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  • 1. Alinezhad, S.
    et al.
    Mirabdollah, A.
    Forgács, Gergely
    University of Borås, School of Engineering.
    Feuk-Lagerstedt, Elisabeth
    University of Borås, School of Engineering.
    Sárvári Horváth, Ilona
    University of Borås, School of Engineering.
    Expression of keratinase gene in Bacillus megaterium using an expression vector of pHIS1525.SPlipA and utilization of the resulting recombinant strain for chicken feather degradation prior to biogas production2009Conference paper (Other academic)
    Abstract [en]

    An increasing quantity of chickens is being utilized annually in the poultry industry, producing a huge volume of chicken feather waste which presents a high quality supply of keratin. Keratinases possessing high level of keratinolytic activity on insoluble keratin play a crucial role in hydrolyzing chicken feathers. Ever since the discovery of proteolytic ability as well as water solubility of keratinase, many industrial processes regarding keratinase application have been developed. A recently invented application to handle poultry waste is to utilize feathers for biogas production. Obviously, large amount of keratinase is required to break down the keratin prior to further conversion to biogas. Previously, several researches have shown that certain bacteria are able to produce keratinase but it is still a challenge to find out which bacteria is the most reliable source for the production with high efficiency. These challenges gave rise to the molecular biologists to bring the focus on gene cloning to develop recombinant strains resulting in overproduction of keratinase. Over the course of various cloning and expression experiments of similar proteins, it was found that Bacillus megaterium could be a susceptible host cell for keratinase production. In our study, the keratinase gene from the chromosomal DNA of Bacillus licheniformis ATCC®53757 was PCR amplified and subsequently cloned into Bacillus megaterium expression vector, pHIS1525.SPlipA. Bacillus megaterium ATCC®14945 strain was transformed with the recombinant plasmid, pKERHIS1525.SPlipA. The KER gene was expressed under xylose inducible promoter, and the product was then purified using Ni-NTA affinity chromatography. After 18 h of incubation an extracellular keratinase activity of 29U ml-1 was achieved (one unit of activity was determined as the amount of enzyme required to an increase of 0.01 in A420 after 30 min of incubation at 37°C). The recombinant strain was further examined for feather degradation using intact chicken feather waste as carbon source. The chopped chicken feathers were partially degraded by the recombinant strain after three days of incubation and the total macroscopic digestion was ultimately observed after seven days resulting in a yellowish peptide rich fermentation broth. The biogas potential of the hydrolysate will be compared with that of untreated feathers by performing anaerobic batch digestion experiments.

  • 2.
    Forgacs, G.
    et al.
    University of Borås, School of Engineering.
    Lundin, M.
    University of Borås, School of Engineering.
    Taherzadeh, M.J.
    University of Borås, School of Engineering.
    Sárvári Horváth, Ilona
    University of Borås, School of Engineering.
    Pretreatment of chicken feather waste for improved biogas production2013In: Applied Biochemistry and Biotechnology, ISSN 0273-2289, E-ISSN 1559-0291, Vol. 169, no 7, p. 2016-2028Article in journal (Refereed)
    Abstract [en]

    This study deals with the utilization of chicken feather waste as a substrate for anaerobic digestion and improving biogas production by degradation of the compact structure of the feather keratin. In order to increase the digestibility of the feather, different pretreatments were investigated, including thermal pretreatment at 120 °C for 10 min, enzymatic hydrolysis with an alkaline endopeptidase [0.53–2.66 mL/g volatile solids (VS) feathers] for 0, 2, or 24 h at 55 °C, as well as a combination of these pretreatments. The effects of the treatments were then evaluated by anaerobic batch digestion assays at 55 °C. The enzymatic pretreatment increased the methane yield to 0.40 Nm3/kg VSadded, which is 122 % improvement compared to the yield of the untreated feathers. The other treatment conditions were less effective, increasing the methane yield by 11–50 %. The long-term effects of anaerobic digestion of feathers were examined by co-digestion of the feather with organic fraction of municipal solid waste performed with and without the addition of enzyme. When enzyme was added together with the feed, CH4 yield of 0.485 Nm3/kg VS−1 d−1 was achieved together with a stable reactor performance, while in the control reactor, a decrease in methane production, together with accumulation of undegraded feather, was observed.

