Biodegradation of deproteinized potato wastewater and glycerol during cultivation of Rhodotorula glutinis yeast Electronic Journal of Biotechnology

Background: Deproteinized potato wastewater and glycerol are two by-products which are dif ﬁ cult to dispose. The objective of this study was to determine the ability of Rhodotorula glutinis to use glycerol and nitrogen compounds present in deproteinized potato wastewater and to evaluate the ability of simultaneous biodegradation of potato wastewater and glycerol via microbiological methods. Results: It has beenfoundthat R.glutinis used glyceroland potatowastewaterasasourceof carbon and nitrogen, respectively. The highest degree of glycerol content (70.6%) reduction was found after cultivation of the investigated strain using a medium with 5% glycerol. In this medium, a signi ﬁ cant reduction in the total protein content, estimated at 61%, was observed. The process of 72 h cultivation of yeast in a medium containing potato wastewater and 5% glycerol reduced the chemical oxygen demand (COD) more than 77%. Supplementation of media with high doses of glycerol (i.e. 20 and 25%) led to decreased metabolic activity in the yeast strain tested. Conclusion: Ithasbeenfoundthatthereisapossibilityofsimultaneousbiodegradation ofpotatowastewaterand glycerol during the cultivation of R. glutinis .


Introduction
In many industrial units, production processes always generate by-products. For these by-products, no rational management concepts exist and their disposal under natural conditions may lead to a progressive degradation of the natural environment. Examples of such wastes are deproteinized potato wastewater generated during the production of potato starch and glycerol fraction occurring during the production of biodiesel. Problems arising during the disposal of these two by-products have led to the search for new biotechnological methods for their treatment.
Potato wastewater is the primary waste generated during the production of potato starch. It has been estimated that during the processing of 1000 tons of potatoes, about 600 m 3 of potato wastewater is produced [1]. Due to the high content of organic substances, potato wastewater from the production of potato starch creates waste disposal problems. To reduce the content of nitrogen compounds, potato-protein precipitation is performed during the thermal-acid coagulation process. Deproteinized potato wastewater contains 2.9-4.3% of dry matter on average, of which protein, sugar and fat (emulsified with water) contents are estimated at 0.93-1.57%, 0.5-0.8% and approximately 0.2%, respectively. It also contains significant amounts of minerals (about 1%), of which potassium (600 mg/L) and phosphorus (about 300 mg/L) are dominant. Moreover, this waste is characterized by a high value of chemical oxygen demand (COD), which is equal to at least 20,000 mg O 2 /L [2]. Potato wastewater is mostly utilized during field sprinkling [3], which may lead to adverse eutrophication of water [2] and soil sealing [3] in the natural environment.
The main by-product generated during the production of biodiesel is a glycerol fraction. This consists mainly of pure glycerol (50-65%), and among the remaining components one can distinguish methanol, free fatty acids, mono-and diacylglycerols, phospholipids, tocopherols, colorants, soaps, water, and catalyst residues [4]. In 2012, biodiesel production was estimated at 23.40 million tons, of which about 40% was produced in the European Union, a leading manufacturer of this fuel for years. In comparison to 2009, the global production of biodiesel has increased by more than 30% [5]. It is expected that in the future, biodiesel production will increase, which in turn will generate higher amounts of waste glycerin. The process of refining crude glycerol to high purity glycerol is energy intensive and expensive; therefore, new methods for the disposal and management of this waste are emerging [6].
Biodegradation is an alternative method of decomposition of pollutants using living organisms [7]. The fundamental issue concerning biotechnological disposal is the selection of micro-organisms, which must decompose contaminants in a short time without producing toxic metabolites [8]. So far, molds have particularly been used for this purpose [9,10,11]. In this paper, an attempt was made to use Rhodotorula glutinis for simultaneous biodegradation of potato wastewater and glycerol.
The objective of this study was to evaluate the ability of R. glutinis yeast to use nitrogen compounds present in deproteinized potato wastewater and glycerol, and to determine the possibility of simultaneous biodegradation of these two wastes via microbiological methods.

