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A novel approach for the production of green biosurfactant from Pseudomonas aeruginosa using renewable forest biomass
Journal Pre-proofs A novel approach for the production of green biosurfactant from Pseudomonas aeruginosa using renewable forest biomass Kateřina Hrůzová, Alok Patel, Jan Masák, Olga Mať átková, Ulrika Rova, Paul Christakopoulos, Leonidas Matsakas PII: DOI: Reference:
S0048-9697(19)35091-0 https://doi.org/10.1016/j.scitotenv.2019.135099 STOTEN 135099
To appear in:
Science of the Total Environment
Received Date: Revised Date: Accepted Date:
18 September 2019 7 October 2019 19 October 2019
Please cite this article as: K. Hrůzová, A. Patel, J. Masák, O. Mať átková, U. Rova, P. Christakopoulos, L. Matsakas, A novel approach for the production of green biosurfactant from Pseudomonas aeruginosa using renewable forest biomass, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.135099
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A novel approach for the production of green biosurfactant from Pseudomonas aeruginosa using renewable forest biomass Kateřina Hrůzová1, Alok Patel1, Jan Masák2, Olga Maťátková2, Ulrika Rova1, Paul Christakopoulos1, Leonidas Matsakas1* 1
Biochemical Process Engineering, Division of Chemical Engineering, Department of Civil,
Environmental and Natural Resources Engineering, Luleå University of Technology, SE-971 87 Luleå, Sweden 2
Department of Biotechnology, University of Chemistry and Technology Prague, Technicka
5, 166 28 Prague, Czech Republic *Corresponding
author: Leonidas Matsakas, Department of Civil, Environmental and
Natural Resources Engineering, SE-971 87 Luleå Sweden, [email protected], tel.: +46 (0) 920 493043
Highlights:
Petrol-based surfactants can be replaced by non-toxic, biodegradable alternatives.
Wood hydrolysates enable cheap and sustainable rhamnolipid (RL) production.
Lignocellulosic biomass has great potential as a carbon source for RL production.
With the use of birch hydrolysate, 2.34 ± 0.17 g/L of RL was obtained after 96 h.
Cultivation on spruce hydrolysate resulted in 2.31 ± 0.10 g/L of RL after 96 h.
A novel approach for the production of green biosurfactant from Pseudomonas aeruginosa using renewable forest biomass
Kateřina Hrůzová1, Alok Patel1, Jan Masák2, Olga Maťátková2, Ulrika Rova1, Paul Christakopoulos1, Leonidas Matsakas1* 1
Biochemical Process Engineering, Division of Chemical Engineering, Department of Civil,
Environmental and Natural Resources Engineering, Luleå University of Technology, SE-971 87 Luleå, Sweden 2
Department of Biotechnology, University of Chemistry and Technology Prague, Technicka
5, 166 28 Prague, Czech Republic *Corresponding
author: Leonidas Matsakas, Department of Civil, Environmental and
Natural Resources Engineering, SE-971 87 Luleå Sweden, [email protected], tel.: +46 (0) 920 493043
Abstract: The rising demand for surfactants by the pharmaceuticals and cosmetic industries has generated vast amounts of petroleum-based synthetic surfactants, which are often toxic and non-degradable. Owing to their low toxicity, stability in extreme conditions, and biodegradability, biosurfactants could represent a sustainable alternative. The present study aimed to maximize the production of rhamnolipids (RL) from Pseudomonas aeruginosa by optimizing glucose concentration, temperature, and C/N and C/P ratios. After 96 h of cultivation at 37 °C, the final RL concentration was 4.18 ± 0.19 g/L with a final yield of 0.214 ± 0.010 g/gglucose when pure glucose was used as a carbon source. At present, the main obstacle towards commercialization of RL production is economic sustainability, due to the high cost of downstream processes and media components. For this reason, a renewable source such as wood hydrolysates (from birch and spruce woodchips) was examined here as a possible source of glucose for RL production. Both hydrolysates proved to be adequate, resulting in 2.34 ± 0.17 and 2.31 ± 0.10 g/L of RL, respectively, and corresponding yields of 0.081 ± 0.006 and 0.089 ± 0.004 g/gsugar after 96 h. These results demonstrate the potential of using renewable biomass for the production of biosurfactants and, to the best of our knowledge, they constitute the first report on the use of wood hydrolysates for RL production.
Keywords: Rhamnolipid, Biosurfactants, Pseudomonas, Wood hydrolysate, Organosolv fractionation Abbreviations: RL, rhamnolipid; BH, Birch hydrolysate; SH, Spruce hydrolysate; HPLC, high-performance liquid chromatography, TLC, thin-layer chromatography, FAME, fatty acid methyl esters; GC-FID, gas chromatography with flame ionization detector.
1. Introduction The global production of surfactants is estimated to surpass 15 million tons per year and is expected to reach more than 24 million tons annually by 2020 (Gudiña et al., 2016). Synthetic surfactants are made from petroleum products, which implies a low cost of production, but a high cost for the environment. To diminish the use of fossil fuels and pollution, eco-friendly alternatives based on renewable resources are required (Chong and Li, 2017a). Rhamnolipids (RL) are the most extensively studied group of biosurfactants with unique physicochemical properties, including low toxicity, high stability in extreme conditions, and biodegradability (Abbasi et al., 2012; Abdel-Mawgoud et al., 2009). These glycolipids are composed of two moieties, one hydrophobic and one hydrophilic. The hydrophobic moiety is made of one or two chains of β-hydroxy fatty acids of varying chain length, most commonly 8–16 carbons. The hydrophilic moiety consists of one or two units of (L)-rhamnose (AbdelMawgoud et al., 2011; Francisca Centeno da Rosa et al., 2010). Some 60 different congeners of RLs are known at present (Abdel-Mawgoud et al., 2010). RLs have a broad range of industrial applications. Their emulsification properties can be used in bioremediation processes to enhance oil recovery, as well as by the food, pharmaceutical, and cosmetic industries as emulsifiers, laundry products, or shampoos (Diab et al., 2013; Rikalovic et al., 2015; Silva et al., 2014; Sinumvayo and Ishimwe, 2015). In addition, RLs can improve treatment of several skin diseases, which further increases their applicability as drugs and cosmetics (Stipcevic et al., 2006). In spite of all the advantages of RLs, their cost of production remains a major bottleneck for their commercialization. To be used at industrial scale, RLs’ price would have to drop by almost 10 fold, from the current 20–25 $/kg to 1–3 $/kg, which is the cost of synthetic surfactants (Chong and Li, 2017b). Downstream processing of RLs contributes to almost 70–80% of total production costs (Sekhon Randhawa
and Rahman, 2014). Despite years of research, no economically convincing downstream technology has been developed yet. Precipitation and organic extraction do not yield a sufficiently pure final product; whereas, the use of preparative HPLC separation would dramatically increase the final price (Sekhon Randhawa and Rahman, 2014). Another factor that impacts heavily on the price of the final product is medium composition and, specifically, the choice of carbon source. In general, high titers and yields are achieved by using hydrophobic carbon sources such as oils (Lee et al., 2004; Zhu et al., 2012), but such substrates are usually more expensive than sugar-based ones (Henkel et al., 2012). In addition, the considerable demand for utilizing renewable resources and waste by-products from the food and agriculture industries has led to extensive work on possible ways of transforming these substrates. As a result, many studies have focused on the use of glycerol for RL production, which is the main by-product of biodiesel production and its output is estimated to reach 4.2 million tons in 2020 (Okoye and Hameed, 2016). Examples of hydrophobic carbon sources include waste frying oil, soapstock from sunflower oil processing, and cotton seed; their yields of RLs are 9.3, 7.3, and 10.4 g/L, respectively (Nitschke et al., 2005; Prabu et al., 2015; Raza et al., 2006). The use of whey as a sugar carbon source leads to a final titer of 0.92 g/L of RLs, whereas molasses from sugar production generate 0.53 g/L of RLs (Dubey and Juwarkar, 2001; Gudiña et al., 2016; Onbasli and Aslim, 2009). The most abundant renewable sugar-based substrate is lignocellulose, which is composed of cellulose, hemicellulose, and lignin. Global production of lignocellulose is estimated at over 500 million tons per year and the cost of the raw material is as low as 40 €/ton, that is more than 10 times cheaper than the sucrose produced from sugar cane, which is around 476 €/ton (Müller et al., 2012). However, lignocellulose feedstocks require pretreatment to remove the barriers that protect cellulose and thus enable cellulose saccharification. Ultimately, this would allow cultivation of the targeted microorganism on the extracted glucose. In that
context, pretreatment of lignocellulose prior to any microbial conversion is a necessity. Over the past years, the focus has shifted from biomass pretreatment to biomass fractionation technologies, whereby biomass is effectively fractionated to cellulose, hemicellulose, and lignin streams. This, in turn, allows for better valorization of these fractions through both biochemical and (thermo) chemical conversion routes. Organosolv pretreatment/fractionation is an excellent strategy to fractionate biomass of various sources, owing to its superior ability to delignify biomass, leaving behind cellulose-rich and low-lignin pretreated solids (Matsakas et al., 2019, 2018). So far, the highest titers of RLs have been obtained by the gram-negative bacterium Pseudomonas aeruginosa. Other strains of the Pseudomonas genus have also been studied, such as Pseudomonas putida or Pseudomonas fluorescens, and additional recombinant strains for RL production have been created. Although the most reported host remains Escherichia coli, the first engineered Saccharomyces cerevisiae strain was constructed in 2018 (Bahia et al., 2018). The aim of the current study was to optimize RL production on synthetic medium supplemented with glucose as a carbon source. Subsequently, birch and spruce hydrolysates were used as renewable sources of glucose for RL production, with the goal of decreasing the cost of the carbon source and, in turn, improve process economics.
