Uptake rate, productivity (Y) and ethanol mole produced per glucose mole for
Abstract
Fermentation technologies have been developed to improve the production of ethanol and an alternative is the immobilization technology, which offers the possibility of efficiently incorporating symbiotic bacteria in the same matrix. This study analyzes the potential use of immobilized and coinmobilized systems on beads of calcium alginate for ethanol production used mango waste (Mangifera indica) by Zymomonas mobilis and Saccharomyces cerevisiae compared with free cells culture and evaluate the effect of glucose concentration on productivity in coimmobilized system using a Chemostat reactor Ommi Culture Plus. For free cell culture, the productivity was higher for Z. mobilis (5.76 g L-1 h-1) than for S. cerevisiae (5.29 g L-1 h-1); while in coimmobilized culture, a higher productivity was obtained (8.80 g L-1 h-1) with respect to immobilized cultures (8.45 g L-1 h-1 - 8.70 g L-1 h-1). The conversion of glucose to ethanol for coimmobilized system was higher (6.91 mol ethanol) with 50 g L-1 of glucose compared to 200 g L-1 of glucose (5.82 mol ethanol); suggesting the immobilized and coimmobilized cultures compared with free cells offer an opportunity for the reuse of organic residues and high alcohol production.
Keywords
- Mangifera indica
- immobilization
- coimmobilization
- ethanol
- Zymomonas mobilis
- Saccharomyces cerevisiae
1. Introduction
1.1. Use of agroindustrial waste in fermentation processes
Alcoholic fermentation is a process by which microorganisms convert hexoses, mainly glucose, fructose, mannose and galactose, in the absence of oxygen and get products as alcohol (ethanol), carbon dioxide and adenosine triphosphate (ATP) molecules. Approximately 70% of the energy is released as heat and the remainder is preserved in two terminal phosphate bonds of ATP, for use in transfer reactions, such as activation of glucose (phosphorylation) and amino acids before of the polymerization. In other words, fermentation is a set of chemical reactions carried out by microorganisms in which an organic compound is oxidized, partially in the absence of oxygen to obtain chemical energy and understood as a partial oxidation when all the carbon atoms of the compound are oxidized to form CO2. It is a process known since antiquity and is currently the only industrial process for the preparation of ethyl alcohol in all countries. The glucose as raw material is not only used, but other types of raw material much cheaper. However, the process of alcoholic fermentation occurs naturally, originated by the activity of some microorganisms through its anaerobic energy cell metabolism; for a large‐scale production process, it is necessary for microorganisms (bacteria, fungi and yeasts) to accelerate the process of alcoholic fermentation and increase the conversion rate [1]. During the twentieth century and until the beginning of the twenty‐first century, alcoholic fermentation has focused exclusively on the improvement of fermentation processes and specifically on the optimization of industrial performance through a good selection of yeast strains, which are the most used microorganisms for the production of ethanol by fermentation, due to its high productivity in the conversion of sugars and better separation of the biomass after fermentation. Yeasts are unicellular (usually spherical) microorganisms of size 2–4 μm and are present naturally in some products such as fruits, cereals and vegetables. Different species of fermentative microorganisms have been identified, among which are mainly
The alcoholic fermentation processes using agroindustrial products present a great challenge given the inconveniences that could arise when using raw material for human consumption or edible vegetable crops for the production of ethanol, and, on the other hand, the change in the use of land destined for the cultivation of vegetables that will be used to produce ethanol and bioethanol, which would sometimes lead to deforestation, food shortages, increase of desert regions and greater inability of soils to retain water, thus disrupting the balance of the hydrological cycle [4].
On a global scale, the use of energy raw materials for energy purposes and in the production of ethanol has led to higher prices for products such as maize or barley, as well as making ethanol production economically unviable. Therefore, it is important to use raw materials that do not compete with food products and that are low cost in the production of biofuels, must also ensure a good profitability and are environmentally sustainable projects. In the energy sector, it has been estimated that the use of all world food surpluses could only produce bioethanol to replace 1% of the oil currently used. Concluding that if food crops were used to produce ethanol, a chain of food imbalances would be generated, which would be unsustainable [5].