Microorganism
R. glutinis LOCK 0051 yeast strain derived from the Pure Culture Collection of the Department of Biotechnology and Food Microbiology, Warsaw University of Life Sciences, constituted the biological material used for the study. Yeasts were stored on slants of YPD medium at 4°C and were subcultured every month.

Potato wastewater
Deproteinized potato wastewater was collected from the technological line of potato starch production (PEPEES S.A., Łomża, Poland). The waste was sterilized (117°C for 10 min, HICLAVE HG-80, HMC Europe) and stored at room temperature. To remove residual solids (starch and cellulose fibers) before use, potato wastewater was centrifuged at 12,900 × g for 10 min (Centrifuge 5804R, Eppendorf). The dry matter content (g/L) was determined by the gravimetric method through drying a certain volume of the sample to constant weight at 105°C for 24 h (SML 32/250, Zatmet). Total protein content was determined by the Kjeldahl method. Samples were subjected to burning in concentrated sulfuric (VI) acid with the addition of a catalyst (Büchi Digestion Unit K-435), followed by alkalinization and distillation with steam water (Büchi Distillation Unit K-355). The resulting product (g/L) was calculated per total protein content using a factor equal to 6.25. The content of reducing sugars (calculated per glucose) in potato wastewater was determined spectrophotometrically (λ = 550 nm) using 3,5-dinitrosalicylic acid [20]. The result was given in g/L. The rate of chemical-oxygen demand (g O 2 /L) in potato wastewater was determined by the dichromate method using the Hach Lange cuvette tests in the Water Center of the Warsaw University of Life Sciences. All the obtained results are listed in Table 1.

Preparation of inoculum
To prepare inoculum, YPD medium containing glucose (20 g/L), peptone (20 g/L), and yeast extract (10 g/L) was used [21]. The initial active acidity was determined at 5.0 ± 0.1. Cultivation was carried out in flat-bottomed flasks containing 100 mL of the medium at 28°C for 24 h at 200 rpm/min (SM-30 Control, Edmund Bühler).

Control and experimental cultures
For submerged yeast cultures, two culture media were used, YPD of an optimal composition for yeasts [21] and non-enriched potato wastewater (PW). For experimental cultures, five experimental media were used containing glycerol as a carbon source (POCH, Poland) and potato wastewater as a source of nitrogen. Glycerol was added to the medium at an amount of 50, 100, 150, 200, and 250 g/L. In later parts of the experiment, culture media with glycerol were denoted by the following abbreviations: PW + G5%, PW + G10%, PW + G15%, PW + G20% and PW + G25%. Inoculation was estimated at 10% (v/v). Cultivation of yeast was carried out on a reciprocating shaker (SM-30 Control, Edmund Bühler) for 72 h, at a speed of 200 cycles/min and temperature equal to 28°C. The experiment was carried out in triplicate.

Growth characteristics of yeast
Yield of dry cellular biomass was determined by the gravimetric method. A specified volume of culture was centrifuged for 10 min at 6000 × g (Centrifuge 5804R, Eppendorf), and rinsed with distilled water. The wet-cell biomass was dried at 85°C (SML 32/250, Zatmet) to a constant weight. The result was given in g d.w. /L.
The optical density of the culture (OD) was determined using the spectrophotometry technique. Two milliliters of culture medium were collected and centrifuged at 5000 g for 5 min (MiniSpin Plus, Eppendorf). Post-culture medium was removed and 2 mL of deionized water was added to the biomass, followed by careful stirring and centrifugation of the solution using the same parameters. The supernatant was decanted and the biomass was suspended in 2 mL of deionized water, followed by the measurement of absorbance of the cell suspension at a wavelength of λ = 600 nm (UV1800 spectrophotometer, Rayleigh) against deionized water.