2. Material and methods 2.1 Microbial growth and inoculum preparation Pseudomonas aeruginosa DBM 3774 was procured from the Culture of Microorganisms of the University of Chemistry and Technology Prague (DBM), Prague, Czech Republic. The strain was stored at -80 °C in 50% w/w glycerol stock solution. The seed culture was prepared
with Luria Broth medium in an Erlenmeyer flask (250 mL) with 100 mL of working solution and the flask was incubated at 30 °C for 24 h.
2.2 Media and cultivation conditions For RL production, P. aeruginosa was cultivated in mineral medium with the following composition in g/L: NaNO3, 9.5; K2HPO4, 3.4; KH2PO4, 4.4; KCl, 1.1; NaCl, 1.1; MgSO4, 0.224; FeSO4, 0.00028; ZnSO4·7H2O, 0.00029; CuSO4·5H2O, 0.00025; MnSO4·H2O, 0.00017; CaCl·4H2O, 0.00024; yeast extract, 0.5; and glucose, 20. The pH of the medium was adjusted to 6.5–6.7, and the medium was autoclaved at 121 °C for 15 min. Flasks were incubated in an orbital shaker for 96 h at 200 rpm. To study the effect of temperature on RL production, P. aeruginosa was incubated at either 30 or 37 °C and with various concentrations of glucose (20, 40, and 60 g/L), with the remaining parameters being the same as mentioned above. To optimize the C/N ratio for maximum RL production, mineral medium with 20 g/L of glucose was used. The concentration of nitrogen source (NaNO3) was adjusted to obtain the desired C/N ratio of 9.2, 13.5, and 18.3, with the concentration of yeast extract (which acts also as a source of vitamins), kept stable. The nitrogen present in the yeast extract was also included in the calculation of the C/N ratio, while the C/P ratio was adjusted to 25 and 50 by varying the concentration of phosphorus sources (K2HPO4 and KH2PO4).
2.3 Wood organosolv pretreatment and enzymatic saccharification After optimizing the cultivation conditions for RL production, pure glucose was replaced with renewable forest biomass from birch (Betula pendula) and spruce (Picea abies). A hybrid organosolv-steam explosion method was used for pretreatment of birch and spruce woodchips (milled <1 mm) (Matsakas et al., 2019, 2018). Both substrates were heated to 200 °C and 1% w/wbiomass H2SO4 was used as a catalyst. Birch was pretreated with 60% v/v ethanol for 15 min and spruce with 52% v/v ethanol for 30 min. After pretreatment, the pretreated solids
were removed by vacuum filtration from the liquor, washed with ethanol, and air-dried. Subsequently, enzymatic saccharification carried out in a 10% w/w solids content solution in 50 mM citrate buffer (pH 5), by using the commercial enzyme solution Cellic CTec2 (Novozymes A/S, Bagsværd, Denmark) at 20 FPU/gsolids enzyme load for 48 h at 50 °C and 180 rpm. The hydrolysate was separated from the un-saccharified solids by centrifugation and analysed by high-performance liquid chromatography (HPLC) to determine the concentration of glucose. The hydrolysate was then sterilized by filtration using a Filtropur BT50 filter with pore size of 0.2 µm (Sarstedt, Nümbrecht, Germany).
2.4 RL production using spruce and birch hydrolysates as a carbon source The same medium components described in section 2.2 (without pure glucose) were dissolved in the birch (BH) and spruce hydrolysates (SH), and diluted with milliQ water to adjust the final concentration of glucose to 20 g/L. An optimized C/N ratio of 13.5 and C/P ratio of 5 was applied for media preparation. The hydrolysates contained also other components, such as hemicellulose sugars (xylose, mannose, galactose) at 8.9 g/L (BH) and 5.9 g/L (SH), and citrate (50 mM buffer) to facilitate saccharification.
2.5 Analytical methods Samples were taken from the cultures every 24 h to analyze microbial growth, residual glucose, and RL production. Cell growth was measured at 400 nm with a spectrophotometer (Genesys 10UV, Thermo Fisher Scientific, Waltham, MA, USA). A calibrations curve was prepared to convert absorbance to dry mass concentration (g/L). Residual glucose from fermentation was measured by HPLC using an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) and a refractive index detector. The column was kept at 65 °C and 5 mM H2SO4 was used as a mobile phase at a rate of 0.6 mL/min (Dobler et al., 2017).
RL production was measured gravimetrically, and verified by the orcinol method and by HPLC. The gravimetric measurement of RL was carried out with 10 mL of sample that was taken from the cultivation flask every 24 h. The biomass was centrifuged at 8000 × g for 10 min. The pH of the supernatant was adjusted to 2 with 1 M HCl and kept in the fridge at 4 °C overnight. The precipitate was obtained by centrifugation at 12000 × g for 15 min and then freeze-dried. The dry precipitate was extracted with a solution of chloroform and methanol (2:1 v/v). RLs were obtained after evaporation of the chloroform-methanol solution and stored in vials at room temperature. RL samples were analyzed by HPLC also for their rhamnose content (Dobler et al., 2017). For this purpose, 100 µL of 10 M H2SO4 was added to 1 mL of the supernatant and incubated at 100 °C for 4 h. After that, the sample was neutralized with 10 M NaOH and filtered with a 0.22-μm filter. Subsequently, the sample was analyzed by HPLC using an Aminex HPX-87H column and calibrated with pure rhamnose (0–10 g/L). The orcinol method (Koch et al., 1991) was carried out with 0.5 mL of supernatant, which was extracted twice with 1 mL of diethyl ether. After evaporation of diethyl ether, the extracted RLs were dissolved in 0.5 mL of milliQ water. Subsequently, 100 µL of the sample was added to 900 µL of the reagent solution (0.19% w/v orcinol in 53% w/w H2SO4). The sample was incubated at 80 °C for 30 min, then cooled down to room temperature. Absorbance was measured at 421 nm in a Genesys 10UV spectrophotometer. Calibration was carried out with pure rhamnose (0–50 mg/L). Thin-layer chromatography (TLC) was used to analyze the extracted RLs as described by Wilhelm et al. (2007), with some modifications (Wilhelm et al., 2007). Briefly, 2 μL of RL solution (chloroform: methanol 1:2) was applied on the silica sheet – silica gel 60RP-18 F254 (Merck, Darmstadt, Germany), 1 cm above the bottom margin. A solution of chloroform:methanol:glacial acetic acid (65:15:02) was prepared as the mobile phase and added until 0.8 cm of the sanding layer was reached. Subsequently, the sheet was dried and
stained with a buffer containing 75 mg orcinol, 4.2 mL concentrated H2SO4, and 21 mL deionized water. The last step included charring for 10 min in a chamber at 100 °C (Tielen et al., 2010). The extracted RLs were transesterified to fatty acid methyl esters (FAMEs) by using an acid catalyst (HCl/MeOH; 5%, v/v). Subsequently, FAMEs were analyzed by gas chromatography with flame ionization detector (GC-FID; Agilent, Santa Clara, CA, USA) using a capillary column (Select FAME; dimensions 50 m × 0.25 mm ID and 0.25 μm film thickness) under operating conditions previously reported by our group (Patel and Matsakas, 2019).
3. Results 3.1 Effect of temperature and glucose concentration on RL production First, we evaluated the effect of different cultivation parameters on the growth of P. aeruginosa and corresponding RL production. P. aeruginosa was cultivated under various concentrations of glucose at two different temperatures (Table 1). After 96 h, the carbon source was not fully utilized when P. aeruginosa was incubated at 30 °C. The concentration of residual glucose in media containing initially 20, 40, and 60 g/L of glucose was 2.86 ± 0.35, 22.55 ± 0.24, and 35.27 ± 0.43 g/L, respectively. At 37 °C, glucose consumption was much higher, with residual glucose after 96 h of cultivation being 0, 16.5 ± 0.79, and 34.2 ± 1.69 g/L when the initial concentration of glucose was 20 g/L, 40 g/L, and 60 g/L, respectively. Extending the cultivation period beyond 96 h did not further improve glucose consumption, RL production, or biomass concentration. As shown in Table 1, RL concentration after 96 h of cultivation at 30 °C was 2.02 ± 0.03 g/L (0.101 ± 0.002 g/gglucose), 2.46 ± 0.13 g/L (0.062 ± 0.003 g/gglucose), and 3.00 ± 0.09 g/L (0.050 ± 0.002 g/gglucose) when an initial glucose concentration of 20, 40, and 60 g/L was used, respectively. Augmenting the temperature to 37 °C led to a significant incease in RL
concentration, which reached 3.42 ± 0.10 g/L (0.171 ± 0.005 g/gglucose) and 3.72 ± 0.46 g/L (0.093 ± 0.011 g/gglucose) with 20 and 40 g/L of initial glucose, respectively. Only an initial glucose concentration of 60 g/L led to lower RL production, with the final concentration amounting to 1.24 ± 0.20 g/L (0.021 ± 0.003 g/gglucose). Microbial growth was comparable at both tested temperatures as well as with different initial glucose concentrations. At 30 °C, biomass was 0.86 ± 0.02, 0.82 ± 0.01, and 0.78 ± 0.02 g/L after 96 h with an initial 20, 40, and 60 g/L of glucose, respectively. Similar values of 0.88 ± 0.06, 0.82 ± 0.02, and 0.80 ± 0.08 g/L of biomass were reached at 37 °C.