An alternative to producing ethanol is through the use of other nontraditional raw materials, which arise as by‐products and/or waste from industrial processes. Propose new technologies that allow the production of ethanol from cane residues, solid waste and those materials containing cellulose and hemicellulose, which allows the revalorization of waste from various industries, converting them into raw material for the production of ethanol.
At present, efforts have been made mainly in the search for cheap raw materials, which replace the traditional ones, in order to achieve greater efficiency in the processes of fermentation, recovery and purification of alcohol produced. The importance of the production of bioethanol has as main interest to compete with the use of fossil fuels since ethanol can be used as fuel for motor vehicles increasing the octane number, and therefore the reduction of consumption and contaminants (10–15% less carbon monoxide and hydrocarbons). Ethanol can be mixed with unleaded gasoline from 10 to 25% without difficulty, although some engines have been able to incorporate 100% alcohol as fuel. Thus, ethanol could substitute for methyl tert‐butyl ether (MTBE), an oxygenation product with which gasolines have been reformulated in Mexico since 1989, which has reduced CO2 emissions. This action is very important since MTBE, being a very stable compound, with low degradation and very soluble in water.
The production of bioethanol lost importance at the end of the first half of the XX century, being replaced by the production of synthetic ethanol, from petroleum derivatives, which is cheaper, but cannot be used in food preparation, alcoholic beverages or medications. The rise in oil prices turned our eyes toward the fermentation route of ethanol production, and today, we work mainly in the search for cheap raw materials, replacing the traditional sugary materials. Studies carried out by different researchers suggest that the by‐products of mango juice, cane juice and molasses are an efficient alternative for the production of ethanol without affecting the food item, besides increasing the productivity and concentration of ethanol in the fermentation medium, and therefore reduce the costs of ethanol production [6].
Historically, the sugar industry in Mexico is one of the most important, characterized by sugarcane harvests throughout the year, with a production of cane of 46,231,229 tons per year, and the remaining residue derived of sugarcane has been exploited as energy biomass and for the production of different biotechnological products by fermentation. Other alternatives of raw material are mango juice and its residues, and the fruit is grown in all the countries of Latin America, Mexico being the main exporting country of this fruit, with an annual production of approximately 1 million 452 thousand tons of mango and of which more than 60% of this production is given to the South‐Southeast region of our country.
The alternative of using residues or products that can replace the raw materials normally used in ethanol production is now a highly promising possibility, because the cost of production of ethanol is closely related and dependent on the cost of the raw material, the volume and the composition of the same. The existing economy in Mexico related to cane cultivation (experience and sugar tradition) and the export of mango types offers technological alternatives that allow the fermentation of cane juice, molasses and mango juice through
1.2. Tecnología de inmovilización
Research has been developed in order to increase the productivity of alcoholic fermentation processes. The productivity, expressed as grams of ethanol produced per hour per unit of fermentation volume, can be increased by optimizing the composition of the culture medium, by the selection of an appropriate microorganism strain or through the adaptation of the design of reactors [7]. One challenge today is to reduce ethanol production costs, and an alternative is to reduce the cost of the culture media, which can represent about 30% of the final production costs of ethanol [8].
Some fermentation technologies have been developed to improve the production of ethanol and its concentration in the culture media [9, 10]. Among these, the immobilization technology offers advantages in contrast to free cell cultures, such as increased retention time in bioreactors, high cellular metabolic activity, high cell load and protection for cells from stress [10, 11]. The immobilization cell technologies have been applied for different purposes as for the production of hydrogen [12] and compounds commercially used in the food industry [13]. Other studies have been developed with immobilized algal cells to remove nutrients (N and P) from wastewater, phenol and hexavalent chromium [14–17]. Similarly, the immobilization of
On the other hand, the immobilization technology provides the possibility of efficiently incorporating symbiotic bacteria [21, 22]. The interaction between two microorganisms in the same matrix is called coimmobilization, and this association can be positive with higher growth and production. However, there are relatively less applications in the ethanol production involving the immobilization of mixed‐culture systems and/or coimmobilized cultures.