Determination of the potential of yeast for biodegradation of wastewater
To determine the ability to use the added glycerol and the protein present in the potato wastewater, their concentrations were determined in the latter hours of cultivation, followed by the determination of COD in selected culture media.
Glycerol content was determined via the chemical method by using oxidative properties of meta-periodate acid. As a result of its activity toward two adjacent hydroxyl groups in a glycerol molecule, decomposition of the main carbon chain is observed. As a result, two molecules of formaldehyde and a formic acid molecule are formed. The latter is titrated with 0.1 M sodium hydroxide in the presence of bromothymol blue. The result was given in g/100 mL [22].
Total protein content in post-culture media was determined by the Kjeldahl method, using a conversion factor equal to 6.25. The results were expressed in g/100 mL.
The rate of chemical-oxygen demand in post-culture media, based on the conducted experiments, was determined using Hach Lange cuvette tests in the Water Center of the WULS and the results were expressed in g O 2 /L.
The experiment was carried out in triplicate and the values represent in the figures and tables are mean ± SD. Based on the obtained results, the degree of utilization of glycerol and nitrogen compounds per total protein, as well as the degree of reduction of COD, were determined.

Statistical analysis of results
The results were analyzed in R statistical software (version i386 2.15.3). One-way analysis of the variance (one-way ANOVA) and Tukey's test at a significance level (α = 0.05) were performed.

Results and discussion
3.1. Growth of R. glutinis in a medium with potato wastewater and glycerol Based on the results of the optical density change of the culture and the biomass yield, it was found that R. glutinis yeasts could be grown in the control medium (YPD) and in all variants of experimental media tested. It can therefore be concluded that glycerol was used as a carbon source and potato wastewater as a nitrogen and minerals source.
Concentrations of compounds which constituted a carbon source acted as factors determining the growth of the yeast strain studied. After analyzing the results of measurements of the optical density of culture biomass yield, it may be concluded that the most favorable condition for the growth of R. glutinis was provided by media containing potato wastewater and glycerol at the concentrations of 5 and 10%, respectively. In both these cases, OD values of the biomass yield were significantly higher compared to the YPD control medium after 72 h (Fig. 1, Table 2). After 72 h of cultivation in PW + G5%, PW + G10% and YPD media, biomass yield was estimated at 20.34, 19.63 and 8.71 g d.w. /L, respectively ( Table 2). During cultivation, in medium containing 15% glycerol, intensive growth of the analyzed yeast strain was observed. After 72 h the value of OD reached the level of 2.4 (Fig. 1), and cell biomass yield was 15.08 g d.w.. /L. These values were slightly higher than those determined after the same time in the YPD control medium. Glycerol at the concentrations of 20 and 25% showed inhibitory effects toward the growth of the yeast strain studied. After 72 h, with such addition of glycerol, cellular biomass yield was estimated at only 6.06 and 4.22 g d.w. /L. Yeast cultured in medium containing potato wastewater demonstrated the highest growth at the 24th h of cultivation. It can be assumed that by this time the cells had exhausted the residual simple sugars present in the potato wastewater (about 0.3%). After depletion of the carbon substrate, proliferation was inhibited and autolysis processes were intensified over time, as indicated by the decreasing OD value and biomass yield.
Schneider et al. [23] grew cultures of R. glutinis ATCC 15125 using media containing wastewater from the brewing industry. As a result of the cultivation which lasted for 168 h, biomass yields of 5.22 g d.w. /L and 7.38 g d.w. /L, respectively, were obtained in media containing wastewater from brewing industry and sewage enriched with 1% glucose. Similar low levels of yeast biomass yield of R. glutinis TISTR 5159 were reported by Saenge [24]. The authors used ammonium sulfate as a nitrogen source, and glycerol at the concentrations of 5%, 9.5% and 14% as a carbon source. Similarly, no significant differences were observed between the results obtained after 72 h of cultivation for media supplemented with 5% and 9.5% of glycerol addition (5.15-5.65 g d.w. /L). However, the concentration of 14% was found to have an inhibitory effect on growth of the analyzed yeast strain (4.30 g d.w. /L). The results of biomass yields cited in the above-mentioned papers were as much as four times lower than the results obtained in the present study (Table 2), which demonstrates the stimulating activity of the potato wastewater on R. glutinis growth.