  • 3.
    Forgács, Gergely
    University of Borås, School of Engineering.
    Biogas Production from Citrus Wastes and Chicken Feather: Pretreatment and Co-digestion2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Anaerobic digestion is a sustainable and economically feasible waste management technology, which lowers the emission of greenhouse gases (GHGs), decreases the soil and water pollution, and reduces the dependence on fossil fuels. The present thesis investigates the anaerobic digestion of waste from food-processing industries, including citrus wastes (CWs) from juice processing and chicken feather from poultry slaughterhouses. Juice processing industries generate 15–25 million tons of citrus wastes every year. Utilization of CWs is not yet resolved, since drying or incineration processes are costly, due to the high moisture content; and biological processes are hindered by its peel oil content, primarily the D-limonene. Anaerobic digestion of untreated CWs consequently results in process failure because of the inhibiting effect of the produced and accumulated VFAs. The current thesis involves the development of a steam explosion pretreatment step. The methane yield increased by 426 % to 0.537 Nm3/kg VS by employing the steam explosion treatment at 150 °C for 20 min, which opened up the compact structure of the CWs and removed 94 % of the D-limonene. The developed process enables a production of 104 m3 methane and 8.4 L limonene from one ton of fresh CWs. Poultry slaughterhouses generate a significant amount of feather every year. Feathers are basically composed of keratin, an extremely strong and resistible structural protein. Methane yield from feather is low, around 0.18 Nm3/kg VS, which corresponds to only one third of the theoretical yield. In the present study, chemical, enzymatic and biological pretreatment methods were investigated to improve the biogas yield of feather waste. Chemical pretreatment with Ca(OH)2 under relatively mild conditions (0.1 g Ca(OH)2/g TSfeather, 100 °C, 30 min) improved the methane yield to 0.40 Nm3/kg VS, corresponding to 80 % of the theoretical yield. However, prior to digestion, the calcium needs to be removed. Enzymatic pretreatment with an alkaline endopeptidase, Savinase®, also increased the methane yield up to 0.40 Nm3/kg VS. Direct enzyme addition to the digester was tested and proved successful, making this process economically more feasible, since no additional pretreatment step is needed. For biological pretreatment, a recombinant Bacillus megaterium strain holding a high keratinase activity was developed. The new strain was able to degrade the feather keratin which resulted in an increase in the methane yield by 122 % during the following anaerobic digestion.

  • 4.
    Forgács, Gergely
    et al.
    University of Borås, School of Engineering.
    Alinezhad, Saeid
    Mirabdollah, Amir
    Feuk-Legerstedt, Elisabeth
    University of Borås, School of Engineering.
    Sávári Horváth, Ilona
    University of Borås, School of Engineering.
    Biological treatment of chicken feather waste for improved biogas production2011In: Journal of Environmental Sciences(China), ISSN 1001-0742, E-ISSN 1878-7320, Vol. 23, no 10, p. 1747-1753Article in journal (Refereed)
    Abstract [en]

    A two-stage system was developed which combines the biological degradation of keratin-rich waste with the production of biogas. Chicken feather waste was treated biologically with a recombinant Bacillus megaterium strain showing keratinase activity prior to biogas production. Chopped, autoclaved chicken feathers (4%, W/V) were completely degraded, resulting in a yellowish fermentation broth with a level of 0.51 mg/mL soluble proteins after 8 days of cultivation of the recombinant strain. During the subsequent anaerobic batch digestion experiments, methane production of 0.35 Nm3/kg dry feathers (i.e., 0.4 Nm3/kg volatile solids of feathers), corresponding to 80% of the theoretical value on proteins, was achieved from the feather hydrolyzates, independently of the pre-hydrolysis time period of 1, 2 or 8 days. Cultivation with a native keratinase producing strain, Bacillus licheniformis resulted in only 0.25 mg/mL soluble proteins in the feather hydrolyzate, which then was digested achieving a maximum accumulated methane production of 0.31 Nm3/kg dry feathers. Feather hydrolyzates treated with the wild type B. megaterium produced 0.21 Nm3 CH4/kg dry feathers as maximum yield.