In a petroleum deficiency situation, bioethanol from yeast and bacterial fermentation has become a promising alternative source for fuel. Agricultural and industrial waste containing sugar, starch and cellulose, such as cassava peels, fruit bunches, and the effluents from sugar and pineapple cannery productions have been successfully applied for the bioethanol production [23, 24]. In this context, the municipality of Ciudad del Carmen, Campeche, Mexico, has an annual production of about 2.868 ha mango (
This study was to determine whether the association between
2. Materials and methods
2.1. Microorganism and medium
The yeast strain
2.2. Preparation of immobilized and coimmobilized cells
For the preparation of immobilized cells, we used the technique described by Tam and Wong [26]. Both microorganisms were harvested by centrifugation at 3500 rpm for 10 min. The bacteria and yeast cells were resuspended in 50 mL of distilled water to form a concentrated cell suspension. The suspension was then mixed with a 4% sodium alginate solution in 1:1 volume ratio to obtain a mixture of 2% microorganism–alginate suspension. The mixture was transferred to a 50‐mL burette, and drops were formed when “titrated” into a calcium chloride solution (2%). This method produced approximately 6500 uniform algal beads of approximately 2.5 mm in diameter with biomass content for
The beads were kept for hardening in the CaCl2 solution for 4 h at 25 ± 2°C and then rinsed with sterile saline solution (0.85% NaCl) and subsequently with distilled water. A concentration of 2.6 beads mL−1 of medium (equivalent to 1:25 bead: medium v/v) was placed in a Chemostat Ommi Culture Plus (Virtis) containing 2 L of culture medium. The reactor was maintained under stirring at 120 rpm and 30°C (Figure 2). A similar procedure was used for coimmobilization, with the difference that the concentrate of bacteria (25 mL) and yeast (25 mL) was mixed and then mixed with 50 mL of alginate; this procedure allowed retaining the same concentration of cells in all experiments.
2.3. Experimental setup and procedure
This study was divided into two parts: (1) the batch experiment consisted of evaluating the growth and ethanol production in a medium equivalent to mango juice in cultures with free cells, immobilized and coimmobilized, and (2) evaluate the effect of glucose concentration in the production of ethanol in the system previously selected in the first experimental part based on the ethanol productivity obtained. Fermentation was performed in a Chemostat Ommi Culture Plus (Virtis) with a volume of 2 L operation, adjusting stirring at 120 rpm and maintaining a temperature of 30°C. The medium equivalent to mango juice was similar to that described by Demirci et al. [25] by adjusting the composition of the medium to a concentration of 200 g L−1 glucose, equivalent to that observed in the mango juice (
The experimental design consisted of triplicate cultures in a Chemostat reactor Ommi Culture Plus for
Ethanol content (% v/v) was obtained using the Anton Paar DMA 4100M instrument, which determines the density of the mixture in relation to the standard OIML‐STD‐90, which can determine the content of distillate ethanol (% v/v); according to the ethanol density recorded, it was possible to obtain an approximate of ethanol content (grams of ethanol per liter of culture) produced for each experiment. Prior to the determination of the ethanol content, a distillation of cultures was conducted with a plate column distiller PS‐DA‐005/PE of four plates, at small scale. The cooling water flow was 3 L h−1 at 15°C. An aliquot of 3 L was distilled for 4 h, maintaining the operating conditions at atmospheric pressure, without reflux and with a temperature ramp in the heating jacket of 30°C up to 80°C.
