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

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

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.

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.