3.2 Effect of the C/N ratio on RL production RL production can be enhanced by as much as 3–5-fold under nitrogen or phosphorus limiting conditions (Abdel-Mawgoud et al., 2011; Nitschke et al., 2011; Wu et al., 2008). Therefore, different C/N ratios (9.2, 13.5, and 18.3) were examined for RL production, while glucose was maintained at 20 g/L. By increasing the C/N ratio, higher RL production and yields, as well as biomass concentration were obtained (Table 2). More specifically, the final concentrations of RLs were 3.98 ± 0.26 g/L (0.204 ± 0.013 g/gglucose), 4.18 ± 0.19 g/L (0.214 ± 0.010 g/gglucose), and 4.08 ± 0.03 g/L (0.209 ± 0.002 g/gglucose) when P. aeruginosa was cultivated at 37 °C for 96 h with C/N ratios of 9.2, 13.5, and 18.3, respectively. Maximum RL production was observed with a C/N ratio of 13.5, which was thus chosen for further experiments.
3.3 Effect of the C/P ratio on RL production Given that the C/P ratio also plays a crucial role in RL production (Abdel-Mawgoud et al., 2011; Nitschke et al., 2011; Wu et al., 2008), media whose phosphorous concentration was adjusted to 20% (C/P 25) and 10% (C/P 50) of the initial value were compared to regular
medium (C/P 5). As shown in Table 3, there was no significant difference in final RL concentration and yield between regular medium and those with a C/P ratio of 50 or 25, with the corresponding values amounting to 4.04 ± 0.03 g/L (0.209 ± 0.003 g/gglucose) and 3.46 ± 0.06 g/L (0.175 ± 0.003 g/gglucose), respectively. As a result, the C/P ratio was maintained at 5 for further experiments.
3.4 RL production on wood hydrolysates After optimizing the cultivation conditions of P. aeruginosa on synthetic media for high RL concentration and yield, replacement of glucose with glucose derived from wood hydrolysates was assessed as a mean to reduce the cost of the carbon source. Use of wood hydrolysates supported growth and RL production by P. aeruginosa, albeit in an uneven way (Table 4). Specifically, biomass was almost double in BH (2.00 ± 0.28 g/L) and approximately 50% higher in SH (1.50 ± 0.07 g/L) cultures compared to when glucose was used (Table 4). We believe this effect could be explained by the presence of additional compounds in the hydrolysates that promoted bacterial growth. The concentration of hemicellulose sugars decreased from 8.9 g/L to 4.3 g/L when BH was used, and during cultivation on SH the concentration decreased from 5.9 g/L to 3.1 g/L. Another significant difference between hydrolysates and glucose regarded the production of RLs, which was lower with hydrolysates. Use of BH and SH media resulted in a final RL concentration of 2.34 ± 0.17 g/L (0.081 ± 0.006 g/gsugar) and 2.31 ± 0.10 g/L (0.089 ± 0.004 g/gsugar), respectively, which corresponded to about half the values obtained with pure glucose. Considering the higher biomass, it appears that compounds present in complex media such as wood hydrolysates, enhanced bacterial growth at the expense of RL formation.
3.5 RL characterization
RLs are always produced as a mixture of different congeners. The RL molecule consists of two moieties – a hydrophilic and a hydrophobic one. The hydrophilic moiety consists of rhamnose, which can be present as one or two units connected to each other and to the hydrophobic moiety. Therefore, RLs are divided into mono-rhamnolipids and di-rhamnolipids based on the number of rhamnose units. The RL mixture can be defined also by the hydrophobic moiety, which consists of one or two 3-hydroxy fatty acid chains. Fatty acid chains differ in length, saturation, and degree of branching. Some 60 congeners have been recorded so far (Abdel-Mawgoud et al., 2010), prompting us to characterize the RLs produced by P. aeruginosa in the current work by TLC and GC.
3.5.1 TLC analysis TLC was used to analyze the mono-/di-rhamnolipid ratio in four selected samples, namely RL produced in the standard medium (see section 2.2), medium with an optimized C/N ratio of 13.5, BH, and SH, all at 37 °C. As shown in Figure 1, modifying medium composition did not significantly alter the mono-/di-rhamnolipid ratio. Both mono- and di-rhamnolipids were present in all the samples.
3.5.2 GC analysis The hydrophobic moiety was analyzed by GC-FID. The fatty acid profiles of four RL samples are shown in Table 5. When medium with an initial 20 g/L of glucose was used, almost all the fatty acid chains (92.6%) in the mixture were saturated fatty acids with carbon chain lengths of 14 to 18 (64.2% of C16:0). However, increasing the C/N ratio from 4.9 to 13.5 led to a significant change in the fatty acid profile and saturated fatty acid content decreased to 36.8%. Instead, mono- and polyunsaturated fatty acids rose to 48.5% and 13.8%, respectively, with C18:1 becoming the main fatty acid in this RL (43.8%). When hydrolysates were used, C16:0
and C15:1 were the main fatty acid chains, and the ratio of mono- and polyunsaturated fatty acids differed. 4. Discussion At the beginning of the study, the effect of temperature and glucose concentration on the growth of the bacterium was studied. P. aeruginosa is usually grown at either 30 °C or 37 °C, however, each strain requires specific conditions. For example, P. aeruginosa S2 produced 5.31 g/L of RL when cultivated at 37 °C in a batch reactor with 40 g/L of initial glucose, but only 2.77 g/L of RL at 30 °C (Chen et al., 2007). In contrast, the optimal temperature for P. aeruginosa UW-1 was 30 °C and 1.2 g/L of RL was generated when 60 g/L of initial glucose was used for flask growth (Sim et al., 1997). Notably, other Pseudomonas strains require even lower temperatures. For example, Pseudomonas chlorapsis NRRL B-30761 produced 1 g/L of RLs when grown in a flask with 20 g/L of initial glucose at 20 °C (Gunther et al., 2005). Similar results were obtained with Pseudomonas putida BD2 and P. putida 21 BN, whereby 0.15 and 1.2 g/L of RLs were generated in flasks at 28 °C with 20 g/L of initial glucose (Janek et al., 2013; Tuleva et al., 2002). In the current work, optimal growth and RL production was observed at 37 °C when 3.42 ± 0.10 g/L (0.171 ± 0.005 g/gglucose) of RL was reached. In addition, the concentration of 20 g/L of initial glucose was chosen for futher investigation since higher initial glucose concetration did not reach significatly higher yields and concentrations of RL. Also, glucose was not fully utilized when media with 40 and 60 g/L were used. Table 6 shows a comparison of present RL production results and previously published ones. Apart from the temperature and the concentration of glucose in the medium, other factors are also important toward optimizing the RL production. Among the different options, limitation in key elements (such as nitrogen or phosphate), which can be studied by varying the C/N and/or C/P ratios in the cultivation medium. In this context, we initially tested the effect of
C/N ratio at a range of 9.2 to 18.3, with the optimal results of RL production to be obtained at 13.5. The effect of C/N was studied in other works as well. For example, Guerra-Santos at el. (1984) tested C/N ratios in the range of 8 to 30 for P. aeruginosa DSM2659 grown in continuous culture with 18.2 g/L of glucose at 37 °C. A C/N ratio of 18 resulted in 1.5 g/L of RLs, whereas lower C/N ratios led to lower RL production and, below a C/N ratio of 11, no RLs were detected (Guerra-Santos et al., 1984). When P. aeruginosa FM1 was cultivated in flasks with 40 g/L of glucose at 37 °C and a C/N ratio of 6.6 to 100, the highest RL concentration (8.6 g/L) was obtained with a C/N ratio of 26 (Wu et al., 2008). Similar results were obtained with a Pseudomonas nitroreducens strain cultivated in flasks with 40 g/L of glucose at 30 °C; after testing a range of C/N ratios between 10 and 100, the optimal was found to be 22, which resulted in 5.46 g/L of RLs (Onwosi and Odibo, 2012). Guerra-Santos at el. (1984) performed trials to optimize the C/N ratio followed by examination of a range of C/P ratios (10 to 32) for P. aeruginosa DSM2659. When a C/P ratio of 16 was used in continuous cultivation, 1.5 g/L of RLs were obtained; above that, RL production decreased steeply (Guerra-Santos et al., 1984). However, in the current work the optimal C/P ratio was 5. Replacment of commercial glucose with glucose derived from renewable resources is importnat towards commercialization of RL production. However, use of lignocellulosic resources for microbial production of RLs is very rare. For example, Prabu et al. (2015) used hydrolysates from pretreated wheat straw for P. aeruginosa ATCC 19429 cultivation in flasks at 30 °C for 72 h. The initial concentration of reducing sugars was 76 g/L, but only 39.52 g/L was utilized (Table 7). The final RL concentration reached 9.43 g/L, a value higher than the one obtained in this study, but the yield of 0.124 g/gglucose is similar to the present one (Prabu et al., 2015). In a recent study that applied organosolv pretreatment, wastewater obtained after lignin removal was used instead of biomass for RL production. Although the wastewater
contained a low concentration of glucose (0.55–1.06 g/L) and xylose (5.69–9.34 g/L), glycerol was the main carbon source for RL production by P. aeruginosa ATCC 27583. The cultivation was carried out at 37 °C for 48 h and RL concentration reached up to 1.24 g/L (de Araújo Padilha et al., 2019). In the current study we suggested the use of wood hydrolysates and more specifically cellulose-rich pretreated solids from the organosolv fractionation of birch (Betula pendula) and spruce (Picea abies) woodchips. Based on our results, the production of RL reached up to 2.34 ± 0.17 g/L (0.081 ± 0.006 g/gsugar) and 2.31 ± 0.10 g/L (0.089 ± 0.004 g/gsugar) with use of BH and SH, respectively. In addition, not only glucose but also hemicellulose sugars (xylose, mannose, and galactose) were utilized by P. aeruginosa for RL production. In the last stage of the study, the obtained RLs were also chemically characterized for their fatty acid profile. It was found that the media composition plays a crucial role in the fatty acid profile of obtained RLs. As shown in Table 5, the use of wood hydrolysates lead to a significant change in fatty acid profile – more monosaturated fatty acids were incorporated in the structure of RL. Moreover, longer fatty acid (C16:0, C15:1 and C18:1) were found in RL produced by P. aeruginosa DBM 3774 when compared to RL commonly reported into the literature where the most reported fatty acids in RLs are C8–C12. For example, Hošková et al. (Hošková et al., 2015) analyzed RLs produced by three diferrent strains: P. aeruginosa B59188, Acinetobacter calcoaceticus B-59190, and Enterobacter asburiae B-59189. A similar mineral medium was used for RL production at 30 °C but citrate (20 g/L) was added as a carbon source. Only five fatty acids were indentified in the study (C8, C10, C10:1, C12, C12:1). Analogous results were observed when P. aeruginosa MA01 was cultivated at 30 °C with soy bean oil as a carbon source. A profile of the generated RLs revealed only three fatty acids: C10, C12, and C12:1 (Abbasi et al., 2012). Incorporation of longer fatty acid chains in
a molecule of RL was reported for Burkholderia thailandensis E264, whereby C10, C12, C14, and C16 were produced, with C14 being the dominant species (Dubeau et al., 2009).
