Giuseppe Squillaci and Alessandra Morana

Research that focuses on the use of high value-added bioproducts for industrial applications is essential for the implementation of sustainable approaches forecasting a bio-based economy. The effective use of biomass feedstocks, particularly lignocellulosic materials, in large-scale applications will evolve from innovative research aimed at the development and implementation of biorefineries established for specific feedstocks. In this context, an important step is the concept of fractionating biomass into its core constituents (cellulose, hemicellulose and lignin) for further enhanced valorization. Contrary to the valorization of cellulose fraction, which has been extensively studied, there is a gap in the valorization of the hemicellulose fraction (xylose-rich substrate) towards bioproducts. In this context, the main goal of this activity, funded by BRISK 2 program, was to optimize the growth of the haloarchaeon Halorhabdus utahensis (DSM-12940 in a bench-top 3L-bioreactor (BioFlo III – New Brunswick Scientific, NJ, USA), available at LNEG (Lisbon, Portugal), for exopolysaccharides and pigments production using xylose as carbon source.  

H. utahensis was firstly grown in shake-flask using the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) suggested medium (DSMZ 927) supplemented with xylose in order to verify its ability to metabolize this sugar. Then, the ability to produce exopolysaccharides (EPS) and pigments was investigated. Thus, H. utahensis was grown in 1 L shake-flasks containing 200 mL culture medium with 0.2% and 1% (w/v) xylose, respectively, at 37°C and 120 rpm agitation. The microorganism was capable to metabolize xylose at both concentrations and to produce orange-pink growth cultures due to the presence of red coloured cells (Figs. 1 and 2). This was a clear indication of the presence of pigments in the cell membrane of the haloarchaeon. The pH of the medium was checked twice a day, as acidification was observed during the growth, and its value was adjusted to the original (7.6) by adding some drops of 5 M NaOH. The growth curves of H. utahensis in 0.2% and 1% xylose are reported in Fig. 3. The microorganism reached an OD600 nm of 1.28 after 93 h in 0.2% xylose, and of 3.11 after 140.5 h in 1% xylose. Under these experimental conditions, H. utahensis was able to synthesize exopolysaccharides with yields of 23.5 mg/L and 47.5 mg/L in 0.2% and 1% xylose, respectively.

Based on these results, the bioreactor assay at LNEG was conducted by inoculating H. utahensis (pellet of 200 mL inoculum previously grown in DSMZ 927 culture medium with 1% xylose) in 1.5 L culture medium containing 1% xylose (OD600 nm initial = 0.102). The culture conditions in bioreactor were: 0.5 VVM aeration, 250 rpm, pH 7.6 and T= 37°C. In these conditions, this halophilic microorganism (270 g/L NaCl) was able to reach an OD600 nm maximal of 2.73 (Thermo Fisher Scientific spectrophotometer, model Genesys 20, USA), corresponding to a biomass production of 6.37 g dry weight/L, with a full consumption of xylose after 264 h (11 days). In addition, 2.5 g/L acetic acid was also produced by the haloarchaeon (Fig. 4). The pigments production in the bioreactor can be observed in Fig. 5. The supernatant of H. utahensis culture containing the EPS was screened for surfactant activity using 1.5 mL supernatant + 0.750 mL n-heptane and the emulsion resulted is presented in Fig. 6, where C is the control with water and  H.u. S. is the test with supernatant of H. utahensis. 

At LNEG facilities, the cell viability of H. utahensis final culture (11 days) was observed using both the epifluorescence microscopy (Olympus BX-60, Tokyo, Japan) (Fig. 7 A, B, C) and the flow cytometry (FACSCalibur Flow Cytometer, BD Biosciencies, San Jose, CA, USA) (Fig. 8 A, B, C, D). For both techniques, cells were stained with 3 fluorescent dyes: carboxyfluorescein diacetate (CFDA), which stains metabolically active cells; propidium iodide (PI), which stains cells with compromised membranes, and Nile red, which stains lipids. In Fig. 7A is presented the culture observed in the epifluorescence microscope without fluorescent dyes. In addition, samples taken from the bioreactor were incubated at 37oC for 30 min with each of the three dyes and observed under the microscope. The results revealed that in these conditions PI was not able of staining the cells. This could be due to interference of the high salt concentrations present in medium (270 g/L). Different PI concentrations and incubation conditions should be tested to allow the correct staining of the cells. Both CFDA and Nile red dyes showed good coloration under these conditions. CFDA stained the inside of the cells and allowed a better observation of the cell morphology. Fig. 7B shows the different shapes present in the culture stained with CFDA. Nile red stained the cell membranes and could indicate the presence of some sort of lipid accumulation since some cells present much greater fluorescence, as seen in Fig. 7C. 

Flow cytometry was tested as technique to assess cellular metabolic state accordingly to the protocol described by Fernandes et al. [Bioprocess Biosyst Eng (2018), 41(2): 143–155]. Without dyes it was possible to clearly detect the population and differentiate between intact and lysed cells (Fig. 8A and 8B, respectively). The intact cells are bigger and more complex and appear in the upper part of graphic. It was also possible to detect metabolically active cells with CFDA (Fig. 8C), however, since PI staining did not work, it was impossible to perform the complete protocol. Nile red was also successfully used (Fig. 8D), and, with further optimization it could possibly be used to quantify lipids accumulation. These results show the potential of flow cytometry to be used with this haloarchaeon microorganism, however, there is a need to considerably optimize the protocols, especially because every step must be performed with the cells immersed in liquid with 270 g/L NaCl concentration, which could hinder the process. 

Ongoing work on IRET (ex IBAF)-CNR:  

Further processing of the supernatant and biomass produced in the batch fermentation, at LNEG facilities, will be carried out for EPS, pigments and potential polyhydroxyalkanoates screening and/or characterization. 

Indeed, a collaboration work between the IRET applicants and the LNEG supervisors is in course, aiming to get a joint publication in international scientific journal, in due time. Nevertheless, for now, the results obtained/derived from this TA will be presented at EUBCE’2019, to be held from 27-30 May in Lisbon, Portugal (http://www.eubce.com/). The abstract submitted to EUBCE’2109 was: “L. Alves, S.M. Paixão, T.P. Silva, G. Squillaci, I. Serino and A. Morana (2019). Fermentation of xylose-rich substrates by the haloarchaeon Halorhabdus utahensis towards high value-added bioproducts”. 

Photos 1 to 4 were addressed to show the LNEG2 facilities used during this TA.