Oleaginous Yeast Cryptococcus curvatus Culture for Biodiesel Feedstock with Wastewater from Anaerobic Bio-hydrogen Production
Zhanyou Chi, Yubin Zheng, Shulin Chen*
Department of Biological Systems Engineering
Washington State University
Pullman, WA
*Corresponding author: Shulin Chen
Department of Biological Systems Engineering
Washington State University
Pullman, WA 99164-6120
Email:
Fax: (509) 335-2722
Keywords: Cryptococcus curvatus; oleaginous yeast; lipid production; biodiesel; biohydrogen; wastewater
1. treated wastewater.
2. poor presentation.
3. optimization of oil production is unnecessary
4. results that deal with shake-flask cultures are of no particular value
5. Fermentations for oil production need to be up to 100 g/L to be economically viable.
6. Food grade materials, including oils, have to be produced from food grade substrates.
7. Why the authors didn't use the waste water?
Abstract
Single cell oil produced from oleaginous yeast culture can be a potential feedstock for biofuel production, but suffers high cost of raw material. In this study, volatile fatty acids (VFA) produced from fermentative hydrogen production process were tested as a cheap carbon source for oleaginous yeast culture. With acetate as carbon source, culture condition of oleaginous yeast Cryptococcus curvatus was optimized with statistical method, and a second-order polynomial equation model was developed to predict the effect of major factors upon cell growth, as well as the interactions between these factors. It was found that ammonium concentration, initial pH, and acetate concentration had significant effects on the cell growth, and there was strong interaction between ammonium concentration and acetate concentration. It is interesting that pH 5.5, which is usually resulting in good growth of C. curvatus when glucose or glycerol used as carbon source, totally inhibited its growth when acetate used as feedstock, and only a pH greater than 8.0 supported fast growth of C. curvatus. Fed batch culture of C. curvatus with fermentative hydrogen production effluent (FHPE) as initial medium resulted in 16.9 g/L dry biomass, indicating no inhibitor produced in the fermentative hydrogen production process, and the effluent can be good feedstock for C. curvatus’ culture. Finally, FHPE from food waste was tested as feedstock for C. curvatus’ culture. To eliminate the inhibition caused by the high concentration ammonium, ammonia stripped FHPE was used as feedstock, and it was proved to be good feedstock for C. curvatus’ culture. This study developed a lipid production process using FHPE as cheap feedstock, which would benefit both waste treatment and biofuel production.
1. Introduction
Currently, biodiesel is produced mainly from traditional oils source such as soybeans, canola oil, animal fat, palm oil, corn oil, waste cooking oil, and jatropha oil (reference). Thus, the source is limited due to the low productivity per unit land area and the concerns over competition with human consumptions of these oils. Single cell oil (SCO) production by oleaginous yeast (Kyle and Ratledge, 1992), fungus (Ratledge and Wynn, 2002), heterotrophic microalgae (Li et al., 2007), and phototrophic microalgae (Chisti, 2007) has recently gained more attention because of its potential advantages either in high productivity or the potential use of organic waste as the raw materials. Compared with phototrophic algae, the heterotrophic process has the advantage of high cell density and without light limitation, but one of its disadvantages is the high cost of feedstock. Thus, cheap feedstock is the key hurdle for the application of single cell oil technology for biofuel production.
A variety of organic waste and wastewater have been investigated as feedstock for dark fermentative hydrogen production (Li and Fang, 2007). In this process, bacteria first produce sugars from the organic wastes, then produce hydrogen from the sugars (Liu et al., 2005). About one-third carbon in sugar is converted to CO2, while the other two-thirds are converted to VFA, mainly acetate or butyrate, which have to be further treated to be discharged into environment without causing pollution. To deal with this problem, some studies have tried to convert residue VFAs to valuable products such as methane, hydrogen or electricity (2002) (Barbosa et al., 2001; Fang et al., 2005). (Liu et al., 2005; Oh and Logan, 2005). However, these technologies have little viability for biofuel production, especially for the production of heavy duty biofuel such as biodiesel. Compared to these, oleaginous yeast culture is a much more mature technology. If the VFA is converted into oil enriched yeast biomass, it not only reduces the biological oxygen demand (BOD), but also provides feedstock for biofuel production. Such a strategy will greatly improve the economical viability of single cell oil production since the feedstock has a negative value.