5. Conclusion The present study demonstrates that optimizing cultivation conditions of P. aeruginosa DBM 3774 and medium composition led to an increase in RL production and RL yield as compared to the initial conditions. The highest RL concentration was 4.18 ± 0.19 g/L with a yield of 0.214 ± 0.010 g/gglucose when pure glucose was used as a carbon source. In an effort to reduce the cost of the carbon source, we evaluated the use of wood hydrolysates as a renewable alternative to glucose for RL production. The final RL concentrations were 2.34 ± 0.17 and 2.31 ± 0.10 g/L with yields of 0.081 ± 0.006 and 0.089 ± 0.004 g/gsugar for birch and spruce hydrolysate, respectively. These findings demonstrate the elevated potential of using forest resources for the production of green biosurfactants.
Acknowledgements We would like to acknowledge Kentaro Umeki and Albert Bach Oller from Energy Engineering, Division of Energy Science, Department of Engineering Sciences and Mathematics, Luleå University of Technology, Luleå, Sweden for providing the GC-FID apparatus. We would also like to thank Sveaskog, Sweden, for providing the birch and spruce wood
chips.
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Table 1. Effect of temperature and glucose concentration on RL production, RL yield, and biomass concentration Temperature (°C) 30
37
Initial Residual RL RL yield glucose glucose concentration (g/gglucose) concentration concentration (g/L) (g/L) (g/L) 20 2.86 ± 0.35 2.02 ± 0.03 0.101 ± 0.002
Biomass (g/L) 0.86 ± 0.02
40
22.55 ± 0.24
2.46 ± 0.13
0.062 ± 0.003
0.82 ± 0.01
60
35.27 ± 0.43
3.00 ± 0.09
0.050 ± 0.002
0.78 ± 0.02
20
0.15 ± 0.02
3.42 ± 0.10
0.171 ± 0.005
0.88 ± 0.06
40
16.5 ± 0.79
3.72 ± 0.46
0.093 ± 0.011
0.82 ± 0.02
60
34.2 ± 1.69
1.24 ± 0.20
0.021 ± 0.003
0.80 ± 0.08
Table 2. Effect of various C/N ratios at 20 g/L of glucose on RL production C/N ratio 4.9
RL concentration (g/L) 3.42 ± 0.10
RL yield (g/gglucose) 0.171 ± 0.005
Biomass concentration (g/L) 0.88 ± 0.06
9.2
3.98 ± 0.26
0.204 ± 0.013
1.10 ± 0.02
13.5
4.18 ± 0.19
0.214 ± 0.010
1.02 ± 0.03
18.3
4.08 ± 0.03
0.209 ± 0.002
1.01 ± 0.01
Table 3. Effect of various C/P ratios at 20 g/L of glucose on RL production C/P ratio
C/N ratio
RL yield (g/gglucose)
Biomass (g/L)
13.5
RL concentration (g/L) 4.18 ± 0.19
5
0.214 ± 0.010
1.02 ± 0.03
25
13.5
3.46 ± 0.06
0.175 ± 0.003
0.76 ± 0.02
50
13.5
4.04 ± 0.03
0.209 ± 0.003
0.91 ± 0.01
Table 4. RL production on wood hydrolysates in comparison to glucose Carbon source
C/N ratio
RL yield (g/gsugar)
Biomass (g/L)
13.5
RL concentration (g/L) 4.18 ± 0.19
Glucose (20 g/L)
0.214 ± 0.010
1.02 ± 0.03
BH (20 g/L)
13.5
2.34 ± 0.17
0.081 ± 0.006
2.00 ± 0.28
SH (20 g/L)
13.5
2.31 ± 0.10
0.089 ± 0.004
1.50 ± 0.07
Table 5. Fatty acid profile of RLs produced at 37 °C using 1) medium with an initial 20 g/L of glucose, 2) medium with a C/N ratio of 13.5, BH – birch hydrolysate; SH – spruce hydrolysate. The numbers represent percentage of the specific fatty acid in the total lipids and is expressed in % w/w. Fatty acid chain 1 2 BH SH C14:0 8.4 1.3 2.3 1.3 C15:0 10.2 3.3 1.0 C16:0 64.2 23.1 33.1 20.2 C17:0 4.2 1.4 C18:0 5.5 10.5 8.6 12.5 C20:0 1.9 C22:0 1.6 SFA 92.6 36.8 50.3 35.1 C15:1 34.6 49.8 C18:1 43.8 1.3 1.7 C20:1 3.5 1.1 4.1 C22:1 1.2 C24:1 1.2 MUFA 0.0 48.5 38.3 55.6 C18:2 n6 8.4 C20:3 n3 3.4 C20:4 n6 2.0 1.4 PUFA 0.0 13.8 1.4 0.0 Total: 92.6 99.0 90.1 90.6 SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsturated fatty acids
Table 6. Comparison of RL production by various Pseudomonas strains cultivated under different temperatures, glucose concentrations, and growth conditions Strain
Glucose
RL yield (g/gglucose)
Temperature (°C)
Cultivation
Tim e (h)
Reference
(g/L)
RL concentration (g/L)
P. chlororaphis NRRL B-30761
20
1.0
0.050
20
Batch-flasks
120
(Gunther et al., 2005)
P. aeruginosa UW-1
60
1.2
0.020
30
Batch - flasks
168
(Sim et al.,
1997) P. aeruginosa DSM2659.
