Algal Research 8 (2015) 95–98 Contents lists available at ScienceDirect Algal Research journal homepage: www.elsevier.com/locate/algal Polyhydroxybutyrate production using a wastewater microalgae based media Asif Rahman a, Ryan J. Putman a, Kadriye Inan b, Fulya Ay Sal c, Ashik Sathish a, Terence Smith a, Chad Nielsen a, Ronald C. Sims a, Charles D. Miller a,⁎ a b c Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322–4105, United States Department of Molecular Biology and Genetics, Karadeniz Technical University, Trabzon, Turkey Department of Biology, Karadeniz Technical University, Trabzon, Turkey a r t i c l e i n f o Article history: Received 11 September 2014 Received in revised form 9 December 2014 Accepted 20 January 2015 Available online xxxx Keywords: Wastewater Microalgae Bioproduct Polyhydroxybutyrate a b s t r a c t Bioproduct production from wastewater microalgae has the potential to contribute to societal needs with value added chemicals. Microalgae can remediate wastewater to remove nitrogen, phosphorus, and heavy metals and can be processed to produce biofuels and bioproducts. It was previously demonstrated that recombinant Escherichia coli could produce polyhydroxybutyrates (PHBs) when cultured on a wastewater microalgae wet lipid extracted media. In this present study, microalgae were harvested from the effluent of a wastewater treatment facility via centrifugation and hydrolyzed to create a liquid medium for recombinant E. coli growth and PHB production. Standard E. coli growth media was supplemented with various concentrations of hydrolyzed algal extract to produce a maximum of 31% PHB of the E. coli dry cell weight. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Microalgae have been well studied for production of biodiesel [1] and recently microalgae have been proposed to be the basis for a biorefinery model where multiple chemicals can be produced simultaneously [2]. Producing several chemicals from the same microalgae feedstock could potentially make the production of multiple commodity chemicals from a biological resource economically viable. The limitations to microalgae culturing are well-documented, including but not limited to: nutrient supply, water scarcity, harvesting, and dewatering [3]. The City of Logan, UT has a 460 acre seven pond facultative lagoon system to treat weak domestic wastewater. Weak domestic wastewater contains approximately 20 mg/L nitrogen and 4 mg/L phosphorus, and is ideal for microalgae growth [4]. Facultative lagoon systems can be used to culture mixed consortia of microalgae to remediate the wastewater by removal of phosphorus and nitrogen. There are a wide range of methods previously employed to harvest microalgae from an open pond system that include: rotating algal biofilm reactor (RABR) [5,6], biological and chemical flocculants [2,7,8], and centrifugation [9]. Harvested microalgae can then be processed and used as a feedstock for production of bioproducts [10,11]. It has been demonstrated that Escherichia coli can be cultured on microalgae based substrates for production of biofuels and bioplastics [12,13] ⁎ Corresponding author. E-mail address: charles.miller@usu.edu (C.D. Miller). http://dx.doi.org/10.1016/j.algal.2015.01.009 2211-9264/© 2015 Elsevier B.V. All rights reserved. E. coli can be easily cultured and has a fast doubling time making it an ideal candidate for production of recombinant bioproducts. Polyhydroxybutyrates (PHBs) are bioplastics that can be recombinantly produced in E. coli [14] and cyanobacteria [15]. PHB is a potentially useful polymer, in addition to being completely biodegradable, it has similar properties to traditional petrochemically derived plastics such as polypropylene and polystyrene [16]. Three genes are needed for the conversion of acetyl-CoA to PHB in E. coli. The pBHR68 plasmid contains the lac promoter and three genes (phaA, phaB, and phaC) needed for production of the short chain length (scl) polymer PHB [17]. Bacterial PHB production is not widespread in part due to the cost of the carbon substrate. It has been estimated that the carbon substrate in a large scale manufacturing context would constitute approximately 37% of the total production cost [18]. Due to the high cost of carbon, an alternative low cost substitute is needed to culture E. coli in order to make PHB production economically viable. In a previous study, it was demonstrated that E. coli harboring the pBHR68 plasmid was able to successfully grow on a Scenedesmus obliquus microalgae based media [2]. In a different study, various harvesting methods were used to collect microalgae grown in photobioreactors [19] and then the harvested