Restructuring the conventional sugar beet industry into a novel

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Restructuring the conventional sugar beet industry into a novel biorefinery: Fractionation and bioconversion of sugar beet pulp into succinic acid and value-added co-products Maria Alexandri, Roland Schneider, Harris Papapostolou, Dimitrios Ladakis, Apostolis A. Koutinas, and Joachim Venus ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b04874 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

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ACS Sustainable Chemistry & Engineering

Restructuring the conventional sugar beet industry into a novel biorefinery: Fractionation and bioconversion of sugar beet pulp into succinic acid and value-added co-products

Maria Alexandri†,‡, Roland Schneider†, Harris Papapostolou‡, Dimitrios Ladakis‡, Apostolis Koutinas‡,*, Joachim Venus†,*

†

Department of Bioengineering, Leibniz Institute for Agricultural Engineering and Bioeconomy (ATB), Max-Eyth Allee 100, Potsdam, 14469, Germany

‡

Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Οdos 75, Athens 11855, Greece

* Equally contributed as corresponding authors e-mails of corresponding authors: [email protected]; [email protected]

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Abstract Sustainable chemical production should rely on the valorisation of crude renewable resources. Waste biomass refining complies with bio-economy and circular economy initiatives. In this regard, sugar beet pulp (SBP) was efficiently fractionated into pectins, phenolic compounds and a sugar-rich hydrolysate that was subsequently used as fermentation feedstock for succinic acid production. Phenolic compounds were separated via acidified aqueous ethanol extraction, while pectins were obtained via sequential treatment with HCl, NaOH and isopropanol. Hydrolysis of cellulose and hemicellulose was optimised in lab- and pilot scales leading to 45 g/L of total sugars with glucose and arabinose being the predominant ones. Labscale fed-batch fermentations were carried out with the bacterial strain Actinobacillus succinogenes cultivated on SBP hydrolysate resulting in the production of 30 g/L of succinic acid concentration with productivity of 0.62 g/L/h and yield of 0.8 g/g. Similar fermentation efficiency was also demonstrated in 50 L bioreactor cultures. Succinic acid crystals were purified from the fermentation broth by two alternative downstream separation processes based on either semi-pilot scale bipolar membrane electrodialysis with product purity and yield of 79% and 21.2% or acidification of succinate salts using cation exchange resins with product purity and yield of 95% and 80.1%, respectively. The novel biorefinery concept led to 78.6 g of phenolic-rich extract, 303.1 g of a pectin-rich isolate, 268 g of succinic acid and 208.4 g of remaining solids with 20% protein content from 1 kg of SBP.

Keywords: sugar beet pulp, biorefinery, pectins, antioxidants, succinic acid, bipolar membrane electrodialysis, Actinobacillus succinogenes

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Introduction Biomass refining involves the valorisation of a renewable resource via its fractionation and bioconversion to value-added products such as fuels, chemicals, polymers and bioactive ingredients.1 The development of integrated biorefineries based on the efficient exploitation of industrial wastes and by-products for the production of value-added products, would maximize profit decreasing at the same time the dependence on petroleum-based commodities.2 The sustainability of the process could be enhanced significantly if the production of chemicals from crude renewable resources is coupled with the extraction of value-added products (e.g. antioxidants, proteins, pectins) that would contribute to the overall process viability.1 Sugar beet constitutes one of the most important crops, as it is number seven commodity worldwide, with annual production of about 270 million t. Germany is one of the most important producers of sugar beet in Europe, with annual production of 28 million t, representing the 10% of the worldwide production.3 Sugar beet pulp (SBP) is the main byproduct of the sugar production industry resulting after sucrose extraction from the crop. It is estimated that its annual production accounts approximately to 5 million t (on dry basis) in Europe and about 1 million t in the USA.4 The current practice involves drying and pelletising the pulp in a cost-intensive process. The dried SBP is then sold as low-value animal feed. However, the production of value-added products from SBP will enhance the profitability of to the conventional sugar production industry. SBP has been widely evaluated for the extraction of pectins.5,6 SBP has also been used as fermentation feedstock for the production of bioethanol,7 acetone-butanol-ethanol,8 lactic acid7 and biogas.9 There are also many studies dealing with the hydrolysis of cellulose and hemicellulose for the production of sugar-rich substrates.10,11 SBP has also been considered as feedstock for biorefinery development. Hamley-Bennet et al. optimized pectin solubilisation



