CC BY
7UDC 67.08/665.127.6:[665.2+665.3]
Mikhail A. Pushkarev1, Grigoriy V. Kozlov2, Alexander V. Garabadzhiu3, Eldar R. Yagmurov4, Daniil Yu. Beliaev5, Angelina A. Agureeva6, Inessa A. Fagradyan7
BIOCATALYTIC CONVERSION OF LIPID-CONTAINING WASTES FROM FISH PROCESSING INDUSTRIES
St. Petersburg State Institute of Technology (Technical University), 26, Moskovsky Pr., St Petersburg, 190013, Russia
e-mail: malexpush@bk.ru
The aim of this study was to develop an ecologically sustainable and economically viable way of utilization of lipid - containing wastes from fish processing with the possibility of obtaining valuable products: biodiesel fuels and concentrate of omega - 3 polyunsaturated fatty acids.
The key technologically important properties of wastes from fish processing industries such as temperature, molar ratio of reagents, presence of inert atmosphere, amount of biocatalysts, were studied for their influence on the catalytic reaction by Novozym 435 as well as on transesterification of fish oil triacylglycerides with ethanol. The method of fatty acids fractionation by using urea complexation has been modified for substrate properties. Correspondingly, the diagrams of fatty acids fractionation during the multi - step urea complexation have been obtained.
The results obtained would allow to create ecologically attractive technological processes that would have simpler equipment standards (temperature 50-60 °C, absence of alkalis and acids, atmospheric pressure) and smaller number of technological steps involved, the excluded steps are: washing and drying fractions of fatty acid esters, evaporation of acyl-acceptor excesses, drying of acyl glycerides, dehydration of acyl-acceptors.) Ecological stability is achieved by biological degradation of all reaction mixture components.
Key words: waste processing, biocatalysis, transesterifica-tion, lipases, wastes from fish processing, fish oils, polyunsaturated fatty acids, ethanolysis, urea fractionation, biodiesel.
DOI 10.15217/issn1998984-9.2016.37.55
Introduction
Fish processing could generate wastes of up to 50 % of the body processed fish's weight based on the body components of interest to the processor [1]. It can be esti-
М.А. Пушкарев, Г.В. Козлов, А.В. Гарабджиу, Э.Р. Ягмуров, Д.Ю. Беляев, А.А. Агуреева,
И.А. Фаградян
БИОКАТАЛИТИЧЕСКАЯ КОНВЕРСИЯ ЛИПИДСОДЕРЖАЩИХ ОТХОДОВ
РЫБОПЕРЕРАБОТКИ
Санкт-Петербургский государственный технологический институт (технический университет), Московский пр., 26, Санкт-Петербург, 190013, Россия e-mail: malexpush@bk.ru
Целью данной работы являлась разработка экологически безопасного и экономически перспективного способа утилизации липидсодержащих отходов рыбопереработки с получением ценных продуктов - концентрата омега-3 полиненасыщенных жирных кислот и биодизельное топливо. Изучены ключевые технологически значимые свойства отходов рыбопереработки профильных предприятий Санкт-Петербурга, влияние важнейших технологических параметров (температура, соотношение реагентов, наличие инертной атмосферы, количество биокатализатора) на протекание, катализируемой иммобилизованной липазой Novozym 435, трансэтерификации триацилглицеридов рыбьего жира с этанолом.
Адаптирован к специфике сырья метод разделения жирных кислот комплексообразованием с мочевиной. Получены диаграммы распределения жирных кислот при многоступенчатом комплексообразовании с мочевиной. Полученные данные позволяют создавать экологически перспективные технологические процессы за счет снижения требований к оборудованию (температура 50-60 °С, отсутствие щелочей и сильных кислот, атмосферное давление) и сокращения количества технологических стадий (исключается промывка и осушка фракции эфиров жирных кислот, отгонка избытка ацил-акцептора, осушка ацилглицеридов и абсолютирование ацил-акцептора).
Экологическая безопасность обеспечивается биоразлагае-мостью всех компонентов реакционной смеси.
