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제지 슬러지로부터 유용 유기산 생산을 위한 복합생물공정개발
Reports NRF is supported by Research Projects( 제지 슬러지로부터 유용 유기산 생산을 위한 복합생물공정개발 | 2004 Year 신청요강 다운로드 PDF다운로드 | 이상목(인하대학교) ) data is submitted to the NRF Project Results
Researcher who has been awarded a research grant by Humanities and Social Studies Support Program of NRF has to submit an end product within 6 months(* depend on the form of business)
사업별 신청요강보기
  • Researchers have entered the information directly to the NRF of Korea research support system
Project Number D00015
Year(selected) 2004 Year
the present condition of Project 종료
State of proposition 재단승인
Completion Date 2006년 05월 03일
Year type 결과보고
Year(final report) 2006년
Research Summary
  • Korean
  • 제지슬러지의 당화공정을 위해서는 cellulase와 β-glucosidase가 필요하다. 효소 생산을 위한 연구를 먼저 진행하였다. 효소 생산을 위한 배양은 혼합 배양 및 순수 배양으로 실행되었으며, 순수 배양시에는 효소 생산 증대를 위해 배양기에서 배양을 하였다. 혼합 배양은 두 개의 균주에서 생산되는 cellulase와 β-glucosidase의 영향 및 소비되는 cellulose 양에 의해서 높은 효율의 효소 활성을 얻을 수 없었다. 이에 순수 배양에서는 A. niger 배양시 약 11U/mL의 β-glucosidase activity를 나타내었으며, fed batch 등을 통하여 보다 높은 β-glucosidase 생산을 할 수 있을 것으로 기대된다. 또한, 본 실험실에서 보유하고 있는 T. reesei의 cellulase 생산 및 활성은 높기 때문에 cellulase의 공급은 문제가 없을 것으로 보인다. 펄프 sludge의 당화 공정은 본 실험실에서 생산한 cellulase와 상업용 효소인 Novozym 188을 이용하여 실험하였다. 반응기에서 당화 공정은 15% 펄프 sludge에서 37 g/L의 당 생성을 보였으며, 이는 펄프 sludge 내에 존재하는 cellulose에 대하여 78% 이상의 당 전환율을 보이고 있다. 유기산 생성을 위한 배양에 충분히 사용 가능한 glucose 농도이다. 또한, 혼합 배양의 단점을 해결하기 위해 배양기를 통한 β-glucosidase 생산 실험을 실행하였다.
    당화과정 후에는 미생물이 제지슬러지에서 생산된 당을 이용하여 젖산을 생산할 수 있도록 하는 연구 진행이 필요하다. 이를 위해 동시당화발효공정을 통한 젖산 생산 공정을 진행하였다. Lactic acid 생산을 위한 공정에서는 당화공정과 발효공정으로 나눌 수 있다. 두 공정의 최적 조건은 다르며, 각 인자의 조율로써 당화 및 발효공정에 적용을 할 수 있을 것이다. 당화 및 발효공정에 최적에 가장 필요한 인자는 온도와 pH의 조절이다. 당화 및 발효공정을 따로 실시한 결과와 SSF공정을 실시한 결과, 현 연구에서는 42℃에서 동시 당화 및 생산을 하는 것이 유리할 것이다. SSF공정은 공정 시간에 단축과 장치에 단순화 및 product inhibition의 억제에 효과적이며, 가수분해 되지 않은 sludge의 불용성 부분은 발효 공정 후에 처리하는 것이 유리하기 때문이다. 이에 따라, SSF fed-batch 공정을 실시하였으며, flask에서 90g/L lactic acid를 생산하였다. 이는 분리공정을 위해서 충분한 농도로 보인다. 또한 fermenter를 이용한 실험에는 최대 81.2g/L lactic acid를 생산하였으나, productivity 측면에서는 fermenter를 이용한 경우는 1.48g/L.h이며, flask인 경우는 0.98g/L.h이다. 즉, fermenter에서 최적화 실험을 통하여 보다 높은 농도의 lactic acid 생산을 할 수 있을 것이다. Pilot-scale에서 동시당화발효공정을 실시하였다.