from SBP via steam pretreatment.11 The cellulose-rich fraction was then utilized as fermentation substrate for the production of 0.48 g of bioethanol per g of glucose. CárdenasFernandez et al. proposed the fractionation of SBP into soluble pectin and a cellulose-rich fraction.12 Enzymatic hydrolysis of cellulose led to a glucose-rich medium that was subsequently fermented into bioethanol, while selective hydrolysis of the pectin-rich fraction could lead to the production of arabinose and D-galacturonic acid-rich streams. The arabinose could be converted into L-gluco-heptulose that could be used in medical applications. Succinic acid is considered an important platform chemical for the development of a sustainable chemical industry, as it can be used as precursor for the production of various bulk chemicals, polymers and resins among others.1 Succinic acid is traditionally produced via catalytic hydrogenation of maleic acid or maleic anhydride, a process that is both expensive and harmful to the environment.13 The market of bio-based succinic acid is growing rapidly with a current annual production capacity of 38,000 t and a market price of 2.9 $ kg-1.14 The reduction of succinic acid production cost via fermentation would lead to its establishment as a major building block in the bioeconomy era for the production of numerous commodity chemicals.1 This could be achieved through the development of novel biorefinery concepts exploiting the full potential of renewable resources. Knoshaug et al. reported a novel biorefinery concept focusing on the utilisation of biomass from the green alga Scenedesmus acutus for the production of renewable diesel blendstock, biogas and succinic acid.15 Alexandri et al. reported a novel biorefinery based on the utilisation of spent sulphite liquor for the production of phenolic-rich extract, lignosulphonates and succinic acid.16 Patsalou et al. proposed the exploitation of citrus peels for the production of essential oils, pectins and succinic acid.17 Although succinic acid is already produced on industrial scale,18 biorefinery development is still in research and development stage.


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The aim of this study is to propose a biorefinery concept based on SBP refining, polysaccharide hydrolysis and bioconversion to succinic acid. The proposed process involves the extraction of an antioxidant-rich extract and pectins from the SBP followed by hydrolysis of cellulose and hemicellulose in order to obtain fermentable sugars. Succinic acid production using SBP derived hydrolysates was carried out in lab-scale and 50 L scale bioreactors. Downstream separation and purification (DSP) of succinic acid crystals has also been carried using two different methods, aiming to provide a complete overview of the process. The novel biorefinery concept presented in this study could restructure the conventional sugar production industry leading to improved profitability with diversified market outlets.

Materials & Methods Sugar Beet Pulp SBP was provided by the industry Pfeifer & Langen GmbH & Co. KG (Germany) in the form of dry pellets. Cellulose was the main structural polymer (23.0 ± 3.3%) followed by hemicellulose (19.5 ± 6.2%), pectins (30.3 ± 2.1%), protein (9.6 ± 0.6 %) and lignin (2.6 ± 0.4%). Extraction of phenolic compounds and pectins The extraction of phenolic compounds was carried out according to the process presented by Burniol-Figols et al.19 Briefly, acidified 60% (v/v) aqueous ethanol were added to 2 g of SBP (dry basis) at a solid-to-liquid ratio of 1:12.5 and 1:25. The mixture was agitated (150 rpm) at 70 oC for 40 min. The extract was filtered, vacuum evaporated, weighted and finally re-dissolved in methanol for subsequent analysis. Pectins were extracted following the modified procedure of Lv et al.

