This study investigated the potential of oleaginous yeast Rhodotorula glutinis utilizing pulp and paper wastewater effluents as cultivation media for the sustainable production of microbial lipids as biodiesel feedstock. R. glutinis is oleaginous yeast, which has the ability to produce significant quantities of intercellular lipids in the form of triacylglycerols. Yeast lipids are a promising potential feedstock for biodiesel production due to similar fatty acid composition to plant oils. The effect of various carbon sources on biomass production, lipid accumulation, substrate utilization, and fatty acid composition using R. glutinis in the pulp and paper wastewater media was studied. The pulp and paper wastewater was supplemented with glucose, xylose, and glycerol as carbon sources under nitrogen-limited conditions. The maximum lipid productions of 1.3 - 2.9 g •L –1, which corresponded to the intracellular lipid contents of 8% - 15% cell dry weight (CDW), were obtained under various carbon substrates. A kinetic study of the batch fermentation was performed in a 3 L aerobic batch fermenter to describe the cell growth, lipid accumulation, and substrate utilization process, and the kinetic parameter was estimated. The fatty acid profile of oleaginous yeast was rich in palmitic, oleic, and linoleic acids and comparable to vegetable oils. Thus, the results of this study indicated that pulp and paper wastewater could be used to produce lipids as biodiesel feedstock.
The utilization of biodiesel has greatly increased over the past two decades due in part to concerns about the environmental consequences of fossil fuel combustion, the low lubricity quality of ultralow sulfur diesel, and the rapid rises in crude oil prices that can occur. Biodiesel, a mixture of fatty acid methyl esters (FAMEs) derived traditionally from vegetable oils and animal fats, is one of the most prominent renewable energy resources [
Glucose is one of the most common carbon substrates for producing microbial biomass and lipids, which has been extensively investigated for use by different oleaginous microorganisms [
Oleaginous microorganisms require high carbon to nitrogen ratios and the fermentation process requires high amounts of water and nutrients in the media to produce biomass with high contents of storage lipids. Various fermentation substrates have been investigated to evaluate the capability of oleaginous microorganisms in terms of growth and product formation, such as starch wastewater, molasses, wastewaters from potato, fruit juice and lettuce processing, olive oil manufacturing wastewater [
The pulp and paper industry is the fifth largest economy in the US and produces a considerable amount of wastewater. Furthermore, the pulp and paper industry can be considered as one of the largest industries in terms of water and energy utilization, consuming between 20,000 and 60,000 gallons of water per ton of product [
The present work focuses on the production of microbial lipid to be used as biodiesel feedstock through batch aerobic fermentation of pulp and paper wastewater. Pulp and paper wastewater has been considered as a fermentation medium in order to reduce some of the cost associated with this process. The effects of the utilization of different carbon sources such as glucose, xylose, and glycerol by Rhodotorula glutinis on the lipid productivity were investigated. Kinetic models were applied to describe the processes of microbial growth, product formation, and substrate utilization, which can be further used for the process scale up and bioreactor design in a similar system.
R. glutinis (ATCC 15125) was obtained from American Type Culture Collection (ATCC). Sub-cultures of R. glutinis (ATCC 15125) was produced by adding an aliquot of R. glutinis stock, which was stored at -80˚C, into the flask of sugar broth media [1 g∙L−1 Yeast Extract, 1 g∙L−1 Na2HPO4∙12 H2O, 1 g∙L−1 KH2PO4, 0.4 g∙L−1 MgSO4∙7H2O, 1.6 g∙L−1 (NH4)2SO4, 10.0 ml Trace Mineral Solution (3.6 g∙L−1 CaCl2∙2H2O, 0.75 g∙L−1 ZnSO4∙7H2O, 0.13 g∙L−1 CuSO4∙5H2O, 0.5 g∙L−1 MnSO4∙H2O, 0.13 g∙L−1 COCl2.6 H2O, 0.17 g∙L−1 Na2MoO4.2 H2O in distilled water), 6.0 ml Iron Solution (FeSO4∙7H2O in distilled water), and 60 g∙L−1 glucose], were incubated for 4 days using a rotary shaker (New Brunswick Scientific Model I26, Edison, New Jersey) with a rotation speed of ~112 rpm at 30˚C. The media compounds were obtained from Fisher Scientific.
