically pre-cultured in CBS medium  for 48 hours prior to each fermentation experiment. For preparation of Saccharomyces cells for inoculation of fermentation reactions, 100 mL of fresh Saccharomyces culture was harvested by centrifugation at 4100 rpm for 10 minutes and re-suspended in 10 mL of 0.9% NaCl solution in water. The cell DW of the yeast suspension was measured and adjusted to 50 g L-1 prior inoculation of the hydrolysates.
Hydrolysis and fermentation of Synechococcusbiomass
Synechococcus cultures to be used for fermentation were harvested by centrifugation for 20 minutes at 10.000 g (50 mL Falcon tubes) and cell pellets were stored at -20°C until use. Cell pellets were thawed and re-suspended to a slurry by the addition of a small volume of medium A using mild sonication (Misonix S-4000; Qsonica, Newtown, Connecticut, United States) with a cup horn (amplitude 50%, 2 minutes processing time, 5 second on/off cycle) to obtain a final DW content of about 100 g L-1 (pH approximately 7). The subsequent enzyme treatments of the biomass were carried out in the cyanobacterial growth medium without the addition of buffers or other chemical agents. Lysozyme (from chicken egg white, L6876, Sigma-Aldrich, St. Louis, United States) was added (100 mg L-1) and the solution was incubated for 3 hours at 37°C. Two alpha-glucanases (Liquozyme® SC DS and Spirizyme® Fuel; Novozymes A/S, Bagsværd, Denmark) were used according to the instructions by the manufacturer. Liquozyme® SC DS (240 α-amylase units per g) was added (0.21% w/w) and the mixture incubated at 85°C for 1.5 hours. Then another aliquot of Liquozyme® SC DS was added (0.14% w/w) and the mixture incubated at 60°C for 0.5 hours. The pH was then adjusted to 5.5–6.0 with 5 M HCl. Finally Spirizyme® Fuel (750 amyloglucosidase units per g) was added (0.08% w/w). This mixture, now denoted ‘hydrolysate’, was then used for fermentation without further treatment. Experiments with varying lysozyme concentrations were carried out in duplicates (Figures 4 and 5).
Hydrolysates (3 mL) were inoculated with freshly concentrated yeast suspension (60 μL) to a final yeast cell concentration of 1 g DW L-1. The fermentations were run in 10 mL glass vials with a pressure resistant lock. The headspace was flushed with nitrogen gas prior to incubation in an orbital shaker (160 rpm) oven at 34°C. Fermentation experiments with pure Synechococcus hydrolysates were carried out in biological triplicates (Figure 6). Fermentation experiments with glucose solutions supplemented with Synechococcus hydrolysates were carried out in biological duplicates (Figure 7).
Cell suspensions were diluted to an OD730 between 0.1 and 0.6 prior to measurement. All measurements were performed with a UV1800 spectrophotometer (Shimadzu, Kyoto, Japan).
Dry weight determination and chemical analyses
The cell DW in Synechococcus cultures was determined by separating the cells from the liquid by filtration (Whatman GF/F filters, GE Healthcare, Little Chalfont, United Kingdom). The filters were pre-dried for 24 hours at 90°C, then loaded with 1 ml of culture (0.5 mL after 48 hours of growth) and oven dried for 24 to 48 hours at 90°C. The DW content in hydrolysates, yeast cultures, and biomass suspensions was determined by drying the material on aluminum pans inserted in a thermogravimetric moisture analyzer (Sartorius, Göttingen, Germany). Carbon and nitrogen elemental analysis was performed on dried biomass using a Flash 2000 NC Soil Analyzer (Thermo Scientific, Waltham, Massachusetts, United States). Nitrate was determined in the supernatant of cell cultures after removal of the cells by centrifugation using a cadmium-reduction-based nitrate test kit (cat. no. 3319, LaMotte, Maryland, United States). Total carbohydrate was determined using the phenol sulfuric acid assay .
The monosaccharide composition of Synechococcus biomass was analyzed by strong acid hydrolysis using the TAPPI (Technical Association of the Pulp and Paper Industry) standard procedure , except that the standard curve samples were treated identically to the biomass samples to correct for sugar degradation . The monosaccharides D-glucose, D-xylose, L-arabinose, D-mannose, and D-galactose were measured on a ICS5000 system (Dionex, Sunnyvale, California, United States) equipped with a CarboPac PA1 2 × 50 mm guard column and 2 × 250 mm separating column (Dionex), operated at a flow of 0.25 mL min-1 and maintained at 30°C. Prior to detection, a post column flow of 0.1 mL min-1 of 0.2 M NaOH solution was mixed together with the flow carrying the separated sugars and analyzed using a PAD gold detector (Dionex).
The fermentation broth was analyzed for glucose, mannose, lactate, acetate, glycerol, and ethanol using an Ultimate 3000 HPLC (Dionex, Germering, Germany) equipped with an RI-101 refractive index detector (Shodex, Yokohama, Japan) and UV detector at 210 nm (Dionex). The separation was performed on a Rezex ROA column (Phenomenex, Torrance, California, United States) at 80°C with 5 mM H2SO4 as eluent at a flow rate of 0.6 mL min-1.
Dry weight determinations and chemical analyses were carried out in technical duplicates.
- Chl a:
weight per weight.
This work was financially supported by Nordic Energy Research project ‘Conversion of solar energy to infrastructure-ready transport fuels using aquatic photobiological organisms as the hydrocarbon feedstock producer – AquaFEED’ granted to N-U Frigaard. D Cannella was financially supported by the Nordic Top-level Research Initiative project TFI-PK-bio 02 ‘High gravity hydrolysis and fermentation of lignocellulosic material for production of bio-fuels’.
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