Ключевые слова: перерабока отходов, биокатализ, трансэтерификация, липазы, отходы рыбопереработки, рыбий жир, полиненасыщенные жирные кислоты, этано-лиз, фракционирование мочевиной, биодизель.
mated that nearly 64 million tons of fish waste are generated annually [2]. Solid fish waste consists of heads, tails, skin, gut, fins and carcass and contains proteins (58 %), ether extract or fat (19 %) and minerals [3]. These wastes can be used to produce fish protein concentrate, fish oils and enzymes (such as pepsin and chymotrypsin) as well as
1 Mikhail A. Pushkarev assistant of the department of technology of microbiological synthesis, e-mail: malexpush@bk.ru Пушкарев Михаил Алексеевич, ассистент кафедры технологии микробиологического синтеза.
2 Grigoriy V. Kozlov, Ph.D, assistant professor of the department of technology of microbiological synthesis,
Козлов Григорий Владимирович, канд. биол. наук, доцент каф. технологии микробиологического синтеза, e- mail: kozlov_gv@mail.ru
3 Alexander V. Garabadzhiu, Dr Sci. (Chem.), professor of the department of technology of microbiological synthesis, e-mail: gar-54@mail.ru Гарабаджиу Александер Васильевич, д-р хим. наук, профессор каф. технологии микробиологического синтеза, проректор по научной работе
4 Eldar R. Yagmurov, student of the the department of technology of microbiological synthesis, e-mail: 95eldar@gmail.com Ягмуров Эльдар Русланович, студ. каф. технологии микробиологического синтеза
5 Daniil Yu. Beliaev, student of the the department of technology of microbiological synthesis, e-mail: dan.beliaev2010@yandex.ru Беляев Даниил Юрьевич, студ. каф. технологии микробиологического синтеза
6 Angelina A. Agureeva, student of the the department of technology of microbiological synthesis, e-mail: aska9494@mail.ru Агуреева Ангелина Алексеевна, студ. кафедры технологии микробиологического синтеза
7 Inessa A. Fagradyan, student of the the department of technology of microbiological synthesis, e-mail: inessa.fagradyan.95@mail.ru Фаградян Инесса Артуровна, студ. каф. технологии микробиологического синтеза
Received December 02, 2016
other valuable products. The fish oil is used for productions of margarine, omega-3 fatty acids and biodiesel.
Fish oils are available sources for long chain polyunsaturated fatty acids which consist of omega-3 fatty acids mainly composed of cis-5,8,11,14,17- eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19- docosahexaenoic acid (DHA). The omega-3 fatty acids have a wide range of biological effects including protection against arrhythmias, prevention of atherosclerosis, benefit to diabetic patients, reduced blood pressure, reduced symptoms in asthma patients, protection against manic-depressive illness, protection against chronic obstructive pulmonary diseases, improving survival of cancer patients, alleviating the symptoms of cystic fibrosis, reduction in cardiovascular disease and improved learning ability [4-8].
Due to the high cost of enzymes high-tech bio-catalytic utilization of fish processing wastes becomes efficient only if the products possess high value. The optimal solution would be enzymatic hydrolysis of protein in the fish processing waste in order to obtain protein hydro-lysates and lipids. With the use of biocatalytic ethanolysis of the lipid fraction to the fatty acid ethyl esters (FAEE) and subsequent fractionation, two useful products can be obtained - concentrate of omega-3 polyunsaturated fatty acids (PUFA), which is used as nutritional supplement, and PUFA-depleted FAEE which can be utilized as biodiesel.
The combination of these processes in complex technology will allow to improve the efficiency of fish processing industry and will make it more environmentally friendly.
In this study, enzymatic ethanolysis and urea fractionation method were combined in order to utilize oil from fish processing wastes to useful products - biodiesel and omega-3 PUFA concentrates.
Materials and methods
Materials. The samples of fish wastes were obtained from various fish processing industries of Saint-Petersburg (Russia). Wastes were mainly composed of mixture of heads, tails, fins, viscera and skin.
Immobilized lipase Novozym® 435 (Candida antarctica lipase B immobilized on macro-porous polyacrylic resin beads) was used for ethanolysis of fish oil.
Oil extraction. The samples of fish wastes were homogenized in a food blender and freeze-dried at a drying temperature of 47 °C and under a vacuum of 0.133 bar. The dried samples were additionally subjected to grinding.
