J Appl Phycol (2009) 21:569–574 DOI 10.1007/s10811-008-9384-7 Fermentation study on Saccharina latissima for bioethanol production considering variable pre-treatments Jessica M. Adams & Joseph A. Gallagher & Iain S. Donnison Received: 30 July 2008 / Revised and accepted: 19 September 2008 / Published online: 21 October 2008 # Springer Science + Business Media B.V. 2008 Abstract Climate change, fuel security and economics are creating a requirement for alternative, renewable fuels. Bioethanol is currently produced from land-based crops but in future marine biomass could be used as an alternative biomass source for bioethanol production. Macroalgae such as Laminaria spp. grow in abundance around the United Kingdom, reaching >4 m in length and containing up to 55% dry weight of the carbohydrates laminarin and mannitol. Using enzymes, these can be hydrolysed and converted to glucose and fructose, which in turn can be utilised by yeasts to produce ethanol. In previous studies on ethanol production from macroalgae, pre-treatment was at 65°C, pH 2 for 1 h prior to fermentation. This paper shows that these pre-treatments are not required for the fermentations conducted, with higher ethanol yields being achieved in untreated fermentations than in those with altered pH or temperature pre-treatments. This result was seen in fresh and defrosted macroalgae samples using Saccharomyces cerevisiae and 1 U kg−1 laminarinase. Keywords Bioethanol . Fermentation . Laminaria . Macroalgae . Saccharina latissima Introduction Climate change, fuel security and economics are driving the use of renewable technologies. Renewable energy sources Paper presented at the 3rd Congress of the International Society for Applied Phycology, Galway. J. M. Adams (*) : J. A. Gallagher : I. S. Donnison Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Aberystwyth, Wales, UK e-mail:
[email protected] such as solar-, wind-, and hydro-energies may be used to generate electricity or heat either directly or indirectly, but biomass is the only renewable energy source capable of producing liquid fuels (biofuels) for storage and as a transport fuel. In the United Kingdom, 5% of transport fuels must be from renewable sources by 2010 as part of the legally binding renewable transport fuel obligation (Department for Transport 2007). The production of bioethanol involves the fermentation of sugars by microorganisms to produce ethanol. As many sugars are not freely available but form part of structural and storage carbohydrates there is a requirement for treatments such as altered temperature, pH and addition of enzymes to hydrolyse the sugars prior to fermentation. Distilled bioethanol can be blended with petrol (gasoline) and used in vehicles without alterations if mixed at 5% (v/v) (The European Parliament and the Council of the European Union 2003) or up to 85% (v/v) in flex-fuel cars currently being produced by several different vehicle manufacturers (NEVC 2008). As the CO2 released from combusted bioethanol was previously removed from the atmosphere by the plant into organic carbon, the return of this gas is considered ‘carbon neutral’. Use of bioethanol additionally reduces the amount of petrol combusted per kilometre, lowering demand and so increasing the security of supply. The main bioethanol feedstocks—sugarcane, maize, wheat (Wheals et al. 1999)—are all land-based crops, with little work to date conducted on marine sources of biomass. As approximately 50% of global biomass is thought to be generated in a marine environment (Carlsson et al. 2007), marine biomass has great potential as a feed stock for future bioethanol generation. In addition, the issues arising with increasing the proportion of land use for biomass crops and the “food versus fuels” debate are not applicable to macroalgae. Materials and methods Saccharina latissima for fresh studies was harvested in September 2008 from Aberystwyth beach (Ordinance . with the remainder generated from extracted hydrocolloids. macroalgae are capable of producing high yields of material when compared to even the most productive land-based plants. respectively. proteins and alginic acid at the beginning of spring. a group including Laminaria.02% of the surface level of light (Sze 1998). seaweed extract was produced from fresh Laminaria hyperborea pre-treated with water at pH 2 and 65°C for 1 h.5–6 billion. when mannitol and laminarin are at a minimum. fertilisers and animal feed additives (McHugh 2003).5–8 million tonnes of naturally growing and cultivated seaweed harvested worldwide. laminarian beds produce 1. especially by China. The main use is as food products for human consumption. Phaeophyta are an algae division of the brown seaweeds.570 J Appl Phycol (2009) 21:569–574 Macroalgae. where minced or finely shredded Laminaria fronds were incubated with 0. but an average of 1.5 m (Hayward et al. The seaweed industry has an estimated total annual value of US $ 5. 