The STATISTICA 7.0 software for statistical analysis and calculated mean and standard deviation for each treatment were used. The covariance analysis (ANCOVA) with
3. Results
3.1. Growth
In free cell cultures, the growth was observed immediately after being inoculated in the reactor of 2 L. Growth kinetics shows an exponential phase for
For immobilized cells, both yeast and bacteria presented immediate growth after adding the beads to the culture medium; in both treatments, the exponential phase of growth reached a maximum of 80 h. It is noteworthy that although both microorganisms were immobilized under the same procedure, the content of biomass per bead at the beginning of treatment was lower for
3.2. Glucose‐substrate removal
The decrease of substrate showed significant differences (
It is a fact that consumed substrate was greater in free culture for
The average consumption analysis based on removal rates determined during the exponential growth for both species showed that free culture
Culture | Cells | Ethanol formed (% v/v) | Y (g L−1 h−1) | Glucose consumed (g L−1) | Uptake rate (g‐substrate removed per g‐biomass d) | |
---|---|---|---|---|---|---|
Free cells | 80.6 | 5.29a | 90 | 2.71a | 2.76 | |
87.7 | 5.76a | 80 | 2.0a | 3.38 | ||
Immobilized cells | 86.0 | 8.45b | 60 | 0.165b | 4.42 | |
87.7 | 8.70b | 43 | 0.056b | 6.29 | ||
Coimmobilized cells | 88.7 | 8.80c | 47 | 0.235c | 5.82 |
In cultures with immobilized cells, the removal rate in the exponential phase (80 h) was greater for
3.3. Effect of initial concentration of glucose on ethanol production
It is a fact that most cultures from fruits may contain a high concentration of fiber solids that cause problems of mixture in the reactor, and consequently a low contact between cells and substrate. Mango juice is no exception. In this study, we evaluated the growth and alcohol production of
For the coimmobilized of
The content of alcohol produced had no significant differences (
C0 | Ethanol formed (% v/v) | Y (g L−1 h−1) | Glucose consumed (g L−1) | Uptake rate (g‐substrate removed per g‐biomass d) | |
---|---|---|---|---|---|
50 | 89.63 | 8.85 | 40.0 | 0.08 | 6.91 |
200 | 88.70 | 8.80 | 47.0 | 0.235 | 5.82 |
4. Growth and productivity
To evaluate the capacity of growth in free and immobilized culture, two microorganisms, yeast and bacteria, were subjected to the same culture conditions (Figure 3A). In free culture, the yeast
Another factor that probably favors the rapid decline in cell density is attributed to the production of CO2 as result of fermentation activity. Studies suggest the adverse effect of CO2 gas, because if the diffusion of CO2 is lower compared to its production, it will accumulate inside of alginate bead [30]. In this study, the CO2 is observed in the reactor as bubbles attached on the surface of the beads, suggesting that the spread of CO2 gas in the first 80 h was not a factor that inhibited growth and the production of alcohol; however, after this time, gas saturation in the reactor was probably high, affecting the diffusion of CO2. This coupled with a limitation in the transport of nutrients and subsequent inhibition of microorganisms and may have caused glucose consumption to be lower compared to free cells (Table 1).
In particular, in free cell culture, the lower percentage of alcohol obtained by yeast during the increasing fermentation culture commonly relates to the fact that this is affected by the high concentration of ethanol in the solution, which may inhibit metabolism and decrease efficiency [31], unlike bacteria
It is evident that ethanol production was not inhibited in immobilized or coimmobilized systems, and even showed higher productivity with respect to free cells (Table 1), suggesting that they are more efficient in the conversion of sugar with respect to time. Krishnan et al. [19] reported lower productivity for
Interestingly, the immobilized systems showed a higher conversion of substrate of 4.42 and 6.29 mole of ethanol per mole of glucose for yeast and bacteria, respectively, compared to the obtained by free cells, from 2.7 to 3.3 mole of ethanol per mole of glucose. In general, treatments with immobilized cells showed a higher output of ethanol per mole of glucose with respect to that reported by Amin and Verachtert [9] for
According to the results, immobilization and coimmobilization exhibited a lower uptake rate compared to free cells; this shows that there was less consumption of substrate (Table 1). Nevertheless, there was greater productivity, which indicates that it is possible to obtain high alcohol content with a lower requirement of substrate, but with the disadvantage of residual glucose in the medium; this problem can be solved with sequenced systems, as suggested by Demirci et al. [25]. Another alternative of solving this problem is to increase the cell number or inoculum size within the reactor. This is reasonable because a high number of cells could create a greater sorption of substrate (glucose) into the cell and eventually consumed substrate. However, Siripattanakul‐Ratpukdi [10] suggested that with different cell yeast loads, the same reduction (>90%) of substrate is obtained at the end of a treatment period of 10 h.