microalgae was processed via the wet lipid extraction procedure (WLEP) to generate a variety of side streams and bioproducts [2,20]. One of the side streams, termed ‘aqueous phase’ was used to culture E. coli and it was established that the upstream harvesting method of S. obliquus affected the growth of the E. coli in the aqueous phase media. The most successful microalgae harvesting method for high levels of E. coli growth after 48 h (1012–1013 CFU/mL) was observed 96 A. Rahman et al. / Algal Research 8 (2015) 95–98 when the S. obliquus was centrifuged [2]. The same experiment was extended to harvesting wastewater mixed culture microalgae from the City of Logan, UT treatment plant pond E. Different harvested wastewater microalgae samples (approximately 11% lipids as fatty acid methyl esters, FAME [20]) were subjected to the WLEP to generate the aqueous phase. Results showed that harvesting via centrifugation gave the best E. coli growth (~1013 CFU/mL) and PHB production (7.8% PHB dry cell weight) [13]. Centrifugation was selected as the preferred harvesting method because centrifuged microalgae processed via the WLEP demonstrated the highest levels of E. coli growth and PHB production. Additionally, the previous studies [2,13] used an unmodified aqueous phase media to culture E. coli, and a subsequent study used a fraction of the aqueous phase with standard E. coli media and obtained promising PHB yields [21]. The main objectives of the study reported here were to demonstrate E. coli growth and PHB production using hydrolyzed microalgae from wastewater effluent of the City of Logan, UT facultative pond treatment facility. 2. Materials and methods All chemicals and reagents were purchased from Thermo Fisher Scientific (Pittsburgh, PA) unless stated otherwise. 2.1. Microalgae harvesting and processing Wastewater microalgae were harvested from the City of Logan, UT wastewater treatment facility from the effluent stream leaving the facultative lagoon system. Microalgae were centrifuged using a continuous centrifuge (Alfa Laval Clara 80, Lund, Sweden) and dried in a temperature controlled oven. After drying, microalgae were stored at −20 °C. A modified microalgae hydrolysis method was used similar to Ellis et al., where hydrolyzed microalgae was used to culture Clostridia to produce Acetone, Butanol, and Ethanol [22]. Briefly, 10 g of dry microalgae was dissolved into 0.5 M (final concentration) sulfuric acid (H2SO4) with a total volume of 100 mL. The solution was placed on a stir plate and heated to 90 °C for 30 min with constant stirring. After cooling to room temperature, the solution was neutralized to pH 7 with sodium hydroxide (NaOH). This neutralized solution was then centrifuged at 3500 rpm for 30 min to clarify the solution. The supernatant was then used for culturing E. coli. 2.2. Bacterial growth Supernatant from hydrolyzed microalgae was used as the sole carbon source and was added to standard E. coli M9 growth media [23] in 1%, 2%, and 3% ratios (weight dry hydrolyzed microalgae to culture volume ratio, where 1% is 0.5 g dry algae in 50 mL). An additional study was conducted that consisted of culturing E. coli in a 10% hydrolyzed microalgae solution (w/v). The hydrolyzed microalgae supernatant was not autoclaved in order to demonstrate that E. coli growth and PHB production could occur from a non-traditional carbon source. In addition to the liquid algal extract, growth media also contained M9 salts (Becton, Dickinson and Co, Sparks, MD), 0.002 M MgSO4, and 50 μg/mL ampicillin (IBI Scientific, Peosta, IA) [24,25]. The E. coli strain, XL1 Blue (Agilent Technologies, Santa Clara, CA) harboring the pBHR68 plasmid [17] was grown in LB media [23] overnight (~ 15 h). Cultures were then used to start larger 50 mL cultures with an initial optical density (OD 600 ) of 0.05. Isopropyl β- D -1thiogalactopyranoside (0.1 mM) (Gold Biotechnology, Inc. St. Louis, MO) was added at 0 h to induce expression of the phaCAB genes. Bacterial growth was measured using optical density (OD600 nm) at 0, 4, 8, 12, 24, and 48 h. 2.3. Sugar analysis Total sugar was determined using a modified phenol–sulfuric acid method [22]. Briefly, 3 μL of 85% (w/v) phenol solution and 150 μL of 12 M sulfuric acid were added to the samples and the mixture was heated for 5 min at 90 °C. After cooling to room temperature for 5 min in an ice bath, absorbance (A490 nm) was measured using a Synergy 2 microtiter plate reader (BioTek, Winooski, VT). Sugar concentrations were calculated based on a glucose standard. 