2000a). 1999)—consists primarily of linear β-1.glucanase] (EC 3. Kelp forests in shallow subtidal regions are amongst the most productive communities on earth. kelps such as Saccharina latissima (Laminaria saccharina) can grow up to 4 m in length. depending on the proportion of branching (Nelson and Lewis 1974). Previous studies in this area (Horn 2000. coinciding with a reduced growth rate. If these are present. in October–November (Horn 2000). (2000a. especially the species Laminaria japonica (kombu). Japan and South Korea. with maximum ash. In addition. To increase the hydrolysis of laminarin in macroalgae. though some larger algae additionally have gas-filled bladders (Sze 1998). which generate approximately US $ 5 billion per year. allowing macroalgae to grow for several metres down the ropes in the water.3 and 11. production figures between 3. Undaria pinnatifida (wakame) and Porphyra species (nori) are extensively farmed in Asia. The lower level for macroalgae is 0. Laminarin—the main storage carbohydrate isolated from phaeophyta (Zvyagintseva et al. Mannitol concentrations then increase again to give the maximum laminarin and mannitol yields of 25% and 20% dry weight. produced from 7. oxygen (active electron transport chain) or transhydrogenase are required. 2003).6). 1999). generating large amounts of organic carbon. based on methods in a book by Percival and McDowell (1967).2. mannitol has the potential to be a valuable carbohydrate source for ethanol production.3(4)-β. these may be soluble or insoluble in cold water. This division includes Fucus species and ‘kelps’. Composition varies throughout the year. with a more rapid decrease in intensity in northern waters as they contain more plankton and suspended material.1.0 kg organic carbon m−2 year−1 is more typical of kelp beds in general (Sze 1998). Saccorhiza and Alaria species. macroalgae may be cultivated in three dimensions rather than in two as on land. Mannitol is an alcohol form of the sugar mannose but is not readily utilised for fermentation by Saccharomyces cerevisiae as it first requires oxidation to fructose by the enzyme mannitol dehydrogenase.6-linkages (Nobe et al. In autumn the reverse is true. The present study considered whether the techniques published by Percival and McDowell for laminarin extraction are the most suitable for pre-treating macroalgae prior to fermentation. which are multicellular with a branched thallus (Zvyagintseva et al. Typically a ~25 polymer chain. b). The methods referred to are for the extraction of laminarin.75 kg organic carbon m−2 year−1. Light decreases exponentially in water. The study also addressed whether different pre-treatments were required depending on whether the seaweed substrate was fresh or previously frozen. The high yields are due partially to macroalgae requiring less energy for the production of supporting tissue than land plants. but may additionally be seeded onto thin weighted strings suspended over a larger horizontal rope (Kelly and Dworjanyn 2007). thus theoretically providing more glucose for yeast fermentation. Little work has been published on the fermentation of macroalgae to ethanol to date apart from the work of Horn et al. 1996). Due to structural differences between macroalgae and land plants. Laminarin may be readily hydrolysed to glucose using laminarinase [endo-1. When considering the dry weight generated. In Nova Scotia. with cultivated species producing higher yields (Horn 2000). Percival and McDowell 1967) proposed a reduction in pH and an increase in temperature to increase solubility of laminarin. the mannitol concentration plateaus. and the capability to take up nutrients over their entire surface. pre-treatments may be conducted prior to fermentation. generating NADH. In July–August. absence of nutrients in the water and the probable period of sporogenesis in Laminaria species (Black 1950). Around the United Kingdom and Ireland. while Saccorhiza polyschides reaches 4.1 kg m−2 year−1 for non-cultured macroalgae are cited (Gao and McKinley 1993). for example in Zymobacter palmae (Horn et al. In these latter papers. Macroalgae can be favourably compared with the other main bioethanol feedstocks as shown in Table 1. Macroalgae seedlings are often grown on nets or strings (Juanich 1988). To regenerate NAD+.3-linked glucose residues with small amounts of β-1.09 M HCl for 30 min at 70°C before being filtered through muslin. The surrounding water provides buoyancy. in addition to growth in the other two dimensions. 