C. curvatus has been used in large scale cocoa butter substitutes production process from cheese whey (Kyle and Ratledge, 1992). It uses a variety of carbon sources such as glucose, xylose, lactose, glycerol, as well as ethanol. When ethanol is used as carbon source, it is usually converted into acetate. Then, acetyle-CoA will be activated from acetate, and feed into glyoxylate and tricarboxylic acid cycles to produce oxaloacetate (Palmieri et al., 1997). Thus, the strains can take ethanol usually can also take acetate as carbon source. Thus, C. curvatus was selected in this study to convert VFAs into lipid. In previous work, tremendous works on culture condition optimization of C. curvatus have been reported. However, our preliminary experiments found that the optimal culture condition of C. curvatus with acetate as carbon source was significantly different from the culture condition with glucose as carbon source. For example, pH 5.5, usually used as optimal pH for glucose culture, completely inhibited the growth with acetate. Thus, culture conditions such as pH, acetate concentration, and nitrogen concentration was optimized with statistical experiment at first. To test if inhibitors to yeast growth are produced in the fermentative hydrogen production process, sucrose, instead of waste stream, was used as raw material. Otherwise, if there is inhibitor found in the yeast culture process, it would be impossible to judge the inhibitor was brought in from waste stream itself, or from the dark fermentation process. After that, food waste was used as an example raw material for fermentative hydrogen production, and the effluent was used as the feedstock for C. curvatus’ culture.
2. Materials and Methods
2.1 Cell strain and medium
Cryptococcus curvatus (ATCC 20509), known previously as Candida curvata, was used in the research. The seed cell was pre-cultured with a medium composed of 20 g/L malt extract and 5 g/L peptone (Sigma, St. Louis, MO), according to the instructions from ATCC. The cells were grown in 250-mL Erlenmeyer flasks, each containing 50 mL of medium and incubated at 25oC in an orbital shaker set to170 rpm. Sub-cultured cells were used as inoculums for future studies. The inoculums constituted 5% of the total liquid volume in each flask.
2.2 The Central Composite Design
The basic media for the cultures in the central composite design contained KH2PO4 2.7 g/L, Na2HPO4 0.95g/L, MgSO4.7H2O 0.2g/L, Yeast extract 0.1g/L, EDTA 0.1g/L, and spores stock solution 10ml/L. The spores stock solution contains CaCl2.2H2O 4.0 g/L, FeSO4.7H2O 0.55 g/L, ZnSO4.7H2O 0.1g/L, MnSO4.H2O 0.076 g/L, 18 M H2SO4 0.1mL. The cells were grown in 250-mL Erlenmeyer flasks, each containing 50 mL of medium and incubated at 30oC in orbital shakers set to170 rpm.
The factors shown in Table 1 were determined as significant with a preliminary experiment (data not shown). The central composite design was used to optimize their levels, and to reveal the interactions between these factors. The design matrix was a 23 full factorial design combined with 5 central points (runs 15-19) and 6 axial points, where one variable was set at an extreme level (±1.68) while other variables were set at their central point level (Table 2). The coded and real values of each parameter were shown in Table 1. Our preliminary experiment showed that pH 5.5, which is usually used for C. curvatus culture on glucose or glycerol (Evans and Ratledge, 1983; Meesters et al., 1996), completely inhibited its growth when acetate is used as carbon source. Thus, higher pH was used in this design.
Table 1. The Coded Levels and Real Values of parameters in the Central Composite Design
A / B / CLevel / Initial pH / KAc a
(g/L) / NH4Cl
(g/L)
1.68 / 8.34 / 83.6 / 1.47
1 / 8.00 / 70.0 / 1.20
0 / 7.50 / 50.0 / 0.80
-1 / 7.00 / 30.0 / 0.40
-1.68 / 6.66 / 16.4 / 0.13
a Potassium Acetate
Based on the experimental results obtained in Table 2, the responses of dry cell weight, lipid content, and lipid yield were correlated as functions of variables by a second-order polynomial equation, i.e.,
Y = β0 + Σβixi + Σβixi2 + Σβijxixj (1)
where Y is the predicted response, β are the coefficients of the equation, and xi and xj are the coded levels of variables i and j, respectively. The software Design-Expert (Stat-Ease Inc., Minneapolis, MN) was used for this correlation through non-liner regression. The F-test was used to evaluate the significance of the models.