The pulp and paper wastewater was obtained from the International Paper Mill in Redwood, MS. Multiple samples of wastewater were collected and stored in plastic container. After collection, the wastewater containers were placed on ice for 24 hours prior to conducting the fermentation experiment. The wastewater was characterized prior to being used as a fermentation medium as discussed below. The pH of the wastewater was measured using a bench top pH meter (Accumet Research AR25 Dual Channel pH/Ion Meter, Fisher Scientific). The COD of the wastewater was determined according to EPA standard method [
The pulp and paper wastewater (2.4 L) was amended with 60 g∙L−1 carbon sources in 4 individual experiments including glucose, xylose, glycerol, and glucose-xylose (2:1) ratio and seeded with a 20% initial inoculum of R. glutinis (600 ml) in 3 L Bioflo 310 fermenter (New Brunswick Scientific, Edison, NJ) with an initial R. glutinis cell concentration of 10 g∙L−1. The pH of the amended wastewater was adjusted to 6.5 using 1M NaOH solution, and the temperature was controlled at 25˚C. The aeration rate was adjusted at 3 volume of air per volume of medium and per minute (vvm). The agitation rate was set at 300 rpm, and adjusted to maintain a Dissolved Oxygen (DO) level no lower than 60% saturation during the experiment. A polypropylene-based antifoam, 1:10 dilution of nonoil (Sigma-Aldrich, St. Louis, MO) was periodically added to prevent foaming in the vessel. Samples were obtained from the aerobic batch fermenter every 12 h interval during the first 48 h of the experiment and then every 24 hours during the 7 days of incubation time. Control experiment was conducted using R. glutinis starter cultured in sugar broth media to compare the potential lipid production by R. glutinis using pulp and paper wastewater and the optimal media components.
For each treatment, 30 ml of culture samples were taken and centrifuged at 3000 rpm, at 25˚C for 10 minutes using a Sorvall® ST 40 Centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) to separate the supernatant from the cell pellets. Cell pellets were placed in the freezer at −20˚C overnight, and then freeze-dried using a Labconco Freezone 2.5 freeze drier followed by gravimetric weight measurements to determine the cell mass concentration. The supernatant was stored in the freezer at −20˚C to be used for the analysis of sugar, and nitrogen.
The intercellular lipid content of the freeze-dried samples was determined based on the modified Bligh and Dyer
Characteristics | Wastewater |
---|---|
Place of origin | Post paper making process |
pH | 2.25 |
COD (mg/l) | 1478 |
Ammonium (mg/l) | 3.95 |
Sulfate (mg/l) | 1400 |
method [
The supernatant samples were analyzed using YSI 2900 Biochemistry analyzer (YSI Inc. Life Sciences, Yellow Springs, OH, USA) equipped with a glucose oxidase, xylose oxidase, and glycerol oxidase membrane probe for the measurement of glucose, xylose, and glycerol, respectively. The residual ammonium-nitrogen level was analyzed using an ICS 3000 ion chromatography (Dionex Corp, Sunnyvale, CA, USA) to monitor the point of nitrogen depletion, and nitrogen level in the wastewater. ICS is equipped with an IonPac CS16 cation exchange analytical column (250 mm × 4 mm) and CG16 guard column (50 mm × 4 mm) and a conductivity detector.
Statistical analysis was carried out using SAS® 9.4 (Copyright © 2015, SAS Institute Inc., Cary, NC, USA). The statistical significance of differences in mean values was analyzed using one-way analysis of variance (ANOVA) with Tukey-Test at 95% confidence intervals.