  • 5.
    Forgács, Gergely
    et al.
    University of Borås, School of Engineering.
    Niklasson, Claes
    Sárvári Horváth, Ilona
    University of Borås, School of Engineering.
    J. Taherzadeh, Mohammad
    University of Borås, School of Engineering.
    Methane production from feather waste pretreated with Ca(OH)2: Process development and economical analysis2014In: Waste and Biomass Valorization, ISSN 1877-2641, E-ISSN 1877-265X, Vol. 5, no 1, p. 65-73Article in journal (Refereed)
    Abstract [en]

    This study investigated the industrial application of feather waste as a substrate for anaerobic digestion. Feather was pretreated with 0–0.2 Ca(OH)2 g/g TSfeather (total solids of feathers) for 30–120 min at 100–120 °C, in order to increase the digestibility, and to enhance the methane yield in a subsequent digestion at 55 °C. Based on the results of the batch digestion, an industrial process was developed, which can achieve 0.40 Nm3/kg VSfeather (volatile solids of feathers) methane yield from the pretreated feathers, while it fulfills the animal by-product hygenization requirements as well. This base case of the industrial pretreatment process was designed using SuperPro Designer® for utilizing 2,500 tons of feathers per year, which is the waste stream from an average slaughterhouse with a capacity of 60,000 broilers per day. The production cost of the methane is estimated to be 0.475 EUR/Nm3, while the investments on the pretreatment unit requires 0.97 million EUR as total capital investment, and 0.25 million EUR/year for operating cost. However, the process is sensitive to the plant capacity. Changing the plant capacity from 625 to 10,000 tons of feather per year, results in reducing the biogas production cost from 1.177 to 0.203 EUR/Nm3. In addition, sensitivity analysis was performed on the base case to investigate the effect of the value of the incoming feather on the overall process profitability. The results showed that the proposed investment could be considered as being financially viable in the case of production of upgraded biomethane even without the current gate fee system.

  • 6.
    Forgács, Gergely
    et al.
    University of Borås, School of Engineering.
    Pourbafrani, Mohammad
    Niklasson, Claes
    Taherzadeh, Mohammad
    University of Borås, School of Engineering.
    Horváth Sárvari, Ilona
    University of Borås, School of Engineering.
    Methane production from citrus wastes: process development and cost estimation2011In: Journal of chemical technology and biotechnology (1986), ISSN 0268-2575, E-ISSN 1097-4660, Vol. 87, no 2, p. 250-255Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Because of its extreme toxicity for microorganisms, the limonene content of citrus wastes (CWs) has been a major obstacle to the conversion of CWs to biofuels. The main objective of this study was to develop a new process for the utilization of CWs that can be economically feasible when the supply of CW is low. RESULTS: Steam explosion pre-treatment was applied to improve the anaerobic digestibility of CWs, resulting in a decrease of initial limonene concentration by 94.3%. A methane potential of 0.537 ± 0.001 m3 kg−1 VS (volatile solids) was obtained during the following batch digestion of treated CWs, corresponding to an increase of 426% compared with that of the untreated samples. Long-term effects of the treatment were further investigated by a semi-continuous co-digestion process. A methane production of 0.555 ± 0.0159 m3 CH4 kg−1 VS day−1 was achieved when treated CWs (corresponding to 30% of the VS load) were co-digested with municipal solid waste. CONCLUSION: The process developed can easily be applied to an existing biogas plant. The equipment cost for this process is estimated to be one million USD when utilizing 10 000 tons CWs year−1. 8.4 L limonene and 107.4 m3 methane can be produced per ton of fresh citrus wastes in this manner.

  • 7. Kabir, Maryam M.
    et al.
    Forgács, Gergely
    Sárvári Horváth, Ilona
    University of Borås, Faculty of Textiles, Engineering and Business.
    Biogas from Lignocellulosic Materials2015In: Lignocellulose-Based Bioproducts, Switzerland: Springer, 2015, p. 207-251Chapter in book (Other academic)
  • 8.
    Kabir, Maryam M.
    et al.
    University of Borås, School of Engineering.
    Forgács, Gergely
    University of Borås, School of Engineering.
    Sárvári Horváth, Ilona
    University of Borås, School of Engineering.
    Pretreatment of wool based textile wastes for enhanced biogas production2012Conference paper (Other academic)
    Abstract [en]

    Two different wool based textile wastes (TW1 and TW2) have been subjected for biogas production. TW1 was composed of 70% wool and 30% polyamide (PA), while TW2 consisted of 70% wool, 18% PA and 12% kermel (protective polyamide-imide fibre). Two pre-treatments: thermal treatment, enzymatic treatment and combinations of these two were performed to enhance the methane yield. Determining the soluble protein concentrations in the treated samples showed that the additional thermal treatment and the enzyme concentration had significant positive effect on the degradation of wool. Samples treated with thermal and combination treatments were therefore selected for anaerobic batch digestion assays. The best results were obtained after combination treatments resulting in methane yields of 0.33-0.43 Nm3/kg VS, and 0.21-0.26 Nm3/kg VS, for TW1 and TW2, respectively, while only 0.21 and 0.05 Nm3/kg VS methane production was measured after the thermal treatment. The methane yields of untreated samples were close to zero.