Oil extraction from 50 g of a sample was carried out by 300 ml of n-pentane using of the Soxhlet method for 5 hours. The pentane extract was filtered and washed three times with 400 ml portions of water in separating funnel. The organic solvent was removed from the extracted oil by a rotary evaporator. And oil was dried under nitrogen flow at 40 °C for about 2 hours.
Oil characterization. The measured oil properties were: acid value, by volumetric titration as reported in ISO 660:2009 [9]; iodine value by volumetric titration using Wijs reagent, according to the standard ISO 3961:2009 [10]; peroxide value, by volumetric titration, according to the standard ISO 3960:2007 [11]; and water content, by coulometric Karl Fischer titration, according to ISO 8534:996 [12].
Gas chromatography analysis. Analysis of fatty acid ethyl esters (FAEE) and fatty acid methyl esters (FAME) was conducted by Gas Chromatography. One microliter of sample was injected into a gas chromatograph fitted with an SE-52 column (30 m • 0.25 mm • 0.25 ^m film thickness) and a flame ionization detector. Helium was used as the carrier gas and the total column flow was 1 ml/min. Injector and detector temperatures were 250 and 230 °C, respectively. The temperature program was as follows: starting position at 100 °C for 4 min and then heating to 180 °C at rate of 10 °C/min, maintaining temperature at
180 °C for 10 min, followed by heating from 180 to 250 °C with rate of 5 °C/min. Finally, the temperature was raised to 260 °C and held at this temperature for 10 min. Identification of the various fatty acids was based on a Supelco 37 Component FAME Mix.
Enzymatic ethanolysis of fish oil. 10 g of a mixture of fish oil containing 8% (w/w) of hexadecane (internal standard) and 1.47 - 4.4 g of ethanol were placed in a 50 ml cylindrical flat-bottomed glass reactor (inner diameter 35 mm) with a heat jacket connected to a liquid thermostat. Reaction mixtures were allowed to reach set temperature (25 - 60 °C) with stirring by means of a magnetic stirrer with a 25 mm cross-head stirrer bar operating at a constant 300 rpm. Then variable amount of Novozym® 435 (from 5 to 50% (w/w oil)) was added and reaction timing began. Reaction were carried out under argon atmosphere for up to 24 hours. The samples (100 ^l) were periodically withdrawn from the medium and analyzed. Parallelly etha-nolysis of fish oil was carried out without inert atmosphere to determine the effect of atmospheric oxygen on the resulting product.
After a time corresponding to one complete reaction cycle, 20 mL chloroform was added to the reaction mixture and the resulting suspension was then filtered. The enzyme was washed with 10 mL chloroform in order to remove the oil and then dried. Chloroform solution of FAEE was washed three times with 50 ml portions of water in a separatory funnel. The organic solvent was removed by a rotary evaporator, and FAEE were dried under nitrogen flow at 40 °C for about 2 hours.
Multi-step urea fractionation of FAEE. 10 g of ethyl esters of fatty acids, 15 ml of cyclohexane, 3 g of urea and 10 ml of methanol were placed in a 100 ml flask. The medium was stoppered under argon atmosphere and stirred with heating on a magnetic stirrer until urea was completely dissolved. The resulting mixture was cooled to room temperature while still being stirred. Then the flask was connected to a rotary evaporator to eliminate the solvents at a reduced pressure.
Then 15 ml of cyclohexane were added to the mixture, and the resulted suspension was vacuum filtered. The precipitate was washed on the filter with two 15-ml portions of cyclohexane.
The filtrates were combined in the 100 ml flask and evaporated to dryness under reduced pressure. Then 15 ml of cyclohexane, 3 g of urea and 10 ml of methanol were added to the flask, and the procedure described above was repeated eight times more.
The precipitate fractions containing urea complexes of FAEE were dissociated with a mixture of 100 ml warm distilled water and 50 ml of n-hexane in a separatory funnel. The organic fractions were washed with distilled water two times, and n-hexane was removed by a rotary evaporator. The remaining FAEE were dried under a nitrogen stream.
The obtained fractions were analyzed by gas chromatography.