Table 2. The Central Composite Design of the Variables (in Coded Levels) with Cell Dry Weight and final pH as Responses
Factors / ResponsesRun / A:pH / B:KAc
(g/L) / C:NH4Cl
(g/L) / Dry cell weight
(g/L) / Lipid yield
(g/L) / Lipid content
(%) / C/N
ratio
1 / -1 / -1 / -1 / 4.2 / 2.28 / 54.7% / 70
2 / 1 / -1 / -1 / 4.5 / 2.38 / 53.1% / 70
3 / -1 / 1 / -1 / 8.7 / 2.44 / 28.0% / 162
4 / 1 / 1 / -1 / 7.0 / 1.58 / 22.6% / 162
5 / -1 / -1 / 1 / 4.9 / 1.86 / 38.1% / 25
6 / 1 / -1 / 1 / 4.7 / 1.87 / 39.6% / 25
7 / -1 / 1 / 1 / 6.6 / 3.74 / 56.6% / 58
8 / 1 / 1 / 1 / 4.1 / 2.02 / 49.8% / 58
9 / -1.68 / 0 / 0 / 4.0 / 1.96 / 49.1% / 61
10 / 1.68 / 0 / 0 / 3.6 / 1.75 / 48.5% / 61
11 / 0 / -1.68 / 0 / 3.2 / 0.63 / 19.5% / 20
12 / 0 / 1.68 / 0 / 8.1 / 1.56 / 19.3% / 102
13 / 0 / 0 / -1.68 / 6.9 / 2.77 / 40.2% / 298
14 / 0 / 0 / 1.68 / 4.8 / 2.77 / 57.2% / 34
15 / 0 / 0 / 0 / 3.7 / 2.00 / 53.4% / 61
16 / 0 / 0 / 0 / 4.0 / 2.48 / 62.4% / 61
17 / 0 / 0 / 0 / 3.9 / 2.39 / 60.8% / 61
18 / 0 / 0 / 0 / 3.4 / 1.67 / 48.6% / 61
19 / 0 / 0 / 0 / 3.9 / 1.76 / 45.5% / 61
Finally, with the developed model, the optimal values of three parameters are predicted and shown in the first column in Table 3. To prove the accuracy of this model, a verification experiment was conducted for the predicted optimal condition for maximum dry cell weight, lipid yield and lipid content.
2.3 pH controlled fed-batch culture of C. curvatus in fermentor
To obtain a more definitive understanding of the effect of pH on the growth of C. curvatus, three cultures in the stirred tank fermentor with precise pH control at 7.50± 0.05, 7.50 ± 0.05, and 8.00 ± 0.05 were conducted. One 1.0-L NBS (Edison, NJ) Bioflo-110 fermentor with work volumes of 0.5 L was used, and another two 5.0-L fermentors with work volume of 2.0 L were used. The temperature was controlled at 30 oC, and the dissolved oxygen was set at 50% saturated air in the solution with being cascaded to agitation speed. The aeration rates for the cultures were all set-up as 1.0 VVM. Liquid acetic acid was fed to the culture continuously to adjust the pH, as well as used as supplemented carbon source.
2. 4 Dark fermentation hydrogen production with synthetic wastewater
Sucrose was used as raw material for fermentative hydrogen production process. This is to exclude the possible inhibitors brought from waste stream. Otherwise, if there is inhibitor found in the yeast culture process, it would be impossible to judge the inhibitor was brought in from waste stream itself, or from the dark fermentation process. Dark fermentation hydrogen production process was same as that described in (Hu et al, 2008). Briefly, anaerobic sewage sludge was pretreated with heat at 105°C for 1 hr. Then the sludge was inoculated to the dark fermentation process for hydrogen production. The pH of the medium was adjusted to 7.5, and placed into a sealed stirred tank, which had an 8.0-L volume and the working volume was 6.0 L. When the hydrogen production process was finished, the FHPE was collected and centrifuged to separate the bacteria, and the supernatant was sterilized with a 0.22-µm pore size filter.
2.5 VFA and sugar analysis
Concentrations of VFA in the broth were analyzed via headspace chromatographic (HS-GC) analysis (Cruwys et al., 2002). A GC unit (Shimadzu Corporation, Kyoto, Japan) equipped with a flame ionization detector and an HP-INNOWax polyethylene glycol (PEG) capillary column (Agilent Technologies, Santa Clara, CA) were used in this analysis. An AOC-5000 auto injector (Shimadzu GC-2014) was used for vial incubation and automatic sampling, and 500-µL samples were injected. The PEG capillary column was first heated from 140°C to 200ºC at 6ºC/min and then held at 200ºC for 1 min. The temperatures of the injector and detector were 200°C and 250ºC, respectively. Nitrogen was used as the carrier gas at a flow rate of 25 mL/min. Total residue sugar was analyzed with an anthrone solution (Fluka Biochemica, Buchs, Switzerland) method (reference).
2.6 C. curvatus culture with FHPE and fed with acetic acid
The FHPE was determined to contain 2.05 g/L acetic acid and 4.06 g/L butyric acid. The residue sugar concentration was 0.16±0.03 g/L. Thus, if there is significant yeast growth, the biomass should be converted from VFA, but not residue sugar. NBS (Edison, NJ) Bioflo-110 fermentors with 5.0-L volume were used, with work volumes of 2.0 L. The temperature was controlled at 30°C and the dissolved oxygen was set at 50% saturated air in the solution with being cascaded to agitation speed. Because consortiums of bacteria grow in the hydrogen production process, some metabolites produced may have inhibition effect to C. curvatus’ growth. If this happens, using FHPE as the medium would not be practical. To test if inhibitor produced, FHPE was used as the minimal medium, and supplemented with all the composites in the basic medium as described in Method 2.2. The set point of pH was 8.00 ± 0.05. No carbon source was added in the initial medium, since the FHPE contains VFA. With cell growth and VFA consumption, the pH increased gradually. Then, acetic acid was continuously fed to the culture to balance pH, as well as used as supplemented carbon source.