Publications

Production of Syngas Suitable to be Used in Fermentation to Obtain Biochemical Added-Value Compounds

Filomena Pinto, Rui Andre, Paula Marques, Ricardo Mata, Marta Pacheco, Patricia Moura, Francisco Girio

Abstract Lignin-rich residues are a side stream in advanced ethanol biorefineries and they are usually used for heat and power production. However, an alternative is the lignin valorisation by thermochemical and biochemical processes that might be economically more suitable. In this work lignin-rich residues were firstly thermochemical converted to syngas in a fluidised bed gasifier, using steam and oxygen as gasifying agents. The obtained results so far showed that the rise of steam/lignin-rich residues ratio favoured the release of H2 and CO2 and led to a decrease in CO and hydrocarbons content, as steam reforming reactions were favoured. On the other hand, the rise of the equivalent ratio clearly favoured partial oxidation reactions and thus the release of CO and CO2, while H2 and hydrocarbons contents were reduced, due to their partial combustion. The increase of temperature favoured the formation of H2, at the expenses of hydrocarbons and tar release, as both decreased. However, it is fundamental to control gasification temperature to prevent ash melting and consequent bed agglomeration, due to the high ash content in lignin-rich residues, generally with high content of silica and alkali metals. The results led to the selection of gasification conditions (800 °C, equivalent ratio of about 0.13 and steam/lignin ratio of around 0.4) that headed to the following gas ratio CO/CO2/H2=1:0.7:0.7, which was tested for further bacterial fermentation. Fermentation tests with Butyribacterium methylotrophicum showed that the above syngas composition strongly influenced the production levels of acetic and butyric acids. Marked differences in the CO, H2 and CO2 consumption rates were also observed and were correlated with cell growth and metabolites production.

Efficient hydrolysis of oligosaccharides using solid acids

Costa, D., Carvalheiro, F., Duarte, L.C., Gírio, F.

Abstract  This work reports on the use of ion exchange resins for the hydrolysis of hemicellulose-derived oligo-saccharides (OS). Four heat-stable cation exchange resins were evaluated using natural OS in order to access their performance and selectivity to catalyse OS hydrolysis with minimal monosaccharide dehydration to furans. Under mild operation temperature (140 ºC), it was possible to achieve high hydrolysis rates and yields (close to 100%), together with limited sugar degradation. Catalyst recyclability was also evaluated and reutilization up to 6 times was feasible, with minor loss of efficiency.

Efficient production of sugars and lignin streams using ethanol-based organosolv pretreatments

Co-authors: F. Carvalheiro, LNEG, Lisbon, PORTUGAL, F. Pires, LNEG, Lisbon, PORTUGAL, L.C. Duarte, LNEG, Lisbon, PORTUGAL, F. Gírio, LNEG, Lisbon, PORTUGAL

Abstract The development of innovative and low-temperature options based on green solvents for the fractionation of biomass is one of the key challenges for biorefineries. This work aims to develop an innovative option aiming at an integrated biomass upgrade concept to improve the production of lignin derivatives to value-added applications (biomaterials, aromatics), and clean sugars streams for e.g. fermentation to biofuels and other value-added compounds. Among the most promising options for industrial implementation are the organosolv-based processes since they may enable an integrated fractionation of biomass and to benefit from a lower CAPEX, specifically if ethanol is the based solvent, as its recovery can be integrated in the most common biorefinery downstream processing concepts. Nevertheless, this approach still lacks selectivity and efficiency on the recovery and separation of the lignin and hemicellulose fractions.

The Effect of the Chemical Character of Ionic Liquids on Biomass Pre-Treatment and Posterior Enzymatic Hydrolysis

Authors: Joana R. Bernardo; Francisco M. Girio and Rafal M Lukasik.