18.2
1.5
0.082
37
Continuous
Nd
(GuerraSantos et al., 1984)
P. nitroreducens
20
5.46
0.273
30
Batch -flasks
168
(Onwosi and Odibo, 2012)
P. aeruginosa EM1
40
8.6
0.215
37
Batch-flasks
120
(Wu et al., 2008)
P. putida BD2
20
0.15
0.007
28
Batch-flasks
168
(Janek et al., 2013)
P. putida 21BN
20
1.2
0.060
28
Batch-flasks
48
(Tuleva et al., 2002)
P. aeruginosa S2
40
5.31
0.132
37
Batch - reactor
90
(Chen et al., 2007)
P. aeruginosa S2
60
9.4
0.156
37
Fed-batch - reactor
360
(Chen et al., 2007)
P. aeruginosa DBM 3774
20
4.18
0.214
37
Batch-flask
96
This study
Nd – no data available;
Table 7. RL production by different P. aeruginosa strains on renewable resources Strain
Carbon source (g/L)
RL concentration (g/L)
RL yield (g/gcarbon source)
Temperature (°C)
Cultivation
Time (h)
Reference
P. aeruginosa NCIM-2036
76 (WSH)
9.43
0.124
30
Batch - reactor
72
(Prabu et al., 2015)
P. aeruginosa ATCC 27583
24 (BL)
1.24
0.052
37
Batch - flasks
48
(de Araújo Padilha et al., 2019)
P. aeruginosa DBM 3774
20 (BH)
2.34
0.081
37
Batch - flasks
96
This study
P. aeruginosa DBM 3774
20 (SH)
2.31
0.089
37
Batch - flasks
96
This study
WSH – wheat straw hydrolysate; BL - black liquor
S0048-9697(19)35091-0 https://doi.org/10.1016/j.scitotenv.2019.135099 STOTEN 135099
To appear in:
Science of the Total Environment
Received Date: Revised Date: Accepted Date:
18 September 2019 7 October 2019 19 October 2019
Please cite this article as: K. Hrůzová, A. Patel, J. Masák, O. Mať átková, U. Rova, P. Christakopoulos, L. Matsakas, A novel approach for the production of green biosurfactant from Pseudomonas aeruginosa using renewable forest biomass, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.135099
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A novel approach for the production of green biosurfactant from Pseudomonas aeruginosa using renewable forest biomass Kateřina Hrůzová1, Alok Patel1, Jan Masák2, Olga Maťátková2, Ulrika Rova1, Paul Christakopoulos1, Leonidas Matsakas1* 1
Biochemical Process Engineering, Division of Chemical Engineering, Department of Civil,
Environmental and Natural Resources Engineering, Luleå University of Technology, SE-971 87 Luleå, Sweden 2
Department of Biotechnology, University of Chemistry and Technology Prague, Technicka
5, 166 28 Prague, Czech Republic *Corresponding
author: Leonidas Matsakas, Department of Civil, Environmental and
Natural Resources Engineering, SE-971 87 Luleå Sweden, [email protected], tel.: +46 (0) 920 493043
Highlights:
Petrol-based surfactants can be replaced by non-toxic, biodegradable alternatives.
Wood hydrolysates enable cheap and sustainable rhamnolipid (RL) production.
Lignocellulosic biomass has great potential as a carbon source for RL production.
With the use of birch hydrolysate, 2.34 ± 0.17 g/L of RL was obtained after 96 h.
Cultivation on spruce hydrolysate resulted in 2.31 ± 0.10 g/L of RL after 96 h.
A novel approach for the production of green biosurfactant from Pseudomonas aeruginosa using renewable forest biomass
Kateřina Hrůzová1, Alok Patel1, Jan Masák2, Olga Maťátková2, Ulrika Rova1, Paul Christakopoulos1, Leonidas Matsakas1* 1
Biochemical Process Engineering, Division of Chemical Engineering, Department of Civil,
Environmental and Natural Resources Engineering, Luleå University of Technology, SE-971 87 Luleå, Sweden 2
Department of Biotechnology, University of Chemistry and Technology Prague, Technicka
5, 166 28 Prague, Czech Republic *Corresponding
author: Leonidas Matsakas, Department of Civil, Environmental and
Natural Resources Engineering, SE-971 87 Luleå Sweden, [email protected], tel.: +46 (0) 920 493043
Abstract: The rising demand for surfactants by the pharmaceuticals and cosmetic industries has generated vast amounts of petroleum-based synthetic surfactants, which are often toxic and non-degradable. Owing to their low toxicity, stability in extreme conditions, and biodegradability, biosurfactants could represent a sustainable alternative. The present study aimed to maximize the production of rhamnolipids (RL) from Pseudomonas aeruginosa by optimizing glucose concentration, temperature, and C/N and C/P ratios. After 96 h of cultivation at 37 °C, the final RL concentration was 4.18 ± 0.19 g/L with a final yield of 0.214 ± 0.010 g/gglucose when pure glucose was used as a carbon source. At present, the main obstacle towards commercialization of RL production is economic sustainability, due to the high cost of downstream processes and media components. For this reason, a renewable source such as wood hydrolysates (from birch and spruce woodchips) was examined here as a possible source of glucose for RL production. Both hydrolysates proved to be adequate, resulting in 2.34 ± 0.17 and 2.31 ± 0.10 g/L of RL, respectively, and corresponding yields of 0.081 ± 0.006 and 0.089 ± 0.004 g/gsugar after 96 h. These results demonstrate the potential of using renewable biomass for the production of biosurfactants and, to the best of our knowledge, they constitute the first report on the use of wood hydrolysates for RL production.
Keywords: Rhamnolipid, Biosurfactants, Pseudomonas, Wood hydrolysate, Organosolv fractionation Abbreviations: RL, rhamnolipid; BH, Birch hydrolysate; SH, Spruce hydrolysate; HPLC, high-performance liquid chromatography, TLC, thin-layer chromatography, FAME, fatty acid methyl esters; GC-FID, gas chromatography with flame ionization detector.
1. Introduction The global production of surfactants is estimated to surpass 15 million tons per year and is expected to reach more than 24 million tons annually by 2020 (Gudiña et al., 2016). Synthetic surfactants are made from petroleum products, which implies a low cost of production, but a high cost for the environment. To diminish the use of fossil fuels and pollution, eco-friendly alternatives based on renewable resources are required (Chong and Li, 2017a). Rhamnolipids (RL) are the most extensively studied group of biosurfactants with unique physicochemical properties, including low toxicity, high stability in extreme conditions, and biodegradability (Abbasi et al., 2012; Abdel-Mawgoud et al., 2009). These glycolipids are composed of two moieties, one hydrophobic and one hydrophilic. The hydrophobic moiety is made of one or two chains of β-hydroxy fatty acids of varying chain length, most commonly 8–16 carbons. The hydrophilic moiety consists of one or two units of (L)-rhamnose (AbdelMawgoud et al., 2011; Francisca Centeno da Rosa et al., 2010). Some 60 different congeners of RLs are known at present (Abdel-Mawgoud et al., 2010). RLs have a broad range of industrial applications. Their emulsification properties can be used in bioremediation processes to enhance oil recovery, as well as by the food, pharmaceutical, and cosmetic industries as emulsifiers, laundry products, or shampoos (Diab et al., 2013; Rikalovic et al., 2015; Silva et al., 2014; Sinumvayo and Ishimwe, 2015). In addition, RLs can improve treatment of several skin diseases, which further increases their applicability as drugs and cosmetics (Stipcevic et al., 2006). In spite of all the advantages of RLs, their cost of production remains a major bottleneck for their commercialization. To be used at industrial scale, RLs’ price would have to drop by almost 10 fold, from the current 20–25 $/kg to 1–3 $/kg, which is the cost of synthetic surfactants (Chong and Li, 2017b). Downstream processing of RLs contributes to almost 70–80% of total production costs (Sekhon Randhawa
and Rahman, 2014). Despite years of research, no economically convincing downstream technology has been developed yet. Precipitation and organic extraction do not yield a sufficiently pure final product; whereas, the use of preparative HPLC separation would dramatically increase the final price (Sekhon Randhawa and Rahman, 2014). Another factor that impacts heavily on the price of the final product is medium composition and, specifically, the choice of carbon source. In general, high titers and yields are achieved by using hydrophobic carbon sources such as oils (Lee et al., 2004; Zhu et al., 2012), but such substrates are usually more expensive than sugar-based ones (Henkel et al., 2012). In addition, the considerable demand for utilizing renewable resources and waste by-products from the food and agriculture industries has led to extensive work on possible ways of transforming these substrates. As a result, many studies have focused on the use of glycerol for RL production, which is the main by-product of biodiesel production and its output is estimated to reach 4.2 million tons in 2020 (Okoye and Hameed, 2016). Examples of hydrophobic carbon sources include waste frying oil, soapstock from sunflower oil processing, and cotton seed; their yields of RLs are 9.3, 7.3, and 10.4 g/L, respectively (Nitschke et al., 2005; Prabu et al., 2015; Raza et al., 2006). The use of whey as a sugar carbon source leads to a final titer of 0.92 g/L of RLs, whereas molasses from sugar production generate 0.53 g/L of RLs (Dubey and Juwarkar, 2001; Gudiña et al., 2016; Onbasli and Aslim, 2009). The most abundant renewable sugar-based substrate is lignocellulose, which is composed of cellulose, hemicellulose, and lignin. Global production of lignocellulose is estimated at over 500 million tons per year and the cost of the raw material is as low as 40 €/ton, that is more than 10 times cheaper than the sucrose produced from sugar cane, which is around 476 €/ton (Müller et al., 2012). However, lignocellulose feedstocks require pretreatment to remove the barriers that protect cellulose and thus enable cellulose saccharification. Ultimately, this would allow cultivation of the targeted microorganism on the extracted glucose. In that
context, pretreatment of lignocellulose prior to any microbial conversion is a necessity. Over the past years, the focus has shifted from biomass pretreatment to biomass fractionation technologies, whereby biomass is effectively fractionated to cellulose, hemicellulose, and lignin streams. This, in turn, allows for better valorization of these fractions through both biochemical and (thermo) chemical conversion routes. Organosolv pretreatment/fractionation is an excellent strategy to fractionate biomass of various sources, owing to its superior ability to delignify biomass, leaving behind cellulose-rich and low-lignin pretreated solids (Matsakas et al., 2019, 2018). So far, the highest titers of RLs have been obtained by the gram-negative bacterium Pseudomonas aeruginosa. Other strains of the Pseudomonas genus have also been studied, such as Pseudomonas putida or Pseudomonas fluorescens, and additional recombinant strains for RL production have been created. Although the most reported host remains Escherichia coli, the first engineered Saccharomyces cerevisiae strain was constructed in 2018 (Bahia et al., 2018). The aim of the current study was to optimize RL production on synthetic medium supplemented with glucose as a carbon source. Subsequently, birch and spruce hydrolysates were used as renewable sources of glucose for RL production, with the goal of decreasing the cost of the carbon source and, in turn, improve process economics.