The low glucose reduction observed in this study in alginate beads can be attributed to the decline in cell density, but it is likely that the diffusion of substrate could have been prevented. Studies have reported that the adsorption of substrate by the matrix was observed in the first hours of treatment, with a possible decrease of substrate diffusion within the matrix in a continuous process [34].
On the other hand, Robinson et al. [35] suggest that the diffusion rate within the alginate matrix depends on the concentration gradient between the culture medium and matrix; this is, when the nutrient concentration in the culture medium decreases, the diffusion rate occurs within the matrix and therefore the removal rate. In this study, during the first hours of treatment, the matrix is probable a partial saturation with substrate (glucose), because the substrate is decreased during the culture time, and cell growth for both microorganisms was continuous. Clearly, the immobilized cell system successfully decreased glucose by adsorption of the matrix (immobilized glucose) and biodegradation (bioconversion of glucose), being the main process the biodegradation. This suggests that the main factor that could limit glucose removal may have been the high concentration of CO2 in the reactor.
4.1. Coimmobilization of Z. mobilis and S. cerevisiae at different glucose concentrations
The biomass content in alginate beads shows that a high concentration of glucose (200 g L−1) leads to a rapid decrease compared to cultures with a low glucose concentration (50 g L−1). This confirms the fact that the high concentration of glucose saturates beads faster, reducing the diffusion between beads and the culture medium; consequently, the diffusion of CO2 produced can be reduced and remains trapped inside the bead, causing a decrease in growth and substrate consumption.
Conversely, the low concentration of substrate of the culture medium indicates the presence of a soft transport and substrate accumulation within the matrix, allowing a proper consumption and growth of bacteria and yeast. Therefore, a low concentration of substrate may actually increase the production of alcohol with minimal residual glucose, reaching values of 6.91 mol of ethanol per mole of glucose, with respect to a high glucose concentration (Table 2).
Previous studies in our laboratories have shown that the fermentation process of mango juice for a coimmobilized system can produce a production ratio of 1.4 L of alcohol (79% v/v ethanol) for every 3 L of mango juice.
5. Conclusions
The present study has shown the existing potential of using coimmobilized systems in the production of ethanol. The association of
In general, the immobilization technology offers an alternative by increasing productivity and conversion of substrate compared to culture systems with free cells. In the present study, the immobilized systems showed high conversion capacity to obtain high alcohol content with a lower requirement of substrate.
The possible substrate inhibition was not a factor affecting cell growth in both organisms; it is clear that the immobilized cell system successfully reduced glucose by the matrix adsorption (immobilized glucose) and biodegradation (bioconversion of glucose), being biodegradation the main process. This suggests that the main factor that could limit further growth was the high concentration of CO2 in the reactor. Furthermore, although no significant differences were detected in the alcohol content in immobilized culture in diluted medium, the conversion from glucose to ethanol is greater in those media with a glucose concentration of 50 g L−1. For practical purposes, it is desirable that the fermentation of waste organic be performed through dilutions to increase the homogeneity of alginate beads within the reactor and consequently allow the diffusion of CO2 and substrate through the beads.
References
- 1.
Mathews KC, Van Holde KE, Kevin GA. Biochemistry, San Francisco, California, editorial Addison Wesley, 3er edition, 2002: P.1368 - 2.