Results and discussion
Waste fish oil characteristics. The amount of moisture content in the fish waste samples was from 66.4 to 89.2% (w/w). The samples were dried and lipids were extracted. To prevent long time heat treatment of the samples, n-pen-tane was used as solvent for extraction due to its low boiling point 36 °C. The yield of extracted lipids was from 11.5 to 38.8% (w/w) of dry sample weight. Water content in extracted oils was 0.16±0.09% (w/w). Iodine value varied from 93 to 164 g I2 100g-1. PUFA content was from 18.7 to 33.2% (w/w).
Acid and peroxide values varied from 0.6 - 26.4 mg KOH g-1 and from 3.2 to 21.6 meq O2 kg-1, respectively. The high AV and PV indicate degradation of some lipids.
Since to the high content of free fatty acids in obtained oils was observed, it was almost impossible (without
significant loss in product yield) to transesterify these oils to FAEE (or FAME) by means of alkaline catalysis.
Oils from the fish wastes were combined and the resulting mixture was used at the next stage of investigation.
Characteristics of the combined oil: iodine value -130 g I2 100g-1, acid value - 14.9 mg KOH g-1 (7.95% (w/w) of free fatty acids) and peroxide values - 14.7 meq O2 kg-1. The fatty acid composition of the combined oil is shown in Table 1. Weighted average molar mass of the oil was calculated according to FA composition and was as high as 941 gmol-1.
Table 1. FA content of the combined fish oil
Fatty acid
С14:0
С16:0
С16:1
С18:0
С18:1
С18:2
С18:3
С20:1
С20:2
С20:5
С22:1
С22:6
Another components
Content, % w/w
8.2
13.69
4.99
1.87
15.5
1.73
5.92
11.17
0.88
6.9
16.23
3.94
Enzymatic ethanolysis of fish oil. Enzymatic transesterification of oils has many advantages: low reaction temperature, low sensitivity to free fatty acids, glycerol obtained during this process is without alkaline or acidic impurities, and immobilized enzyme is easily separated from the reaction mixture. Consequently, the use of enzymatic transesterification in industrial scale allows to reduce the number of process steps and to reduce the requirements for the process equipment. The negative aspect of the enzymatic process is long time reaction.
Effect of temperature. The effect of temperature on the transesterification of fish waste oil was determined. Several trials of ethanolysis with an enzyme loading of 5% (w/ w) were conducted at temperatures of 25, 35, 50 and 60 °C. Molar ratio of ethanol to oil 3:1 was chosen to minimize inactivating effect.
The conversions obtained in reactions catalyzed by lipase increased with increasing reaction temperature in the range of 25-60 °C (Figure. 1).
Figure 1. Time courses for the fatty acid ethyl esters production at different temperatures. Molar ratio ethanol/oil = 3/1; enzyme amount 5 % (w/w).
One aspect of the ecological technology is to minimize the energy consumption. Obviously, biocatalytic processes can occur at temperatures that can be achieved in several regions of the world without any kind of additional heating.
Temperature 25 °C was selected for subsequent experiments not only for economic reasons, but also to trigger an inactivating effect of the acyl acceptor on the lipase (which should increase with higher temperatures).
Effect of ethanol:oil molar ratio. Ethanolysis reactions of fish waste oil with different molar ratios of ethanol to oil (3, 6 and 9) were studied in the presence of 5 % (w/w) enzyme with respect to the oil. After 3 h reaction, enzyme displayed the amount of oil conversion of 11.1%, 15.3% and 15.8% (w/w) for molar ratios of ethanol to oil 3, 6 and 9, respectively. It is obvious that an increase in the conversion is insignificant while molar ratio increases from 6 to 9. The etha-nol seems to produce a positive effect on the reaction kinetics by facilitating formation of a homogeneous suspension of the reactants and the biocatalyst.
Effect of enzyme loading. Several trials were conducted to find out the effect of loading of immobilized lipase on the production of FAEE in a batch reactor. Plots of conversion (after 0.5; 1 and 1.5 hours) versus loading of Novozym® 435 for the ethanolysis of fish waste oil are shown in Figure 2a. Evidently, there is linear dependence between enzyme loading and yield of FAEE obtained during the process. Figure 2b represents time periods for the FAEE production with four different amounts of enzyme loading.