20

More

specifically, a suspension of SBP with a solid-to-liquid ratio of 1:20 (w/v) was placed in a beaker at 94 oC under agitation at 200 rpm. The pH of the suspension was adjusted to 1.2



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using 12 M HCl. The resulting slurry was cooled down, the pH was adjusted to 4.5 using 25% (w/w) NH3·H2O and filtered under vacuum. Pectins were precipitated and recovered from the supernatant using two volumes of 100% ethanol and dried at room temperature. Hydrolysis of SBP SBP was initially subjected to acid pretreatment and subsequently to enzymatic hydrolysis. The experiments were carried out in triplicates. The hydrolysis yield was evaluated according to the total amount of sugars released in comparison to the theoretical amount of sugars that could be derived from the cellulose and hemicellulose content of SBP. For cellulose the conversion factor used was 0.9 and for hemicellulose it was 0.88.21 Total hydrolysis yield for both cellulose and hemicelluloses was calculated according to the following equation:

𝑇𝑜𝑡𝑎𝑙 ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝑦𝑖𝑒𝑙𝑑 (%) =

total released monosaccharides (g) x 0.9 x 100 hemicellulose (g) + 𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 (𝑔)

eq. (1)

Preliminary evaluation of SBP pretreatment A set of preliminary experiments was initially carried out according to the study of Bellido et al.8 A suspension of 6% SBP (w/v) in water placed in 250 mL Erlenmeyer flasks was pretreated using four different methods: autohydrolysis, autohydrolysis at pH 4, pretreatment with 0.5% (v/v) H2SO4 and pretreatment with 0.5% (v/v) HCl. Hydrolysis of hemicellulose was carried out at 121 oC for 15 min. The remaining solids were subjected to enzymatic hydrolysis, after adjusting the pH to 5, with the addition of 20% (w/w) NaOH using Accellerase 1500 (Genencor). Enzyme loading was 275 carboxymethylcellulose activity units (CMC U) per g of cellulose (0.1 mL enzyme per g of cellulose), and hydrolysis was carried out at 50 oC for 24 h under stirring (150 rpm).


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Evaluation of different solid-to-liquid ratios Acid hydrolysis was evaluated at four solid-to-liquid ratios (6%, 7.5%, 10% and 15%, w/v) using 0.5 % (v/v) H2SO4 at 121 oC for 15 min or 30 min. After hemicelluloses hydrolysis, the pH was adjusted to 5 and Accellerase 1500 was added at 275 CMC U (0.1 mL/g cellulose) and 1375 CMC U (0.5 mL/g cellulose) per g of cellulose. The final slurry was centrifuged in order to remove the solids and the supernatant was analysed for sugars and byproducts. Microorganism and inoculum preparation The strain Actinobacillus succinogenes 130Z (DSM 22257) was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. Inoculum preparation was carried out in 30 g/L Tryptic Soya Broth and incubated at 37 οC for 12 h. Lab- and pilot-scale fermentations Batch and fed-batch fermentations in lab-scale were conducted in 5 L bench-top BIOSTAT bioreactor (Sartorius AG, Germany) with 3 L working volume. Fed-batch fermentations in pilot-scale were carried out in a 50 L bioreactor (B-Braun Biotech, Germany) with 30 L working volume. Stirring was kept constant at 300 rpm in lab-scale fermentations and 200 rpm in pilot-scale cultures. The pH was adjusted to 6.6-6.8 by adding 20% (w/v) NaOH. The medium also contained 5 g/L yeast extract (Ohly KAT, Deutsche Hefewerke GmbH & Co. OHG, Germany), 10 g/L MgCO3, 1.16 g/L NaH2PO4•H2O, 0.31 g/L Na2HPO4, 1 g/L NaCl, 0.2 g/L MgCl2•6H2O, 0.2 g/L CaCl2•2H2O and antifoam. The experiments were carried out under continuous sparging of CO2 (0.1 vvm) at 37 οC. The fermentation medium was pasteurised at 70 οC for 1 h. The inoculum was 10% (v/v) in all fermentations. The experiments were repeated twice. Succinic acid yield during batch and fed-batch fermentations was calculated according to equation 2 and 3, respectively: 𝑆𝐴 𝑌𝑖𝑒𝑙𝑑 =