2.4. Polyhydroxybutyrate analysis PHB analysis was conducted on samples after 48 h of bacterial culturing. PHB concentration was determined from a 1H NMR/GC correlation as described previously [26]. Briefly, approximately 15 mg of lyophilized sample were dissolved in 1 mL deuterated chloroform (CDCl3 with 0.03% TMS (v/v), Cambridge Isotope Laboratories, Inc. Andover, MA) and 5% sodium hypochlorite solution. Samples were vortexed, incubated, and centrifuged to promote phase separation. PHB phase was run on a Jeol ECX-300 NMR (Jeol USA, Inc. Peabody, MA) and a standard NMR/ GC correlation was used for PHB quantification. 2.5. Statistical analysis Data was processed with Statistical Analysis Software (SAS 9.4, SAS Institute Inc., Cary, NC). One-way analysis of variance (ANOVA) was conducted on the data collected at 48 h for % PHB for the three different media types. REGWQ post hoc comparison was performed on significant results with a confidence level of 95%. All % PHB experiments were conducted in triplicate. 3. Results and discussion The steps conducted in this study to remove microalgae from wastewater effluent of the City of Logan, UT facultative pond treatment facility, to hydrolyze the dried microalgae, and to grow E. coli to produce the PHB polymer are depicted in Fig. 1. E. coli harboring the pBHR68 plasmid was grown on 1%, 2%, 3%, and 10% microalgae-M9 media (w/v). The maximum optical density (OD600) for E. coli grown in 1% media was approximately 1.3, where stationary phase was reached at approximately 12 h post-induction (Fig. 2). The 2%, 3%, and 10% samples reached stationary phase at 24 h and achieved a maximum OD600 of 2.5, 3.61, and 7.6 respectively. E. coli growth in microalgae-M9 media was typical of that observed in traditional glucose-M9 media in other studies [25]. Cultures were allowed to continue growing until 48 h to allow time for PHB accumulation. Results of the total sugar analysis indicated that simple sugars were present in the microalgae extract and were consumed during the course of bacterial growth. The 1% sample had 152 mg/L total sugar at 0 h and after 48 h of bacterial growth had 132 mg/L. The 2% sample had 320 mg/L sugar at 0 h and after 48 h had 267 mg/L. The 3% sample had 445 mg/L sugar at time 0 and 287 mg/mL after 48 h. The 10% sample had 1890 mg/L total sugar and finished with 1344 mg/L sugar. The amount of sugar consumed by the bacteria in each experiment (1%, 2%, 3%, and 10%) was comparable to the growth observed in Fig. 2. Comparing PHB yields 48 h post-induction demonstrated that the M9 media containing 1% and 2% microalgae extract had the most PHB (as a percentage of E. coli dry cell weight). The 1% and 2% samples produced 31 ± 8.9% and 28.2 ± 2.1% PHB, respectively (Fig. 3). In comparison, E. coli cultured in 3% and 10% microalgae extract media that had an average PHB accumulation of 11.2 ± 2.6% and 4.6 ± 0.7%, respectively. The statistical analysis yielded a p-value of 0.0026 indicating significant results (confidence of 95%, p b 0.05). Post hoc comparison using REGWQ showed that the PHB production levels at 1% and 2% were not A. Rahman et al. / Algal Research 8 (2015) 95–98 97 Fig. 1. Schematic for production of polyhydroxybutyrates in Escherichia coli from wastewater microalgae. significantly different from each other (indicated with ‘a’ in Fig. 3). Additionally, the statistical analysis also demonstrated that PHB accumulation in 3% and 10% samples were not statistically different from each other (indicated with ‘b’ in Fig. 3). The PHB production from 1% and 2% media samples was both significantly different from the PHB yields observed from E. coli grown in 3% and 10% algal extract. These results demonstrate that there is a drop in PHB production from the 2% to the 3% microalgae media. The drop in PHB production at the higher percent microalgae media could be attributed to the increase in salts present in the media. In this study microalgae media was neutralized from a low pH with sodium hydroxide, thereby generating sodium sulfate. In a previous study, wastewater microalgae harvested with Aluminum Sulfate (Alum) and used to culture E. coli yielded no PHB [13]. As described, E. coli growth in the 3% and 10% samples reached stationary phase later than then 1% and 2% samples, suggesting that E. coli in the 3% and 10% samples could also have had less time to accumulate PHB. It has been suggested previously that acetyl-CoA is required for cell synthesis in log phase but is diverted to PHB production in stationary phase [27]. Future work could be conducted to best determine if extending the culturing time of the 3% and 10% experiments improves yields of PHB and whether or not this is economically viable. The percentage of PHB accumulated in bacterial cells cultured in the 1% and 2% microalgae-M9 media