2. Material and methods 2.1 Microbial growth and inoculum preparation Pseudomonas aeruginosa DBM 3774 was procured from the Culture of Microorganisms of the University of Chemistry and Technology Prague (DBM), Prague, Czech Republic. The strain was stored at -80 °C in 50% w/w glycerol stock solution. The seed culture was prepared
with Luria Broth medium in an Erlenmeyer flask (250 mL) with 100 mL of working solution and the flask was incubated at 30 °C for 24 h.
2.2 Media and cultivation conditions For RL production, P. aeruginosa was cultivated in mineral medium with the following composition in g/L: NaNO3, 9.5; K2HPO4, 3.4; KH2PO4, 4.4; KCl, 1.1; NaCl, 1.1; MgSO4, 0.224; FeSO4, 0.00028; ZnSO4·7H2O, 0.00029; CuSO4·5H2O, 0.00025; MnSO4·H2O, 0.00017; CaCl·4H2O, 0.00024; yeast extract, 0.5; and glucose, 20. The pH of the medium was adjusted to 6.5–6.7, and the medium was autoclaved at 121 °C for 15 min. Flasks were incubated in an orbital shaker for 96 h at 200 rpm. To study the effect of temperature on RL production, P. aeruginosa was incubated at either 30 or 37 °C and with various concentrations of glucose (20, 40, and 60 g/L), with the remaining parameters being the same as mentioned above. To optimize the C/N ratio for maximum RL production, mineral medium with 20 g/L of glucose was used. The concentration of nitrogen source (NaNO3) was adjusted to obtain the desired C/N ratio of 9.2, 13.5, and 18.3, with the concentration of yeast extract (which acts also as a source of vitamins), kept stable. The nitrogen present in the yeast extract was also included in the calculation of the C/N ratio, while the C/P ratio was adjusted to 25 and 50 by varying the concentration of phosphorus sources (K2HPO4 and KH2PO4).
2.3 Wood organosolv pretreatment and enzymatic saccharification After optimizing the cultivation conditions for RL production, pure glucose was replaced with renewable forest biomass from birch (Betula pendula) and spruce (Picea abies). A hybrid organosolv-steam explosion method was used for pretreatment of birch and spruce woodchips (milled <1 mm) (Matsakas et al., 2019, 2018). Both substrates were heated to 200 °C and 1% w/wbiomass H2SO4 was used as a catalyst. Birch was pretreated with 60% v/v ethanol for 15 min and spruce with 52% v/v ethanol for 30 min. After pretreatment, the pretreated solids
were removed by vacuum filtration from the liquor, washed with ethanol, and air-dried. Subsequently, enzymatic saccharification carried out in a 10% w/w solids content solution in 50 mM citrate buffer (pH 5), by using the commercial enzyme solution Cellic CTec2 (Novozymes A/S, Bagsværd, Denmark) at 20 FPU/gsolids enzyme load for 48 h at 50 °C and 180 rpm. The hydrolysate was separated from the un-saccharified solids by centrifugation and analysed by high-performance liquid chromatography (HPLC) to determine the concentration of glucose. The hydrolysate was then sterilized by filtration using a Filtropur BT50 filter with pore size of 0.2 µm (Sarstedt, Nümbrecht, Germany).
2.4 RL production using spruce and birch hydrolysates as a carbon source The same medium components described in section 2.2 (without pure glucose) were dissolved in the birch (BH) and spruce hydrolysates (SH), and diluted with milliQ water to adjust the final concentration of glucose to 20 g/L. An optimized C/N ratio of 13.5 and C/P ratio of 5 was applied for media preparation. The hydrolysates contained also other components, such as hemicellulose sugars (xylose, mannose, galactose) at 8.9 g/L (BH) and 5.9 g/L (SH), and citrate (50 mM buffer) to facilitate saccharification.
2.5 Analytical methods Samples were taken from the cultures every 24 h to analyze microbial growth, residual glucose, and RL production. Cell growth was measured at 400 nm with a spectrophotometer (Genesys 10UV, Thermo Fisher Scientific, Waltham, MA, USA). A calibrations curve was prepared to convert absorbance to dry mass concentration (g/L). Residual glucose from fermentation was measured by HPLC using an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) and a refractive index detector. The column was kept at 65 °C and 5 mM H2SO4 was used as a mobile phase at a rate of 0.6 mL/min (Dobler et al., 2017).
RL production was measured gravimetrically, and verified by the orcinol method and by HPLC. The gravimetric measurement of RL was carried out with 10 mL of sample that was taken from the cultivation flask every 24 h. The biomass was centrifuged at 8000 × g for 10 min. The pH of the supernatant was adjusted to 2 with 1 M HCl and kept in the fridge at 4 °C overnight. The precipitate was obtained by centrifugation at 12000 × g for 15 min and then freeze-dried. The dry precipitate was extracted with a solution of chloroform and methanol (2:1 v/v). RLs were obtained after evaporation of the chloroform-methanol solution and stored in vials at room temperature. RL samples were analyzed by HPLC also for their rhamnose content (Dobler et al., 2017). For this purpose, 100 µL of 10 M H2SO4 was added to 1 mL of the supernatant and incubated at 100 °C for 4 h. After that, the sample was neutralized with 10 M NaOH and filtered with a 0.22-μm filter. Subsequently, the sample was analyzed by HPLC using an Aminex HPX-87H column and calibrated with pure rhamnose (0–10 g/L). The orcinol method (Koch et al., 1991) was carried out with 0.5 mL of supernatant, which was extracted twice with 1 mL of diethyl ether. After evaporation of diethyl ether, the extracted RLs were dissolved in 0.5 mL of milliQ water. Subsequently, 100 µL of the sample was added to 900 µL of the reagent solution (0.19% w/v orcinol in 53% w/w H2SO4). The sample was incubated at 80 °C for 30 min, then cooled down to room temperature. Absorbance was measured at 421 nm in a Genesys 10UV spectrophotometer. Calibration was carried out with pure rhamnose (0–50 mg/L). Thin-layer chromatography (TLC) was used to analyze the extracted RLs as described by Wilhelm et al. (2007), with some modifications (Wilhelm et al., 2007). Briefly, 2 μL of RL solution (chloroform: methanol 1:2) was applied on the silica sheet – silica gel 60RP-18 F254 (Merck, Darmstadt, Germany), 1 cm above the bottom margin. A solution of chloroform:methanol:glacial acetic acid (65:15:02) was prepared as the mobile phase and added until 0.8 cm of the sanding layer was reached. Subsequently, the sheet was dried and
stained with a buffer containing 75 mg orcinol, 4.2 mL concentrated H2SO4, and 21 mL deionized water. The last step included charring for 10 min in a chamber at 100 °C (Tielen et al., 2010). The extracted RLs were transesterified to fatty acid methyl esters (FAMEs) by using an acid catalyst (HCl/MeOH; 5%, v/v). Subsequently, FAMEs were analyzed by gas chromatography with flame ionization detector (GC-FID; Agilent, Santa Clara, CA, USA) using a capillary column (Select FAME; dimensions 50 m × 0.25 mm ID and 0.25 μm film thickness) under operating conditions previously reported by our group (Patel and Matsakas, 2019).
3. Results 3.1 Effect of temperature and glucose concentration on RL production First, we evaluated the effect of different cultivation parameters on the growth of P. aeruginosa and corresponding RL production. P. aeruginosa was cultivated under various concentrations of glucose at two different temperatures (Table 1). After 96 h, the carbon source was not fully utilized when P. aeruginosa was incubated at 30 °C. The concentration of residual glucose in media containing initially 20, 40, and 60 g/L of glucose was 2.86 ± 0.35, 22.55 ± 0.24, and 35.27 ± 0.43 g/L, respectively. At 37 °C, glucose consumption was much higher, with residual glucose after 96 h of cultivation being 0, 16.5 ± 0.79, and 34.2 ± 1.69 g/L when the initial concentration of glucose was 20 g/L, 40 g/L, and 60 g/L, respectively. Extending the cultivation period beyond 96 h did not further improve glucose consumption, RL production, or biomass concentration. As shown in Table 1, RL concentration after 96 h of cultivation at 30 °C was 2.02 ± 0.03 g/L (0.101 ± 0.002 g/gglucose), 2.46 ± 0.13 g/L (0.062 ± 0.003 g/gglucose), and 3.00 ± 0.09 g/L (0.050 ± 0.002 g/gglucose) when an initial glucose concentration of 20, 40, and 60 g/L was used, respectively. Augmenting the temperature to 37 °C led to a significant incease in RL
concentration, which reached 3.42 ± 0.10 g/L (0.171 ± 0.005 g/gglucose) and 3.72 ± 0.46 g/L (0.093 ± 0.011 g/gglucose) with 20 and 40 g/L of initial glucose, respectively. Only an initial glucose concentration of 60 g/L led to lower RL production, with the final concentration amounting to 1.24 ± 0.20 g/L (0.021 ± 0.003 g/gglucose). Microbial growth was comparable at both tested temperatures as well as with different initial glucose concentrations. At 30 °C, biomass was 0.86 ± 0.02, 0.82 ± 0.01, and 0.78 ± 0.02 g/L after 96 h with an initial 20, 40, and 60 g/L of glucose, respectively. Similar values of 0.88 ± 0.06, 0.82 ± 0.02, and 0.80 ± 0.08 g/L of biomass were reached at 37 °C.