Abstract Ionic liquids have been recognised as interesting solvents applicable in efficient lignocellulosic biomass valorisation, especially in biomass fractionation into individual polymeric components or direct hydrolysis of some biomass fractions. Considering the chemical character of ionic liquids, two different approaches paved the way for the fractionation of biomass. The first strategy integrated a pre-treatment, hydrolysis and conversion of biomass through the employment of hydrogen-bond acidic 1-ethyl-3-methyimidazolim hydrogen sulphate ionic liquid. The second strategy relied on the use of a three-step fractionation process with hydrogen-bond basic 1-ethyl-3-methylimidazolium acetate to produce high purity cellulose, hemicellulose and lignin fractions. The proposed approaches were scrutinised for wheat straw and eucalyptus residues. These different biomasses enabled an understanding that enzymatic hydrolysis yields are dependent on the crystallinity of the pre-treated biomass. The use of acetate based ionic liquid allowed crystalline cellulose I to change to cellulose II and consequently enhanced the glucan to glucose yield to 93.1 ± 4.1 mol% and 82.9 ± 1.2 mol% for wheat straw and eucalyptus, respectively. However, for hydrogen sulphate ionic liquid, the same enzymatic hydrolysis yields were 61.6 ± 0.2 mol% for wheat straw and only 7.9 ± 0.3 mol% for eucalyptus residues. These results demonstrate the importance of both ionic liquid character and biomass type for efficient biomass processing.

Pretreatment and Fractionation of Wheat Straw by Organosolv in Biphasic System

Authors: Viola, E., Morgana, M., Zimbardi, F., Cerone, N., Romanelli, A., Valerio, V.

Abstract In this work we investigated the pretreatment of wheat straw in a biphasic system constituted by an organic solvent (butanol) and oxalic acid solution. The pretreatments were carried out in a 300 mL Parr reactor at total solid load of 5 wt%; temperature 140-180 °C, time 30-90 min and load of oxalic acid 0-10%. The pretreated straw was fractionated in the main constituents: cellulose, hemicellulose and lignin. The cellulose fraction has undergone enzymatic hydrolysis tests with commercial mix of enzymes in shaken flasks at 50°C up to 72h. The Organosolv treatment was optimized to maximize the purity of the cellulose and hemicellulose fractions and the glucose yields after enzymatic hydrolysis, by using an experimental design and response surface methodology. The results showed that the treatment is highly efficient to fractionate and deconstruct the straw, since its conversion into glucose, by enzymatic hydrolysis, reached the yield of 98%.

Gasification of lignin-rich residues for the production of biofuels via syngas fermentation: Comparison of gasification technologies

Eleni T. Liakakou, Berend Vreugdenhil, Nadia Cerone, Francesco Zimbardi, Filomena Pinto, R. Andre, P. Marques, R. Mata, Francisco Manuel Girio

This paper reports the use of lignin-rich residues from second generation bioethanol production, to produce syngas that can be applied in the gas fermentation process. Three gasification technologies at a different scale were considered in this study. Fixed bed updraft gasification of about 30 kg/h solid feed, bubbling fluidized bed gasification of about 0.3 kg/h solid feed and indirect gasification of about 3 kg/h solid feed. Two lignin-rich residues with different properties were tested and the results were evaluated in terms of feedstock pretreatment (grinding, drying and pelleting) and syngas quality requirements for the fermentation process. The molar H 2 /CO ratio (ranging from 0.6 to 1.0) and the tar yield (18–108 g/Nm ³ ) obtained from the three gasification technologies was quite different. For the syngas fermentation process, low H 2 to CO ratio is preferred, as most of the organisms grow better on CO than H 2 . Furthermore, different contents of impurities that can reduce the fermentability of the gas (such as hydrocarbons, HCN, HCl, NH 3 , COS and other organic S- compounds) were detected in the product gas. The concentration of these compounds in the syngas is related to the content of the corresponding compounds in the original feedstock. The different characteristics of the lignin-rich feedstocks are related to the specific pre-treatment technologies for the (hemi)cellulose extraction. By tuning the pre-treatment technology, the properties of the feedstock can be improved, making it a suitable for gasification. Tar and unsaturated hydrocarbon compounds need to be removed to very low levels prior to the fermentation process. As a next step, the combination of the gasification and the appropriate product gas cleaning, with the syngas fermentation process for the production of bio-alcohols will be evaluated and the overall efficiency of the gasification-fermentation process will be assessed.

Genome-wide systematic identification of methyltransferase recognition and modification patterns

Torbjørn Ølshøj Jensen, Christian TellgrenRoth, Stephanie Redl, Jérôme Maury, Simo Abdessamad Baallal Jacobsen, Lasse Ebdrup Pedersen & Alex Toftgaard Nielsen 

Abstract

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.

Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions

Jensen TØ, Pogrebnyakov I, Falkenberg KB, Redl S, Nielsen AT

Abstract

Use of thermophilic organisms has a range of advantages, but the significant lack of engineering tools limits their applications. Here we show that β-galactosidase from Geobacillus stearothermophilus (BgaB) can be applicable in a range of conditions, including different temperatures and oxygen concentrations. This protein functions both as a marker, promoting colony color development in the presence of a lactose analogue S-gal, and as a reporter enabling quantitative measurement by a simple colorimetric assay. Optimal performance was observed at 70 °C and pH 6.4. The gene was introduced into G. thermoglucosidans. The combination of BgaB expressed from promoters of varying strength with S-gal produced distinct black colonies in aerobic and anaerobic conditions at temperatures ranging from 37 to 60 °C. It showed an important advantage over the conventional β-galactosidase (LacZ) and substrate X-gal, which were inactive at high temperature and under anaerobic conditions. To demonstrate the versatility of the reporter, a promoter library was constructed by randomizing sequences around −35 and −10 regions in a wild type groES promoter from Geobacillus sp. GHH01. The library contained 28 promoter variants and encompassed fivefold variation. The experimental pipeline allowed construction and measurement of expression levels of the library in just 4 days. This β-galactosidase provides a promising tool for engineering of aerobic, anaerobic, and thermophilic production organisms such as Geobacillus species.

Air-steam and oxy-steam gasification of hydrolytic residues from biorefinery

Nadia Cerone, Francesco Zimbardi, Luca Contuzzi, Mauro Prestipino, Massimo O. Carnevale, Vito Valerio

Abstract

This paper reports the use of lignin-rich solid, derived from enzymatic hydrolysis of lignocellulosic biomass, to produce syngas and pyrolysis oil. The tested process was an updraft gasification carried out at pilot scale of about 20 kg/h as solid feed. The reactivity of two residues, one from straw and one from cane, was investigated by TGA in air, oxygen, nitrogen, using a heating program simulating the thermal gradient in the gasifiers. Below 400 °C the residues completely burned in air or oxygen with an apparent reaction order of 0.1–0.2. The 75%–80% of the organic mass was pyrolysed at 700 °C, when the gasification with H2O and CO2 started. In the plant tests, the residue was completely converted in gaseous and liquid energy carriers with an overall energy efficiency of up to 87%. Ten conditions were examined with different air flow (19.0, 25.5, 26.5 kg/h), O2 (4.0, 4.5, 5.5 kg/h) or H2O (as steam at 160 °C: 1, 2.5, 4.0, 4.5, 5.5, 8.5 kg/h). The experimental data were analyzed using the Response Surface Analysis (RSA) in order to highlight the dependence on the Equivalence Ratios of oxidation. The molar ratio H2/CO in the syngas increased by using steam as co-gasification agent, and reached the value of 2.08 in oxy-steam gasification. Steam was necessary to stabilize the process when using oxygen as it was effective in lowering the average temperature in the gasifier. Another positive effect of using steam was the shift of the temperature maximum far from the grate where ash melting could occur. Oxy-steam gasification provided the best results in terms of syngas heating value (LHV 10.9 MJ/m3) and highest thermal power output of the plant (67 kWth). The tar yield was inversely correlated with the residence time of the gas in the bed, in according with a zero order reaction for tar cracking into incondensable hydrocarbons and hydrogen.

Efficient Fractionation of Corn Stover by Organosolv Pretreatment and Enzymatic Hydrolysis of the Obtained Cellulosic Residue

Zimbardi, F., Viola, E., Gallo, V., Valerio, V.

Abstract

Corn stover was pretreated in a two phase system water/butanol acidified by oxalic acid. The purpose was to realize an efficient biomass fractionation and saccharification of the treated solid. The pretreatment was carried out in a 200 ml stirred autoclave at 140 °C and under pressure of CO2. After the treatment, the washed solid residue was hydrolyzed with enzymes. The analysis of the two liquid stream and solid residue pointed out that 1) Cellulose was quantitatively recovered as separate solid; 2) About two thirds of the xylan were recovered in the aqueous phase; 3) Most of lignin was solubilized and recovered in the organic phase; 4) The saccharification efficiency of the cellulose contained in the solid residue was complete.