5 L blender (Kenwood. The remaining flasks. Tokyo.800a 1. Hants. bagged and frozen within 3 h of collection. Samples were cut.560c. For frozen substrate: samples of S.1 U laminarinase (Sigma) (unless stated) and yeast preparation at a 0.400 2. .150 Sugar cane 68. producing a finely shredded slurry. and then the pH was adjusted to pH 6 (if required). mixed with an equal weight of water.000b 40. fucose. then twice a day for the fermentation period. Flasks were placed in a waterbath at 32°C on a multiple point stirring plate. Yeast solution preparation A solution of 5% (w/w) dried Ethanol Red yeast (Fermentis. Saccharina latissima for frozen studies was harvested in November 2007 at Barnacarry beach (Ordinance survey reference NM809227) in Argyll and Bute. Pre-treatment Slurries were placed on a Telesystem HP multiple point stirring block (Variomag.0 g of this preparation were added to replicate 100 mL Erlenmeyer flasks and made to 100 g dilute slurries with tap water. Daytona Beach.0 mL slurry were removed six times during the fermentation period. methanol and ethanol were determined from RI detected peaks. flasks at 70°C were cooled on ice.J Appl Phycol (2009) 21:569–574 Table 1 A comparison between the major bioethanol crops and macroalgae Wheat (grain) Average world yield (kg ha−1 year−1) Dry weight of hydrolysable carbohydrates (kg ha−1 year−1) Potential volume of ethanol (L ha−1 year−1) h a 571 Maize (kernel) 4. producing a finely shredded slurry.815a 3. Agilent Technologies. latissima were cut into ~5 cm2 pieces. 2×pH 6 remained on the stirring block and were incubated at 23°C for 30 min. then flasks at pH 2 for both temperature pre-treatments were returned to pH 6 using 5 M NaOH. sealed with a crimp cap and stored at 5–8°C prior to high performance liquid chromatography (HPLC) analysis. Substrate preparation For fresh substrate: samples of S. Santa Clara CA). Japan) into 0. and cut using a Kenwood 1. latissima were defrosted. bagged and processed as below within 2 h of collection.010 Sugar beet 47. UK) for 5 min. FL) at room temperature for 5 min. Samples were cut.6 mL min−1 (Jasco.756 Macroalgae 730. Millipore.260a 11. glucose.M.800 g for 5 min.5 mL glass vials. the relative concentrations of fermentation components laminarin.5% (v/v) acetonitrile solution in 5 mM H2SO4.150g 23.46 b survey reference SN 581823) in Ceredigion. Havant.2. Seattle.45 µm Durapore (PVDF) syringe-driven filter unit (Millex-HV. 2×pH 2. Using the HPLC software (EZChrom Elite Version 3. glycerol.5 L blender (Kenwood) for 5 min. This data was imported into Microsoft Office Excel 2007 (Microsoft.d 1. Scotland (UK). Samples were syringed through a 0.600f 6. 2007) e Boyer and Hannah (2001) f CEDUS website (2008) g Horn (2000) h Assuming conversion rate of 0.5% (v/v) final fermentation volume were then added to the slurry following pretreatment. with samples taken at 0 h. Aliquots of 75. 50 µL supernatant was then removed and added to 450 μL 0. Fermentation An aliquot of 0. France) in deionised water was prepared and stirred on a multiple point stirring block in a pre-heated waterbath (32°C) for 30–60 min prior to use. UK). mannitol.825f 5. removed and vortexed every 5 min to reduce settling. lactic acid. then mixed with water in a 1:2 ratio and cut using a Kenwood 1. Following pre-treatment. Essex. Samples were centrifuged at 14.e 2. Great Dunmow. Aliquots of 50. This was conducted on a Resex ROA–organic acid H+ column run at 30°C in 5 mM H2SO4 mobile phase at 0. Adams (unpublished data. Scientific Software. Sampling methodology Aliquots of 1. WA) and used to plot component concentrations over the fermentation period. succinic acid. Wales (UK).100d.010 FAO STAT (2008) Gao and McKinley (1993) c Henry and Kettlewell (1996) d J. acetic acid.070a 8. Four flasks were then adjusted to pH 2 using 2 M HCl. Two of these flasks plus two at pH 6 were incubated at 70°C for 30 min. Marcq-en-Baroeul.0 g of this preparation were added to replicate 100 mL Erlenmeyer flasks and made to 100 g dilute slurries with tap water. 