3.2 Effect of the C/N ratio on RL production RL production can be enhanced by as much as 3–5-fold under nitrogen or phosphorus limiting conditions (Abdel-Mawgoud et al., 2011; Nitschke et al., 2011; Wu et al., 2008). Therefore, different C/N ratios (9.2, 13.5, and 18.3) were examined for RL production, while glucose was maintained at 20 g/L. By increasing the C/N ratio, higher RL production and yields, as well as biomass concentration were obtained (Table 2). More specifically, the final concentrations of RLs were 3.98 ± 0.26 g/L (0.204 ± 0.013 g/gglucose), 4.18 ± 0.19 g/L (0.214 ± 0.010 g/gglucose), and 4.08 ± 0.03 g/L (0.209 ± 0.002 g/gglucose) when P. aeruginosa was cultivated at 37 °C for 96 h with C/N ratios of 9.2, 13.5, and 18.3, respectively. Maximum RL production was observed with a C/N ratio of 13.5, which was thus chosen for further experiments.
3.3 Effect of the C/P ratio on RL production Given that the C/P ratio also plays a crucial role in RL production (Abdel-Mawgoud et al., 2011; Nitschke et al., 2011; Wu et al., 2008), media whose phosphorous concentration was adjusted to 20% (C/P 25) and 10% (C/P 50) of the initial value were compared to regular
medium (C/P 5). As shown in Table 3, there was no significant difference in final RL concentration and yield between regular medium and those with a C/P ratio of 50 or 25, with the corresponding values amounting to 4.04 ± 0.03 g/L (0.209 ± 0.003 g/gglucose) and 3.46 ± 0.06 g/L (0.175 ± 0.003 g/gglucose), respectively. As a result, the C/P ratio was maintained at 5 for further experiments.
3.4 RL production on wood hydrolysates After optimizing the cultivation conditions of P. aeruginosa on synthetic media for high RL concentration and yield, replacement of glucose with glucose derived from wood hydrolysates was assessed as a mean to reduce the cost of the carbon source. Use of wood hydrolysates supported growth and RL production by P. aeruginosa, albeit in an uneven way (Table 4). Specifically, biomass was almost double in BH (2.00 ± 0.28 g/L) and approximately 50% higher in SH (1.50 ± 0.07 g/L) cultures compared to when glucose was used (Table 4). We believe this effect could be explained by the presence of additional compounds in the hydrolysates that promoted bacterial growth. The concentration of hemicellulose sugars decreased from 8.9 g/L to 4.3 g/L when BH was used, and during cultivation on SH the concentration decreased from 5.9 g/L to 3.1 g/L. Another significant difference between hydrolysates and glucose regarded the production of RLs, which was lower with hydrolysates. Use of BH and SH media resulted in a final RL concentration of 2.34 ± 0.17 g/L (0.081 ± 0.006 g/gsugar) and 2.31 ± 0.10 g/L (0.089 ± 0.004 g/gsugar), respectively, which corresponded to about half the values obtained with pure glucose. Considering the higher biomass, it appears that compounds present in complex media such as wood hydrolysates, enhanced bacterial growth at the expense of RL formation.
3.5 RL characterization
RLs are always produced as a mixture of different congeners. The RL molecule consists of two moieties – a hydrophilic and a hydrophobic one. The hydrophilic moiety consists of rhamnose, which can be present as one or two units connected to each other and to the hydrophobic moiety. Therefore, RLs are divided into mono-rhamnolipids and di-rhamnolipids based on the number of rhamnose units. The RL mixture can be defined also by the hydrophobic moiety, which consists of one or two 3-hydroxy fatty acid chains. Fatty acid chains differ in length, saturation, and degree of branching. Some 60 congeners have been recorded so far (Abdel-Mawgoud et al., 2010), prompting us to characterize the RLs produced by P. aeruginosa in the current work by TLC and GC.
3.5.1 TLC analysis TLC was used to analyze the mono-/di-rhamnolipid ratio in four selected samples, namely RL produced in the standard medium (see section 2.2), medium with an optimized C/N ratio of 13.5, BH, and SH, all at 37 °C. As shown in Figure 1, modifying medium composition did not significantly alter the mono-/di-rhamnolipid ratio. Both mono- and di-rhamnolipids were present in all the samples.
3.5.2 GC analysis The hydrophobic moiety was analyzed by GC-FID. The fatty acid profiles of four RL samples are shown in Table 5. When medium with an initial 20 g/L of glucose was used, almost all the fatty acid chains (92.6%) in the mixture were saturated fatty acids with carbon chain lengths of 14 to 18 (64.2% of C16:0). However, increasing the C/N ratio from 4.9 to 13.5 led to a significant change in the fatty acid profile and saturated fatty acid content decreased to 36.8%. Instead, mono- and polyunsaturated fatty acids rose to 48.5% and 13.8%, respectively, with C18:1 becoming the main fatty acid in this RL (43.8%). When hydrolysates were used, C16:0
and C15:1 were the main fatty acid chains, and the ratio of mono- and polyunsaturated fatty acids differed. 4. Discussion At the beginning of the study, the effect of temperature and glucose concentration on the growth of the bacterium was studied. P. aeruginosa is usually grown at either 30 °C or 37 °C, however, each strain requires specific conditions. For example, P. aeruginosa S2 produced 5.31 g/L of RL when cultivated at 37 °C in a batch reactor with 40 g/L of initial glucose, but only 2.77 g/L of RL at 30 °C (Chen et al., 2007). In contrast, the optimal temperature for P. aeruginosa UW-1 was 30 °C and 1.2 g/L of RL was generated when 60 g/L of initial glucose was used for flask growth (Sim et al., 1997). Notably, other Pseudomonas strains require even lower temperatures. For example, Pseudomonas chlorapsis NRRL B-30761 produced 1 g/L of RLs when grown in a flask with 20 g/L of initial glucose at 20 °C (Gunther et al., 2005). Similar results were obtained with Pseudomonas putida BD2 and P. putida 21 BN, whereby 0.15 and 1.2 g/L of RLs were generated in flasks at 28 °C with 20 g/L of initial glucose (Janek et al., 2013; Tuleva et al., 2002). In the current work, optimal growth and RL production was observed at 37 °C when 3.42 ± 0.10 g/L (0.171 ± 0.005 g/gglucose) of RL was reached. In addition, the concentration of 20 g/L of initial glucose was chosen for futher investigation since higher initial glucose concetration did not reach significatly higher yields and concentrations of RL. Also, glucose was not fully utilized when media with 40 and 60 g/L were used. Table 6 shows a comparison of present RL production results and previously published ones. Apart from the temperature and the concentration of glucose in the medium, other factors are also important toward optimizing the RL production. Among the different options, limitation in key elements (such as nitrogen or phosphate), which can be studied by varying the C/N and/or C/P ratios in the cultivation medium. In this context, we initially tested the effect of
C/N ratio at a range of 9.2 to 18.3, with the optimal results of RL production to be obtained at 13.5. The effect of C/N was studied in other works as well. For example, Guerra-Santos at el. (1984) tested C/N ratios in the range of 8 to 30 for P. aeruginosa DSM2659 grown in continuous culture with 18.2 g/L of glucose at 37 °C. A C/N ratio of 18 resulted in 1.5 g/L of RLs, whereas lower C/N ratios led to lower RL production and, below a C/N ratio of 11, no RLs were detected (Guerra-Santos et al., 1984). When P. aeruginosa FM1 was cultivated in flasks with 40 g/L of glucose at 37 °C and a C/N ratio of 6.6 to 100, the highest RL concentration (8.6 g/L) was obtained with a C/N ratio of 26 (Wu et al., 2008). Similar results were obtained with a Pseudomonas nitroreducens strain cultivated in flasks with 40 g/L of glucose at 30 °C; after testing a range of C/N ratios between 10 and 100, the optimal was found to be 22, which resulted in 5.46 g/L of RLs (Onwosi and Odibo, 2012). Guerra-Santos at el. (1984) performed trials to optimize the C/N ratio followed by examination of a range of C/P ratios (10 to 32) for P. aeruginosa DSM2659. When a C/P ratio of 16 was used in continuous cultivation, 1.5 g/L of RLs were obtained; above that, RL production decreased steeply (Guerra-Santos et al., 1984). However, in the current work the optimal C/P ratio was 5. Replacment of commercial glucose with glucose derived from renewable resources is importnat towards commercialization of RL production. However, use of lignocellulosic resources for microbial production of RLs is very rare. For example, Prabu et al. (2015) used hydrolysates from pretreated wheat straw for P. aeruginosa ATCC 19429 cultivation in flasks at 30 °C for 72 h. The initial concentration of reducing sugars was 76 g/L, but only 39.52 g/L was utilized (Table 7). The final RL concentration reached 9.43 g/L, a value higher than the one obtained in this study, but the yield of 0.124 g/gglucose is similar to the present one (Prabu et al., 2015). In a recent study that applied organosolv pretreatment, wastewater obtained after lignin removal was used instead of biomass for RL production. Although the wastewater
contained a low concentration of glucose (0.55–1.06 g/L) and xylose (5.69–9.34 g/L), glycerol was the main carbon source for RL production by P. aeruginosa ATCC 27583. The cultivation was carried out at 37 °C for 48 h and RL concentration reached up to 1.24 g/L (de Araújo Padilha et al., 2019). In the current study we suggested the use of wood hydrolysates and more specifically cellulose-rich pretreated solids from the organosolv fractionation of birch (Betula pendula) and spruce (Picea abies) woodchips. Based on our results, the production of RL reached up to 2.34 ± 0.17 g/L (0.081 ± 0.006 g/gsugar) and 2.31 ± 0.10 g/L (0.089 ± 0.004 g/gsugar) with use of BH and SH, respectively. In addition, not only glucose but also hemicellulose sugars (xylose, mannose, and galactose) were utilized by P. aeruginosa for RL production. In the last stage of the study, the obtained RLs were also chemically characterized for their fatty acid profile. It was found that the media composition plays a crucial role in the fatty acid profile of obtained RLs. As shown in Table 5, the use of wood hydrolysates lead to a significant change in fatty acid profile – more monosaturated fatty acids were incorporated in the structure of RL. Moreover, longer fatty acid (C16:0, C15:1 and C18:1) were found in RL produced by P. aeruginosa DBM 3774 when compared to RL commonly reported into the literature where the most reported fatty acids in RLs are C8–C12. For example, Hošková et al. (Hošková et al., 2015) analyzed RLs produced by three diferrent strains: P. aeruginosa B59188, Acinetobacter calcoaceticus B-59190, and Enterobacter asburiae B-59189. A similar mineral medium was used for RL production at 30 °C but citrate (20 g/L) was added as a carbon source. Only five fatty acids were indentified in the study (C8, C10, C10:1, C12, C12:1). Analogous results were observed when P. aeruginosa MA01 was cultivated at 30 °C with soy bean oil as a carbon source. A profile of the generated RLs revealed only three fatty acids: C10, C12, and C12:1 (Abbasi et al., 2012). Incorporation of longer fatty acid chains in
a molecule of RL was reported for Burkholderia thailandensis E264, whereby C10, C12, C14, and C16 were produced, with C14 being the dominant species (Dubeau et al., 2009).