It was hypothesized that the oleaginous yeast R. glutinis can produce microbial lipids from pulp and paper wastewaters amended with carbon sources, which can be further converted to biodiesel. In this study, carbon to nitrogen ratio of 70:1 with initial nitrogen and carbon source concentrations of 1.3 and 60 g∙L−1 was used, respectively. The fermentation profiles were plotted and presented in
However, in the control experiment under similar fermentation conditions, R. glutinis accumulated lipids up to 27% ± 4.6% on a cellular biomass basis with 21 ± 3.12 gL−1 biomass production within 6 days, which was significantly higher than all the treatments in terms of biomass and lipid production. Lower lipid content of R. glutinis cultivated in pulp and paper wastewater could be due to the presence of organic material and lignin breakdown products in the wastewater, which acts as inhibitory compounds on lipid synthesis. However, in the control experiment, a combination of (NH4)2SO4 and yeast extract was used as a nitrogen source, but in the
experiment using wastewater as a medium, NH4Cl was replaced as a nitrogen source to discard the negative effects of sulfate on the final wastewater. It is reported that organic nitrogenous compounds such as yeast extract and peptone have a positive effect on lipid production, but negatively affect the cell growth; conversely, inorganic nitrogen sources like (NH4)2SO4 and NH4Cl are effective for cell growth, but not favorable for lipid production [
R. glutinis was able to completely deplete the supplied carbon sources in 7 days of fermentation except in the treatments where xylose was supplied (
Monod or logistic equations can be used to explain the growth process of most microorganisms [
where
biomass concentration at any time t (h);
Product formation was modeled by a Luedeking-Piret equation [
on the instantaneous fat-free biomass (X) and fat-free biomass rate
where
coefficient and growth correlation coefficient. After substituting
tegrating assuming at t = 0, the concentration of lipid is equal to its initial concentration
The substrate was utilized primarily for cell growth, along with product formation and cell maintenance. The substrate utilization was described by Luedeking-Piret equation [
as a function of instantaneous fat-free biomass rate
coefficient term:
where
and
where
After substituting
pressing the substrate concentration:
where S0 is the initial substrate concentration at the beginning of the fermentation (t = 0).
In this study, the kinetic models expressing cell growth, lipid accumulation, and substrate utilization were fitted to the experimental data for estimation of the model kinetic parameters. The kinetic equations were solved using a nonlinear regression method. Matlab (MATLAB Release 2014a, The MathWorks, Inc., Natick, Massachusetts, United States) was used for data analysis using nonlinear least squares method (Levenberg-Marquardt algorithm) for minimizing residual sum of square of errors (RSSE). The kinetic parameters related to biomass growth (
The values of maximum specific growth rate
Parameters | Substrates | |||
---|---|---|---|---|
Glucose | Xylose | Glucose/Xylose | Glycerol | |
Fat-free biomass | ||||
X0 (g∙L−1) | 2.97 | 2.7 | 4.26 | 3.18 |
μmax (h−1) | 0.044 | 0.027 | 0.028 | 0.05 |
Xmax (g∙L−1) | 14.51 | 14.71 | 18.13 | 15.47 |
R2 | 0.99 | 0.99 | 0.97 | 0.98 |
Lipid | ||||
m | 0.027 | 0.026 | 0.05 | 0.0005 |
n | 0.0007 | 0.0009 | 0.0003 | 0.0015 |
R2 | 0.99 | 0.98 | 0.98 | 0.97 |
Substrate | ||||
S0 | 60.68 | 63.13 | 63.88 | 62.74 |
a | 2.84 | 3.85 | 2.76 | 2.087 |
b | 0.012 | 0.0031 | 0.006 | 0.018 |
0.42 | 0.266 | 0.44 | 0.48 | |
0.058 | 0.297 | 0.1 | 0.12 | |
0.000001 | 0.0001 | 0.003 | 0.0057 | |
R2 | 0.99 | 0.98 | 0.98 | 0.99 |
The fatty acid composition of oleaginous yeasts was greatly dependent on the type of medium used and the growth condition. Some cultivation conditions such as temperature, pH of the medium, the type of the substrate, C:N ratio of the culture, and available oxygen for microorganisms can affect the accumulated lipids and derived fatty acids profile. The results of the characterization of total extracted lipid and FAMEs yield by transesterification of the lipids within 7 days of fermentation were shown in
In this investigation, the fatty acid profile of R. glutinis was determined using FAMEs analysis in order to evaluate the potential of using this oleaginous yeast as biodiesel feedstock.