1. laminarinase [β-(1. The laminarin proportion present in the slurries through the fermentation period is seen in Fig.4 0. Prior to fermentation.1 0.3)glucanase] was added.5 2 1.2 0. Prior to fermentation.05 0 0. pH 2.3 J Appl Phycol (2009) 21:569–574 Ethanol present (% v/v) Ethanol present (% v/v) 0.● 23°C. Pre-treatments: ■70°C. Prior to fermentation. Pre-treatments: ■70°C. Preparations of S. Preparations of S. slurries were incubated for 30 min at the temperatures and pH values indicated.6 0 10 20 30 40 Fermentation period (h) 50 60 70 0 10 20 30 40 50 Fermentation period (h) 60 70 Fig. pH 6. Following the varied pre-treatments. The feedstock. latissima following various pH and heat pre-treatments sampled over time. 1 Ethanol concentration in fermentations of fresh Saccharina latissima following various pH and heat pre-treatments sampled over time. latissima were then incubated at 32°C.35 0. 2. pH 6. n=2 except for 70° C. pH 2. Saccharina latissima. pH 6 on a multipoint stirring block with 0. latissima were then incubated at 32°C. pH 2. n=2. latissima following various pH and heat pre-treatments sampled over time. error bars standard error Fig. pH 2. slurries were incubated for 30 min at the temperatures and pH values indicated. The mean concentration of ethanol in each fermentation following the varied pre-treatments on fresh S. pH 6.8 1. △ 70°C. Preparations of S.1 0 0 10 20 30 40 50 Fermentation period (h) 60 70 0 10 20 30 40 50 60 70 Fermentation period (h) Fig. The ethanol percentage (v/v) was calculated from each sample taken throughout the fermentation and the ‘success’ of the fermentations was ascribed to ethanol produced during the fermentation period and the proportion of laminarin remaining in the slurry.2 1 0.1 U laminarinase. then Saccharomyces cerevisiae to initiate fermentation. 3 Ethanol concentration in fermentations of defrosted S. 4 Laminarin concentration in fermentations of defrosted S.2 0. slurries were incubated for 30 min at the temperatures and pH values indicated. latissima following various pH and heat pre-treatments sampled over time. △ 70°C. latissima were then incubated at 32°C. pH 2. slurries were incubated for 30 min at the temperatures and pH values indicated.4 1.1 U laminarinase. pH 2.5% (v/v) yeast and 0. pH 6. △ 70°C. n=2. pH 6. These changes were run simultaneously with controls for each parameter. latissima is seen in Fig. which was considered a possible additional requirement for the slurry. error bars standard error .3 0. Pre-treating at 70°C for 30 min prior to rapid cooling was additionally a pasteurisation of the fermentation slurry. pH 6. Pre-treatments: ■70°C.5 1 Black (1950). to give four different pre-treatments in total. pH 2.5 0. t=17 h where n= 1. pH 2. pH 6.6 1. pH 6 pre-treatment. Preparations of S.8 0. ◊23°C. ◊23°C. ◊23°C. error bars standard error Fig. 2. pH 6 on a multipoint stirring block with 0.5% (v/v) yeast and 0. 2 Laminarin concentration in fermentations of fresh S.● 23°C.5% (v/v) yeast and 0. latissima were then incubated at 32°C.25 0.1 U laminarinase. was harvested in September and November to contain high proportions of laminarin and mannitol in the dry material as determined by 4 3.5% (v/v) yeast and 0.● 23°C. error bars standard error Results and discussion Fresh and defrosted macroalgae samples were pre-treated by altering the pH and temperature. Pre-treatments: ■70°C. pH 6 on a multipoint stirring block with 0. Prior to fermentation. △ 70°C.5 Laminarin present (% w/v) 3 2. pH 6 on a multipoint stirring block with 0.● 23°C.15 0. pH 6.572 0.2 2 Laminarin present (% w/v) 1.1 U laminarinase. ◊23°C. n=2. J Sci Food Agric 5:176–183. Pre-treatmentat pH 6. As this pre-treatment was based on increasing laminarin solubility. respectively. 4). which yielded 0. 70°C with both fresh and previously frozen feedstock (Figs. Results show highest ethanol yields of 0. 3 and 4. 1 and 3 fermentations overall proved this a disadvantageous pre-treatment. 2. Conclusion The current demand for bioethanol outstrips supply and with rising oil prices. improved technologies and tax breaks. pH 2. 2.2740050404 . with implications that future fermentations conducted on defrosted macroalgae would be comparable to those conducted on fresh macroalgae. 