was slightly lower than that seen in E. coli harboring pBHR68 grown in M9-glucose media (Table 1). In a previous study, it was found that E. coli harboring the pBHR68 plasmid grown in M9 media supplemented with 1.5% glucose could accumulate up to 47.24 ± 6.0%, 48 h post-induction [24]. Achieving approximately 31 ± 8.9% demonstrates the potential of using microalgae as the sole carbon source in media for E. coli culturing and bioproduct production. Addition of an external carbon substrate such as glucose to the microalgae based media could potentially increase the growth and yields of PHB in E. coli. It was demonstrated in a previous study that 1% glucose addition to a microalgae based media to culture Clostridia tripled the yield of solvent production [22]. Comparing the 10% microalgae extract media (with no M9 addition) to that of a similar study [13], it was found that this 10% media did not perform as well as the media used in the previous study. In the previous study, approximately 9.6 g of microalgae (dry weight equivalent) was extracted (via centrifugation) from the City of Logan, UT wastewater treatment facility pond E and subjected to the wet lipid extraction procedure (WLEP) to produce approximately 7.8% PHB [13]. In the present study, microalgae extracted from the effluent of the wastewater treatment plant generated 4.6% PHB. The lower PHB yield could be attributed to the fact that in this study the microalgae harvested could have already been lysed, resulting in a lower yield of sugars extracted. There could have also been some inhibitory effect from the media that was more concentrated, thus reducing the production capacity of the E. coli. Table 1 shows the yields of PHB obtained from the different experiments carried out in this study compared to that of another study with the same strain of E. coli [24]. In addition to PHB% as a fraction of dry cell weight, the total carbon substrate needed to produce 1 kg PHB was also estimated. It was determined that using the 1% microalgae 9 8 7 1% 6 OD600 2% 5 3% 4 10% 3 2 1 0 0 10 20 Time (h) 30 40 50 Fig. 2. Growth of E. coli harboring the pBHR68 plasmid on 1%, 2%, 3%, and 10% microalgae media. Fig. 3. Polyhydroxybutyrate production from different microalgae based cultures (where % PHB is a proportion of E. coli dry cell weight). Data sets with same letter (a or b) demonstrated no statistically significant difference (p N 0.05), error bars represent standard deviation (n = 3). 98 A. Rahman et al. / Algal Research 8 (2015) 95–98 Table 1 Production and yields of PHB after 48 h from Escherichia coli XL1 Blue harboring the pBHR68 plasmid grown in M9 media supplemented with wastewater microalgae hydrolyzed fraction. Standard deviations are based on triplicates (n = 3). Carbon source in M9 media PHB % g PHB/L g PHB/g carbon substrate Carbon needed (kg) to produce 1 kg PHB Reference 1% microalgae media 2% microalgae media 3% microalgae media 10% microalgae media 1.5% glucose 30.97 ± 8.9 a 28.19 ± 5.1 a 11.24 ± 2.6 b 4.60 ± 0.7 b 47.24 ± 6.0 2.30 ± 1 2.09 ± 0.5 0.77 ± 0.5 0.32 ± 0.1 5.43 ± 1.7 0.232 0.104 0.026 0.003 0.40 4.3 9.5 38.7 305.2 2.5 This study This study This study This study [24] Data sets with same letter (a or b) demonstrated no statistical significant difference (p N 0.05). in M9 media would need approximately 4.3 kg of dry microalgae to produce 1 kg of PHB. This is comparable to a standard 1.5% glucose M9 media in the previous study that was predicted to need 2.5 kg of glucose to make 1 kg of PHB. This estimate assumed a linear scaling from shaker flask volume of 50 mL to a large scale bioreactor, however, in order to get a more accurate measurement additional parameters would need to be considered. 4. Conclusions This study built upon work previous work that demonstrated the production of PHB from a microalgae feedstock. From this study, growth of recombinant E. coli harboring the pBHR68 plasmid (containing the phaCAB operon) on wastewater microalgae based media was observed for all samples. It was found that the maximum PHB accumulation in E. coli was approximately 31 ± 8.9% seen on the 1% microalgae-M9 media 48 h post-induction. Future work could include determining the effects of production of additional bioproducts using recombinant E. coli grown on wastewater microalgae media and the addition of a traditional carbon source such as glucose or a carbon-rich waste to the microalgae based media. Additionally, a technoeconomic analysis could be conducted to determine the most cost effect means of production of various recombinant products in E. coli from a wastewater microalgae feedstock. 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