An industrial strain of S. cerevisiae was used (CCUG53310, Culture Collection University of Göteborg, Göteborg, Sweden) . This strain was originally isolated from an industrial ethanol production plant, Domsjö Fabriker AB, Örnsköldsvik, Sweden. Lactic acid bacteria were obtained from a culture collection, Lactobacillus fermentum (ATCC14931) or isolated from Domsjö Fabriker AB, L. buchneri, L. plantarum. Acetic acid bacteria, Acetobacter tropicalis, A. syzygii, were isolated from the same industrial plant. The isolated bacteria were species determined by a combination of API test (bioMerieux, France) and 16S RNA gene sequencing.
Inoculum cultures were grown in YPD medium (10 g/l yeast extract (Sigma-Aldrich St. Louis, USA), 20 g/l peptone (Nordic Biolabs, Taby, Sweden), 20 g/l glucose (Merck, Darmstadt, Germany)) for yeast and MRS medium (Oxoid, Hampshire, England) for bacteria for 1 to 1.5 days at 30°C in falcon tubes or shake flasks depending on culture volume.
The cultivations were performed in a lignocellulosic hydrolysate of spruce chips pretreated with dilute acid, a composition determined previously . The pH was adjusted to 5.0 with ammonia (Merck, Darmstadt, Germany) and the hydrolysate medium was filter sterilized before usage.
For cocultivations on a small scale in 50 ml falcon tubes, the cells in the inocula were harvested after 1 day by centrifugation, resuspended in sterile water and the optical density at 610 nm (OD610) was measured. The hydrolysate (6 or 10 ml) was inoculated with cells (yeast and/or bacteria) and water in a total of 45 μl/ml hydrolysate to give an initial OD610 of 0.05 for yeast and at 0.09 for bacteria. The lid was closed and the tubes were incubated at 30°C in a rotary shaker and monitored for up to 4 days. Samples for medium analyses were centrifuged (2 min, at minimum 14,000 g) and stored at -20°C before analysis.
Cocultivations on a large bench scale were performed with 1 l of medium using 3 l bioreactors (Belach Bioteknik AB, Stockholm, Sweden) operated at 30°C with a stirring rate of 300 rpm and no gas inlet. The initial pH was set to 5.0 with ammonia (Merck, Darmstadt, Germany) during the preparation of medium and the decrease during cultivations was always less than 0.5 pH units.
Determination of bacteria and yeast viability at an industrial production plant
The start inoculum was a mixture of microorganisms harvested from the Domsjö Fabriker industrial ethanol production plant located in Örnsköldsvik, Sweden. This mixture (sludge) contained the complete microbiological community existing in an industrial ethanol fermentation plant: mainly yeast, lactic acid bacteria and acetic acid bacteria.
This microbiological community was cultivated for 32 h at 30°C in spent sulfite liquor supplemented with 10.2 ml 25% ammonium (Merck, Darmstadt, Germany) and 171 mg/l KH2PO4 (Merck, Darmstadt, Germany) with and without addition of NaCl (Merck, Darmstadt, Germany) (25 g/l) + ethanol (VWR, Leuven, Belgium) (12.5 g/l). The pH was adjusted to 5.0 by addition of 5 M NaOH (Merck, Darmstadt, Germany) prior to fermentation. The cultivations were performed in 300 ml Erlenmeyer flasks with a total volume of 200 ml.
Measurements of the cell viability were performed by colony forming unit (CFU) count.
Metabolites in the medium (ethanol, acetic acid, lactic acid) were analyzed using commercial enzymatic kits assays (R-Biopharm GmbH, Darmstadt, Germany) with adapted volumes in microtiter plates. Absorbance was measured with a Fluostar Galaxy plate reader (BMG Labtechnologies, Offenburg, Germany).
CFU determinations were performed on agar plates with YPD for yeast (when bacteria was present in large numbers, 20 μl of 50 g/l ampicillin (AppliChem, Darmstadt, Germany) was added to each plate) and MRS for bacteria with 0.1 g/l cycloheximide (Merck, Darmstadt, Germany)to suppress growth of yeast. Each dilution was spread on two or three plates. The plates were incubated at 30°C for 2 days for yeast and for 3 days for bacteria to establish distinct colonies before counting.
Multiple regression analysis of the specific growth rate as a function of pH, temperature, and concentrations of NaCl, glucose, ethanol and lactic acid was performed using the software Modde 9.0 (Umetrics AB, Umea, Sweden).