Figure 2. Effect of enzyme loading(a), and time courses for the fatty acid ethyl esters production with different amount of enzyme loading (b). Temperature = 25 °C; molar ratio ethanol/oil = 9/1. Enzyme amount, % w/w: 1-5 2 - 10 3 - 20 4 - 50 %.
Trials involving 50 % (w/w) load of Novozym® 435 and a molar ratio of ethanol to oil 9:1 demonstrated the highest (nearly quantitative) yields of FAEE after 7 hours - 98 %. And 20 % (w/w) enzyme load was sufficient to produced 97.5 % of FAEE after 24 hours of process.
Effect of inert atmosphere. Since target component of fish waste oils - omega-3 PUFAs is very sensitive to oxidation, it is necessary to prevent oxidation processes at all stages of processing of raw materials to the final product. This also applies to the transesterification stage.
Ethanolysis of fish oil with 20% (w/w) enzyme load, 9:1 molar ratio of ethanol to oil was carried out at 25 °C temperature in different environment - air and argon. Peroxide values of FAEEs obtained under different atmospheres were determined - 15.1 meq O2 kg-1 and 18.1 meq O2 kg-1, respectively to argon and air atmospheres.
Urea fractionation of FAEE. Urea complexation is a classical way in which to concentrate polyunsaturated fatty acids. Fatty acids with shorter chain lengths or containing double bonds are less likely to form urea complexes than those containing longer chain lengths of saturated fatty acids [13].
It is well known that urea binds fatty acids and their derivatives in weight to weight ratio of approximately 2.73.0:1. Multi-step complexation was carried out 9 times with urea:FAEE weight ratio 0.3:1, total ratio was 2.7:1. Distribution of FA between fractions was determined (figure 3).
Figure 3. Distribution of FA between fractions: urea complex fractions are from 1 to 9; non-urea complex fraction -10. (a) - saturated acids; (b) - mono- and diunsaturated acids; (c) - polyunsaturated acids.
Table 2. Fatty acid content in non-urea complex fraction
Fatty acid Content, % w/w
С14:0 0
С16:0 0.09
С16:1 0.93
С18:0 0
С18:1 3.51
С18:2 1.00
С18:3 22.28
С20:1 0.23
С20:2 3.90
С20:5 21.23
С22:1 0.01
С22:6 33.42
Another components 13.4
The highest content (% w/w) of the following acids was in the fractions: C18:0 - 1, C16:0 - 2, C14:0 - 2 and 3, C22:1 - 5, C20:1 - 6, C18:1 - 6 and 7, C16:1 - 7, C18:2 - 8, C20:2 - 9, C22:6 and C20:5 and C18:3 - 10 (non-urea complexed fraction). Consequently, this fractionation method may allow to separate individual fatty acids and their esters, if the numbers of steps were increased. Despite this, the aim of this work was to obtain two fractions of FAEE: concentrate of PUFA ethyl esters and PUFA-depleted biodiesel (Table 2 and Table 3, respectively). Yield of non-urea complexed fraction was 22.2% (w/w).
Table 3. Fatty acid content in urea complex fraction
Fatty acid Content, % w/w
С14:0 10.55
С16:0 17.6
С16:1 6.15
С18:0 2.4
С18:1 18.92
С18:2 1.94
С18:3 1.25
С20:1 18.92
С20:2 1.94
С20:5 2.8
С22:1 14.3
С22:6 1.99
Another components 1.26
Conclusions
The use of Novozym® 435 allowed obtaining FAEE from waste fish oil with a near quantitative yield at low temperature in 24 hours. An inert atmosphere of the process was determined to be is necessary since the desired product is susceptible to oxidation. Multi-step urea fractionation yielded the concentrate of PUFA containing minimum amount of saturated and monounsaturated FA.
Combination of enzymatic ethanolysis and urea fractionation had demonstrated the ability to create technologies for the processing of fish waste oils with minimal requirements of equipment and energy consumption.
Acknowledgments
The authors thank Ministry of Education and Science of the Republic of Kazakhstan for the support of research.
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