4).45% (v/v) (Fig.3–11. there was only a small increase in laminarin (at t=0 h) following pre-treatment at the higher temperature (Figs. Interestingly. J Mar Biol Assoc 29:45–72 Black W. demand for land use is increasing and the conversion of land use from food to fuel is a contentious issue. different locations around the United Kingdom to be analysed at the same time. which may be fermented to ethanol. this increase was small and the salt inhibition seen in Figs. 1 and 3.13% (v/v) ethanol compared with fermentations containing neither condition. these issues are negated. the production of bioethanol is becoming increasingly viable. The lower yields generated by the slurries with altered pH may be due to an increase in salt concentration in the slurry. Using frozen macroalgae has distinct advantages.g. 3). Similar yield patterns were seen in fermentations with fresh and defrosted macroalgae. grant number GR/ S28204. However. which reduced the efficiency of the laminarinase on the yeast. The slurries initially contained low salt concentrations from the seawater retained on the macroalgae and ions within the algae. thus releasing the cellular contents. which was included in the macroalgae weight. The fresh macroalgae would have additionally had a higher proportion of laminarin present due to the time of year of harvest (Black 1950).32% (v/v) to 0. Dewar E (1954) The properties of the algal chemicals. 23°C. so negating these pre-treatments in future. Fig. latissima following the pre-treatments are seen in Figs. the importance of this treatment regarding fermentation was questioned.45% (v/v) and the largest reduction in laminarin when neither pre-treatment was employed in both fresh and defrosted macroalgae substrates. Acknowledgements This work was supported by the Engineering and Physical Sciences Research Council (EPSRC). II Some derivatives of Laminarin. Reductions in pH also reduced the ethanol yield from 0. with pre-treatment involving both a pH change and heated incubation yielding 0. with phaeophyta producing 3. but that a 0. In practice. 23°C clearly produced the most ethanol. the two lower-yielding fermentations had the heat pre-treatment. nor used the mannitol component. Lowered pH as a pre-treatment was assumed to have been used to disrupt the cells. but with the adjustment to pH 2 and back to pH 6 with HCl and NaOH.32% (v/v) yield of ethanol was achieved without. the ethanol concentration and laminarin proportion in fermentations using defrosted S. Nelson and Lewis 1974) so in theory aid hydrolysis by laminarinase.24% (v/v) and 0. 3 shows that a yield of 0. followed by pre-treatments pH 2. References Black W (1950) The seasonal variation in weight and chemical composition of the common British Laminariaceae. Mannitol concentrations remained constant throughout the fermentation period following all pre-treatments in fresh and defrosted fermentation slurries (data not shown). Similarly. By considering marine biomass as a source of carbon for bioethanol production. Oban for the supply of the frozen Sacchorina latissima samples. potentially causing inhibition of the yeast and thus reducing ethanol production.J Appl Phycol (2009) 21:569–574 573 Similarly. with previously conducted fermentations receiving a pretreatment of a pH reduction to pH 2 and heating to 65°C for 1 h. 1).1002/jsfa. 70°C. possibly due to the increased solubilisation of inhibitory compounds. the salt concentration would have been increased further. Heating has been shown to solubilise the cold water-insoluble fraction of laminarin (Black and Dewar 1954. subsequent fermentations will increase the ethanol yield further. As the fermentations reported here were not optimised. for fermentations with pretreatments at room temperature. Thanks to Maeve Kelly and Lars Brunner of the Scottish Association for Marine Sciences (SAMS). An autumn harvest can accrue over 50% dry weight as sugars.45% (v/v) to 0.03 % (v/v) ethanol was seen in fermentations with altered temperature and pH pre-treatments. This will reduce the number of steps in the fermentation and so lower processing costs if conducted commercially in future. This is almost the opposite of that expected. and pH 6. doi:10. improving the efficiency of the process without spending energy on unnecessary pre-treatments. However. although there was an increase in laminarin yields in slurries with pH 2 pre-treatments (Figs. The ethanol yield in the 70°C pre-treatment fermentations was lower than in those pre-treated at 23°C.19% (v/v) in Figs. 1. Laminarin was lower in the defrosted macroalgae fermentations due to residual water being frozen with the sample. 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