  • 9.
    Kabir, Maryam M.
    et al.
    University of Borås, School of Engineering.
    Forgácsa, Gergely
    University of Borås, School of Engineering. Chalmers University of Technology.
    Sárvári Horváth, Ilona
    University of Borås, School of Engineering.
    Enhanced methane production from wool textile residues by thermal and enzymatic pretreatment2013In: Process Biochemistry, ISSN 1359-5113, E-ISSN 1873-3298, Vol. 48, no 4, p. 575-580Article in journal (Refereed)
    Abstract [en]

    Methane production from two types of wool textile wastes (TW1 and TW2) was investigated. To improve the digestibility of these textiles, different pretreatments were applied, and comprised thermal treatment (at 120 ◦C for 10 min), enzymatic hydrolysis (using an alkaline endopeptidase at different levels of enzymatic loading, at 55 ◦C for 0, 2, and 8 h), and a combination of these two treatments. Soluble protein concentration and sCOD (soluble chemical oxygen demand) were measured to evaluate the effectivity of the different pretreatment conditions to degrade wool keratin. The sCOD as well as the soluble protein content had increased in both textile samples in comparison to untreated samples, as a response to the different pretreatments indicating breakdown of the wool keratin structure. The combined treatments and the thermal treatments were further evaluated by anaerobic batch digestion assays at 55 ◦C. Combined thermal and enzymatic treatment of TW1 and TW2 resulted in methane productions of 0.43 N m3/kg VS and 0.27 N m3/kg VS, i.e., 20 and 10 times higher yields, respectively, than that gained from untreated samples. The application of thermal treatment by itself was less effective and resulted in increasing the methane production by 10-fold for TW1 and showing no significant improvement for TW2.

  • 10.
    Pourbafrani, Mohammad
    et al.
    University of Borås, Faculty of Textiles, Engineering and Business.
    Forgacs, Gergely
    University of Borås, Faculty of Textiles, Engineering and Business.
    Sárvári Horváth, Ilona
    University of Borås, Faculty of Textiles, Engineering and Business.
    Taherzadeh, Mohammad J.
    University of Borås, Faculty of Textiles, Engineering and Business.
    Framställning av mångahanda biprodukter från fasta citrusrester2011Patent (Other (popular science, discussion, etc.))
  • 11.
    Teghammar, A.
    et al.
    University of Borås, School of Engineering.
    Forgacs, G.
    University of Borås, School of Engineering.
    Sárvári Horváth, Ilona
    University of Borås, School of Engineering.
    Taherzadeh, M.J.
    University of Borås, School of Engineering.
    Techno-economic study of NMMO pretreatment and biogas production from forest residues2014In: Applied Energy, ISSN 0306-2619, E-ISSN 1872-9118, Vol. 116, p. 125-133Article in journal (Refereed)
    Abstract [en]

    Biogas is nowadays getting more attention as a means for converting wastes and lignocelluloses to green fuels for cars and electricity production. The process of biogas production from N-methylmorpholine oxide (NMMO) pretreated forest residues used in a co-digestion process was economically evaluated. The co-digestion occurs together with the organic fraction of municipal solid waste (OFMSW). The process simulated the milling of the lignocelluloses, NMMO pretreatment unit, washing and filtration of the feedstock, followed by an anaerobic co-digestion, upgrading of the biogas and de-watering of the digestate. The process also took into consideration the utilization of 100,000 DW (dried weight) tons of forest residues and 200,000 DW tons of OFMSW per year. It resulted in an internal rate of return (IRR) of 24.14% prior to taxes, which might be attractive economically. The cost of the chemical NMMO treatment was regarded as the most challenging operating cost, followed by the evaporation of the washing water. Sensitivity analysis was performed on different plant size capacities, treating and digesting between 25,000 and 400,000 DW tons forest residues per year. It shows that the minimum plant capacity of 50,000 DW tons forest residues per year is financially viable. Moreover, different co-digestion scenarios were evaluated. The co-digestion of forest residues together with sewage sludge instead of OFMSW, and the digestion of forest residues only were shown to be non-feasible solutions with too low IRR. Furthermore, biogas production from forest residues was compared with the energy produced during combustion.

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