Beltran G, Torija MJ, Novo M, Ferrer N, Poblet M, Guillamon JM, Rozes N, Mas A. Analysis of yeast populations during alcoholic fermentation: A six year follow‐up study. Systematic and Applied Microbiology. 2002;25(2):287-293 - 3.
Doelle HW, Kirk L, Crittenden R, Toh H, Doelle MB. Zymomonas mobilis —science and industrial application. Critical Reviews in Biotechnology. 1993;13(1):57-98 - 4.
Sheehan J, Himmel M. Enzymes, energy, and the environment: A strategic perspective on the U.S. Department of Energy’s research and development activities for bioethanol. Biotechnology Progress. 1999;15(5):817-827 - 5.
Christen P, Minier M, Renon H. Ethanol extraction by supported liquid membrane fermentation. Biotechnology & Bioengineering. 1990;36:116-123 - 6.
Mejía‐Giraldo LF, Martínez‐Correa HA, Betancourt‐Gutiérrez JE, Castrillón Castaño CE. Usage of the common mango agroindustrial waste ( Mangifera indica L. ) in the destruction of fermentable sugars. Ingeniería y Ciencia. 2007;3(6):41-62 - 7.
Amin GV, Eynde D, Verachtert E. Determination of by‐products formed during the ethanolic fermentation, using batch and immobilized cell systems of Zymomonas mobilis andSaccharomyces bayanus . European Journal of Applied Microbiology and Biotechnology. 1983;18:1-5 - 8.
Sivers MV, Zacchi G, Olsson L, Hahn‐Hagerdal B. Cost analysis of ethanol production from willow using recombinant Escherichia coli . Biotechnology Progress. 1994;10:555-560 - 9.
Amin G, Verachtert H. Comparative study of ethanol production by immobilized‐cell systems using Zymomonas mobilis orSaccharomyces bayanus . European Journal of Applied Microbiology and Biotechnology. 1982;14:59-63 - 10.
Siripattanakul‐Ratpukdi S. Ethanol production potential from fermented rice noodle wastewater treatment using entrapped yeast cell sequencing batch reactor. Applied Water Science. 2012. DOI: 10.1007/s13201‐011‐0024‐z - 11.
Tam NFY, Lau PS, Wong YS. Wastewater inorganic N and P removal by immobilized Chlorella vulgaris . Water Science & Technology. 1994;30:369-374 - 12.
Wu SY, Lin CN, Chang JS. Hydrogen production with immobilized sewage sludge in three‐phase fluidized‐bed bioreactors. Biotechnology Progress. 2003;19:828-832 - 13.
Kawaguti HY, Manrich E, Sato HH. Production of isomaltulose using Erwinia sp . D12 cells: Culture medium optimization and cell immobilization in alginate. Biochemical Engineering Journal. 2006;29:270-277 - 14.
Bandhyopadhyay K, Das D, Bhattacharyya P, Maiti BR. Reaction engineering studies on biodegradation of phenol by Pseudomonas putida MTCC 1194 immobilized on calcium alginate. Biochemical Engineering Journal. 2001;8:179-186 - 15.
Park DH, Chab JM, Ryua HW, Lee GW, Yu EY, Rheea JI, Park JJ, Kimd SW, Joe YI, Ryu YW, Hur BK, Park JK, Park K. Hydrogen sulfide removal utilizing immobilized Thiobacillus sp . IW with Ca‐alginate bead. Biochemical Engineering Journal. 2002;11:167-173 - 16.
Humphries AC, Nott KP, Hall LD, Macaskie LE. Reduction of Cr (VI) by immobilized cells of Desulfovibrio vulgaris NCIMB 8303 andMicrobacterium sp . NCIMB 13776. Biotechnology & Bioengineering. 2005;90(5):597-605 - 17.
Ruiz‐Marin A, Mendoza‐Espinosa GL, Stephenson T. Growth and nutrient removal in free and immobilized green algae in batch and semi‐continuous cultures treating real wastewater. Bioresource Technology. 2010;101:58-59 - 18.