The composition of the FAMEs was comparable to the fatty acid profile of the soybean and rapeseed oil, which are the commonly used biodiesel feedstocks in the US and the EU, respectively. Comparing the fatty acid composition of R. glutinis in this study with soybean and rapeseed oil indicates that the FAMEs derived from transesterification of lipids from R. glutinis were more saturated (
Biodiesel | Soybean33 | Rapeseed33 | Palm33 | Sunflower33 | R. glutinis |
---|---|---|---|---|---|
Saturated | 14.2 | 7.1 | 51 | 11 | 37.7 ± 0.6 |
Monounsaturated | 23.2 | 58.6 | 39 | 20 | 31.9 ± 0.28 |
Polyunsaturated | 57.8 | 29.6 | 10 | 69 | 29.6 ± 0.15 |
Cetane number (CN) | 45 - 60 | 44 - 65 | 58 - 70 | 49 - 61.2 | 57.2 ± 0.15 |
Iodine value (IV) (g iodine/100g oil) | 120.52 | 108 | 59 | 136 | 92.9 ± 0.3 |
Saponification value (mg KOH/g oil) | 194.61 | 197 | 205 | 193 | 195 |
Higher heating value (MJ/Kg) | 39.63 | 39.73 | 41 | 39.45 | 40 ± 0.004 |
oxide, and increasing oxidative stability. On the other hand, the unsaturated fatty acids are responsible for improving cold flow property of the biodiesel [
To evaluate the quality of the biodiesel, which can be produced from R. glutinis lipids, some fuel properties such as cetane number, iodine value, saponification value, and higher heating value were estimated using available published models. Bamgboye and Hansen showed that the cetane number can be predicted by developing an equation, which relates the cetane number of biodiesel fuels with FAMEs composition of the feedstock used for biodiesel production [
where,
Cultivation of oleaginous yeast R. glutinis in pulp and paper wastewater indicated that this oleaginous microorganism is able to grow in pulp and paper wastewater for producing microbial lipid. The oleaginous yeast used in this study was able to convert pure glycerol and the mixture of glucose and xylose very efficiently, demonstrating the potential utilization of industrial waste glycerol and xylose containing wastes such as lignocellulosic wastes. The fatty acid composition of the oleaginous yeast was similar to the fatty acid profile of vegetable oils which makes its chemical characteristic similar to plant oils suitable for biodiesel production. The cetane number, saponification value, and higher heating value of the derived biodiesel (FAMEs) were quite similar to those observed for the biodiesel produced from common vegetable oils with some improved quality. The applied mathematical model was able to successfully simulate the growth and lipid accumulation process.
This work was financially supported by the US department of energy, Office of Energy Efficiency and Renewable Energy (Grant No: DE-FG36-06GO86025). The authors thank Magan Green for the technical assistance. The authors are grateful to the International Paper Company for their assistance.
MartaAmirsadeghi,SaraShields-Menard,W. ToddFrench,RafaelHernandez, (2015) Lipid Production by Rhodotorula glutinis from Pulp and Paper Wastewater for Biodiesel Production. Journal of Sustainable Bioenergy Systems,05,114-125. doi: 10.4236/jsbs.2015.53011