(g SA produced + g SA in samples)

(eq. 2)

(g initial TS) ― (g TS in samples)



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(g SA produced + g SA in samples)

(eq. 3)

𝑆𝐴 𝑌𝑖𝑒𝑙𝑑 = (g initial TS) + (g TS in feeding) ― (g TS in samples) ― (g final TS) where SA stands for succinic acid and TS for total sugars. Pilot-scale DSP based on bipolar membrane electrodialysis

Succinic acid produced during fed-batch fermentation in the pilot scale bioreactor was subsequently separated and purified using a series of downstream steps (Figure 1S). The process is described in detail by Neu et al. 22, besides the step of spray drying. The purified stream produced after chromatography was introduced in a spray-dryer GEA Miro, type GEA Mobile Minor. After this step, succinic acid was collected in powder form, weighted and analysed for impurities. DSP based on acidification using cation exchange resins The separation and purification of succinic acid derived from the lab-scale fed-batch fermentation was according to the process presented by Lin et al.

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The cationic resin

employed was EXO8. Succinic acid yield and crystal purity were calculated according to the following equations: 𝑌𝑖𝑒𝑙𝑑 (%) =

𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑒𝑑 𝑠𝑢𝑐𝑐𝑖𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 (𝑔) 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑢𝑐𝑐𝑖𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 𝑖𝑛 𝑡ℎ𝑒 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑙𝑖𝑞𝑢𝑖𝑑 𝑚𝑒𝑑𝑖𝑢𝑚 (𝑔)

𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑒𝑑 𝑠𝑢𝑐𝑐𝑖𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 (𝑔)

𝑃𝑢𝑟𝑖𝑡𝑦 (%) = 𝑇𝑜𝑡𝑎𝑙 𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑔)

(eq. 4)

(eq.5)

Analytical Methods Determination of sugars and organic acids by HPLC Quantitative analysis of monosaccharides and organic acids was carried out by HPLC (DIONEX, USA), as described by Neu et al.22 Determination of furfural and HMF in the SBP-derived hydrolysates The concentration of furfural and HMF in the hydrolysates was carried out using HPLC (ICS-3000, ThermoFisher) with a UV detector. The method followed was according to DIN 32645.24 7 ACS Paragon Plus Environment

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Determination of individual phenolic compounds by HPLC-DAD Chromatographic analysis was carried out using HPLC coupled to a diode array detector (MD-910 Jasco). The conditions followed are described in detail in the study of Alexandri et al.25 Spectrophotometric determination of total phenolic content of the extract Determination of Total Phenolic Content (TPC) was carried out using the FolinCiocalteu colorimetric method as described by Faustino et al.26 The concentration of total phenolic compounds was expressed as mg of gallic acid equivalents per gram of dry SBP (mg GAE/g). All determinations were performed in triplicates. Determination of antioxidant activity of the extract Antioxidant activity of the extracts was determined according to the DPPH• scavenging radical method following the protocol developed by Scherer and Godoy.27 The radical scavenging activity (I%) was calculated using the following equation: I% = [(Abs0-Abs1)/Abs0] ˟ 100

(eq. 6)

where Abs0 was the absorbance of the blank sample and Abs1 the absorbance of the extract. The antioxidant activity index (AAI) was calculated by dividing the final concentration of DPPH with the IC50 of the extract. When AAI < 0.5, the extracts exhibit low antioxidant activity, when AAI has values between 0.5 and 1.0 then the extracts are considered as moderate antioxidants, when 1.0

Restructuring the conventional sugar beet industry into a novel

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