Vallejo‐Becerra V, Vazquez‐Bahena JM, Santiago‐Hernández JA, Hidalgo‐Lara ME. Immobilization of the recombinant invertase INVB from Zymomonas mobilis on nylon‐6. Journal of Industrial Microbiology and Biotechnology. 2008;35:1289-1295 - 19.
Krishnan MS, Blanco M, Shattuck KC, Nghiem PN, Davison BH. Ethanol production from glucose and xylose by immobilized Zymomonas mobilis CP4 (pZB5). Applied Biochemistry and Biotechnology. 2000;84-86 :525-541 - 20.
Yu J, Yue G, Zhong J, Zhang X, Tan T. Immobilization of Saccharomyces cerevisiae to modified bagasse for ethanol production. Renewable Energy. 2010;35:1130-1134 - 21.
Travieso L, Benitez F, Weiland P, Sánchez E, Dupeyrón R, Dominguez AR. Experiments on immobilization of microalgae for nutrient removal in wastewater treatments. Bioresource Technology. 1996;55:181-186 - 22.
De‐Bashan LE, Moreno M, Hernández JP, Bashan Y. Removal of ammonium and phosphorus ions from synthetic wastewater by the microalgae Chlorella vulgaris coimmobilized in alginate beads with the microalgae growth promoting bacteriumAzospirillum brasilense . Water Research. 2002;36:2941-2948 - 23.
Nigan JN. Continuous ethanol production from pineapple cannery waste using immobilized yeast cells. Journal of Biotechnology. 2000;80:189-193 - 24.
Kassim MA, Kheang LS, Bakar NA, Aziz AA, Som RM. Bioethanol production from enzymatically saccharified empty fruit bunches hydrolysate using Saccharomyces cerevisiae . Research Journal of Environmental Sciences. 2011;5:573-586 - 25.
Demirci A, Pometto AL, Ho KLG. Ethanol production by Saccharomyces cerevisiae in biofilm reactors. Journal of Industrial Microbiology & Biotechnology. 1997;19:299-304 - 26.
Tam NFY, Wong YS. Effect of immobilized microalgal bead concentrations on wastewater nutrient removal. Environmental Pollution. 2000;107(1):145-151 - 27.
Cohen Y. Biofiltration—the treatment of fluids by microorganisms immobilized into the filter bedding material. Bioresource Technology. 2000;77:257-274 - 28.
Mallick N. Biotechnological potential of immobilized algae for wastewater N, P and metal removal: A review. BioMetals. 2002;15:377-390 - 29.
Uemoto H, Saiki H. Distribution of Nitrosomonas europaea andParacoccus denitrificans immobilized in tubular polymeric gel for nitrogen removal. Applied and Environmental Microbiology. 2000;66(2):816-819 - 30.
Kim BG, Choi CY. A study on the ethanol production by immobilized cells of Zymomonas mobilis . Korean Journal of Chemical Engineering. 1984;1(1):13-19 - 31.
Nuwamanya E, Chiwona‐Karltun L, Robert SK, Yona B. Bio‐ethanol production from non‐food parts of Cassava ( Manihot esculenta Crantz). Ambio. 2011;41(3):262-270 - 32.
Gunasekaran P, Karunakaran T, Kasthuribai M. Fermentation pattern of Zymomonas mobilis strains on different substrate: A comparative study. Journal of Biosciences. 1986;10(2):181-186 - 33.
Rogers PL, Jon JL, Tribe DE. Kinetics of alcohol production by Zymomonas mobilis at high sugar concentration. Biotechnology Letters. 1979;1:165 - 34.
Siripattanakul S, Khan E. Fundamentals and applications of entrapped cell bioaugmentation for contaminant removal. In: Shah V, editor. Emerging Environmental Technologies. Vol. 2. Berlin: Springer; 2010. pp. 147-169 - 35.
Robinson PK, Reeve JO, Goulding KH. Phosphorus uptake kinetics of immobilized Chlorella in bath and continuous‐flow culture. Enzyme and Microbial Technology. 1989;11:590-596