5. Conclusion The present study demonstrates that optimizing cultivation conditions of P. aeruginosa DBM 3774 and medium composition led to an increase in RL production and RL yield as compared to the initial conditions. The highest RL concentration was 4.18 ± 0.19 g/L with a yield of 0.214 ± 0.010 g/gglucose when pure glucose was used as a carbon source. In an effort to reduce the cost of the carbon source, we evaluated the use of wood hydrolysates as a renewable alternative to glucose for RL production. The final RL concentrations were 2.34 ± 0.17 and 2.31 ± 0.10 g/L with yields of 0.081 ± 0.006 and 0.089 ± 0.004 g/gsugar for birch and spruce hydrolysate, respectively. These findings demonstrate the elevated potential of using forest resources for the production of green biosurfactants.
Acknowledgements We would like to acknowledge Kentaro Umeki and Albert Bach Oller from Energy Engineering, Division of Energy Science, Department of Engineering Sciences and Mathematics, Luleå University of Technology, Luleå, Sweden for providing the GC-FID apparatus. We would also like to thank Sveaskog, Sweden, for providing the birch and spruce wood
chips.
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Table 1. Effect of temperature and glucose concentration on RL production, RL yield, and biomass concentration Temperature (°C) 30
37
Initial Residual RL RL yield glucose glucose concentration (g/gglucose) concentration concentration (g/L) (g/L) (g/L) 20 2.86 ± 0.35 2.02 ± 0.03 0.101 ± 0.002
Biomass (g/L) 0.86 ± 0.02
40
22.55 ± 0.24
2.46 ± 0.13
0.062 ± 0.003
0.82 ± 0.01
60
35.27 ± 0.43
3.00 ± 0.09
0.050 ± 0.002
0.78 ± 0.02
20
0.15 ± 0.02
3.42 ± 0.10
0.171 ± 0.005
0.88 ± 0.06
40
16.5 ± 0.79
3.72 ± 0.46
0.093 ± 0.011
0.82 ± 0.02
60
34.2 ± 1.69
1.24 ± 0.20
0.021 ± 0.003
0.80 ± 0.08
Table 2. Effect of various C/N ratios at 20 g/L of glucose on RL production C/N ratio 4.9
RL concentration (g/L) 3.42 ± 0.10
RL yield (g/gglucose) 0.171 ± 0.005
Biomass concentration (g/L) 0.88 ± 0.06
9.2
3.98 ± 0.26
0.204 ± 0.013
1.10 ± 0.02
13.5
4.18 ± 0.19
0.214 ± 0.010
1.02 ± 0.03
18.3
4.08 ± 0.03
0.209 ± 0.002
1.01 ± 0.01
Table 3. Effect of various C/P ratios at 20 g/L of glucose on RL production C/P ratio
C/N ratio
RL yield (g/gglucose)
Biomass (g/L)
13.5
RL concentration (g/L) 4.18 ± 0.19
5
0.214 ± 0.010
1.02 ± 0.03
25
13.5
3.46 ± 0.06
0.175 ± 0.003
0.76 ± 0.02
50
13.5
4.04 ± 0.03
0.209 ± 0.003
0.91 ± 0.01
Table 4. RL production on wood hydrolysates in comparison to glucose Carbon source
C/N ratio
RL yield (g/gsugar)
Biomass (g/L)
13.5
RL concentration (g/L) 4.18 ± 0.19
Glucose (20 g/L)
0.214 ± 0.010
1.02 ± 0.03
BH (20 g/L)
13.5
2.34 ± 0.17
0.081 ± 0.006
2.00 ± 0.28
SH (20 g/L)
13.5
2.31 ± 0.10
0.089 ± 0.004
1.50 ± 0.07
Table 5. Fatty acid profile of RLs produced at 37 °C using 1) medium with an initial 20 g/L of glucose, 2) medium with a C/N ratio of 13.5, BH – birch hydrolysate; SH – spruce hydrolysate. The numbers represent percentage of the specific fatty acid in the total lipids and is expressed in % w/w. Fatty acid chain 1 2 BH SH C14:0 8.4 1.3 2.3 1.3 C15:0 10.2 3.3 1.0 C16:0 64.2 23.1 33.1 20.2 C17:0 4.2 1.4 C18:0 5.5 10.5 8.6 12.5 C20:0 1.9 C22:0 1.6 SFA 92.6 36.8 50.3 35.1 C15:1 34.6 49.8 C18:1 43.8 1.3 1.7 C20:1 3.5 1.1 4.1 C22:1 1.2 C24:1 1.2 MUFA 0.0 48.5 38.3 55.6 C18:2 n6 8.4 C20:3 n3 3.4 C20:4 n6 2.0 1.4 PUFA 0.0 13.8 1.4 0.0 Total: 92.6 99.0 90.1 90.6 SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsturated fatty acids
Table 6. Comparison of RL production by various Pseudomonas strains cultivated under different temperatures, glucose concentrations, and growth conditions Strain
Glucose
RL yield (g/gglucose)
Temperature (°C)
Cultivation
Tim e (h)
Reference
(g/L)
RL concentration (g/L)
P. chlororaphis NRRL B-30761
20
1.0
0.050
20
Batch-flasks
120
(Gunther et al., 2005)
P. aeruginosa UW-1
60
1.2
0.020
30
Batch - flasks
168
(Sim et al.,
1997) P. aeruginosa DSM2659.
18.2
1.5
0.082
37
Continuous
Nd
(GuerraSantos et al., 1984)
P. nitroreducens
20
5.46
0.273
30
Batch -flasks
168
(Onwosi and Odibo, 2012)
P. aeruginosa EM1
40
8.6
0.215
37
Batch-flasks
120
(Wu et al., 2008)
P. putida BD2
20
0.15
0.007
28
Batch-flasks
168
(Janek et al., 2013)
P. putida 21BN
20
1.2
0.060
28
Batch-flasks
48
(Tuleva et al., 2002)
P. aeruginosa S2
40
5.31
0.132
37
Batch - reactor
90
(Chen et al., 2007)
P. aeruginosa S2
60
9.4
0.156
37
Fed-batch - reactor
360
(Chen et al., 2007)
P. aeruginosa DBM 3774
20
4.18
0.214
37
Batch-flask
96
This study
Nd – no data available;
Table 7. RL production by different P. aeruginosa strains on renewable resources Strain
Carbon source (g/L)
RL concentration (g/L)
RL yield (g/gcarbon source)
Temperature (°C)
Cultivation
Time (h)
Reference
P. aeruginosa NCIM-2036
76 (WSH)
9.43
0.124
30
Batch - reactor
72
(Prabu et al., 2015)
P. aeruginosa ATCC 27583
24 (BL)
1.24
0.052
37
Batch - flasks
48
(de Araújo Padilha et al., 2019)
P. aeruginosa DBM 3774
20 (BH)
2.34
0.081
37
Batch - flasks
96
This study
P. aeruginosa DBM 3774
20 (SH)
2.31
0.089
37
Batch - flasks
96
This study
WSH – wheat straw hydrolysate; BL - black liquor