Determination of biodegradation potential of R. glutinis yeast
R. glutinis yeast demonstrated the ability to utilize glycerol as a carbon source. After 72 h of cultivation in PW + G5% medium, the glycerol content decreased to 1.5%. The total degree of utilization of glycerol was above 70%, while in terms of other experimental cultures, it did not exceed 35% (Table 3). Based on the results obtained, it was found that the microbial biodegradation process of glycerol by R. glutinis runs most efficiently, if its concentration in the environment is not higher than 5%.
Different results for the degree of reduction for the same concentrations of glycerol in media with potato wastewater were obtained by Błażejak et al. [25]. They used a R. gracilis yeast strain derived from the Pure Culture Collection of the University of Life Sciences in Lublin. In medium supplemented with 20% of carbon substrate yeast utilized almost 59% of its content. This indicates that tested in this work R. glutinis yeast strain exhibit higher sensitivity to increased glycerol content in the environment.
The maximum reduction of nitrogen compounds from the medium was detected during the cultivation in media supplemented with glycerol at concentrations of 5 and 10%, respectively. After 72nd h of cultivation in PW + G5% medium, over 0.3 g/100 mL of total protein still remained, and the degree of reduction was estimated at 61%. If cultivation was conducted using PW + 10% medium after 72 h, total protein content was reduced to 0.3 g/100 mL, and the total degree was 63%. In terms of cultivations conducted in media supplemented with the highest doses of glycerol, i.e. 20 and 25%, degree of reduction  of protein content was negligible, and was estimated at 15 and 9%, respectively (Fig. 2). It was found that the concentration of carbon substrates determined the metabolic activity of the yeast strain tested. At its high concentrations (20% and 25%), yeasts practically did not utilize nutrients present in the medium, which could be a result of the increased osmotic pressure of the environment. The osmotic pressure of the culture medium is in fact one of the most important parameters determining microbial growth and propagation [26]. Glycerol, as a substance characterized by a high viscosity [27], may also, to some extent, adhere to yeast cells, hampering the use of nutrients present in the environment. In medium containing only potato wastewater, yeasts did not efficiently use nitrogen compounds due to the low content of simple sugars constituting a source of carbon.
In the last part of the study, chemical rate of oxygen demand was determined in the selected post-culture media. Cultivation carried out in a medium containing only the potato wastewater led to a reduction of the COD of 37.4 ± 2.8 to 17.38 ± 1.75 g O 2 /L after 72 h of process (53% reduction rate). Nowak et al. [11] found that Aspergillus niger 334 and Rhizopus oligosporus 2710 mold strains were capable of reducing the COD value of the potato wastewater by 58 and 52%, respectively, during the 72 h of cultivation.
The chemical-oxygen demand indicator was also determined in the medium with potato wastewater supplemented with 5% of glycerol (99.52 ± 3.63 g O 2 /L), because in this medium yeasts showed highest growth and metabolic activity (Fig. 3). It was observed that 72 h cultivation of R. glutinis yeast in the PW + G5% medium caused reduction of the chemical oxygen demand indicator by 77% and the value of the COD to 22.58 ± 1.20 g O 2 /L. This amount was higher than in PW, what was probably a result of the glycerol residues. Because of the higher degree of utilization of nitrogen compounds from the medium PW + G5%, it can be concluded that cultivation yeast in medium containing additional carbon source has enabled more efficient use of ingredient potato wastewater. The cultivation in the medium PW + G5% time should be extended for another day to metabolize glycerol completely.

Conclusions
R. glutinis yeasts exhibited the ability to metabolize protein substances in the deproteinized potato wastewater, and to assimilate carbon originating from glycerol for cell biomass proliferation. It was found that there is a possibility of simultaneous biodegradation of these two wastes via microbiological methods. The efficiency of waste disposal was dependent on the concentration of compounds constituting a source of carbon for yeasts. The enrichment of potato wastewater in an additional carbon source at the level of 5% allowed the efficient use of glycerin (70%) and nitrogen substances per protein amount (61%) and caused a significant decrease in COD (77%). High doses of glycerol inhibited growth and metabolism of the yeast strain studied, which resulted in the reduction of nutrient utilization from the environment.
From the literature data, it can be concluded that R. glutinis yeasts are capable of synthesis of intracellular metabolites, such as fat and carotenoids, and this may find an application in the food industry. Future studies should involve the determination of these two components in yeast cell biomass obtained after cultivation in media prepared from these waste materials.