    Sludge를 이용한 SSF 공정을 위해서는 cellulase 및 β-glucosidase의 효소 첨가가 중요하다. 즉 β-glucosidase는 상업용 효소를 이용하고 있기 때문에 상당한 비용이 필요하다. 이 β-glucosidase를 본 연구실에서 생산하면, 보다 나은 SSF 공정으로 개선할 수 있을 것이다.

    최종적으로는, 동시당화발효공정에서 생산된 젖산을 분리하는 공정을 진행하였다. 침전과 반응증류를 혼합한 공정, Amberite resin을 이용한 흡착공정, SMB를 통해서 SSF공정을 통해 생산한 젖산을 분리할 수 있다. 침전과 반응증류를 혼합한 공정에서는 에탄올을 사용하여 기존 젖산 분리공정의 문제점(Ca(LA)2)의 높은 용해도)를 해결할 수 있었다. 생산성을 높이기 위해서 침전공정후의 여액을 재사용하였다. 침전과 반응증류를 이용한 젖산 분리의 수율과 순도는 각각 87.1%, 99.3%이었다. Amberite resin IRA-92를 이용한 흡착공정을 통해 얻은 젖산 분리의 수율과 순도는 82.6%, 96.2%로 측정되었다. SMB 공정을 통해서 얻은 젖산 분리의 수율과 순도는 각각 99.6%, 96.4%이었다. 단, SMB공정을 통해서 분리한 젖산의 순도와 수율은 acetic acid와 lactic acid 단 두 유기산만 존재하는 모델 용액을 만들어서 실험한 결과를 토대로 해서 구한 것이다. 실제 SSF 공정을 통해서 생산한 발효액으로부터 젖산을 정제하기 위해서는 다른 불순물들을 제거 할 수 있는 다른 정제 공정이 많이 필요할 것으로 보인다.
  • English
  • Biotechnology is often defined as a clean technology, which produces valuable products from renewable resources. Cellulose waste from the pulp and paper industries is a fine biomass resource, from which valuable products such as ethanol and organic acids, can be fermented. Production of lactic acid from paper sludge was carried out using complex biological processes.
    The effects of water soluble materials in paper sludge (sludge for short) on cellulase and beta-glucosidase activities and the effects of pre-drying of sludge and initial glucose concentrations on the enzymatic sludge hydrolysis were studied. The optimization of enzyme system for hydrolysis of the sludge for production of glucose was also made. Water soluble materials in the sludge showed stimulatory effect on carboxymethyl cellulose (CMC) activity, inhibitory effect on filter paper (FP) activity and no effect on avicelase and beta-glucosidase activities. Production of lactic acid from paper sludge was carried out using simultaneous saccharification and fermentation (SSF). The SSF process design was based upon the experimental data obtained from cellulose hydrolysis and fermentation. The SSF process was employed to avoid excessively dense solution, when the sludge content in the feed is higher than 15%, which is one of the several benefits of SSF. The final product concentration by SSF was observed to be limited by the cellulose content in the system, which can probably be resolved by the intermittent feeding of paper sludge. The SSF of paper sludge via a fed-batch mode, with intermittent feeding, produced lactic acid at 162g/l, with an yield of 74% and a productivity of 1.4g/lh. The modified bioreactor had improved lactic acid production performance after removing indigestible solid materials from the upper compartment, increasing paper sludges feed. Modeling and simulation for simultaneous saccharification and fermentation (SSF) process in bioconversion of paper sludge to lactic acid was carried out. SSF process combined the enzymatic hydrolysis of paper sludge into glucose and the fermentation of glucose into lactic acid in one reactor.
    This paper first reports the purification results of lactic acid from the fermentation broth with paper sludge as a cellulosic feedstock using weak anion exchanger Amberlite IRA-92. Some factors such as flow rate, sample volume loaded, pH, and column were systematically examined to improve the purity, yield and productivity in lactic acid purification. Results indicate that in purification process the increase of pH of the fermentation broth ranging from 5.0 to 6.0 can significantly enhance the recovery yield and purity but apparently reduce the productivity. In addition, the scale-up of purification process in laboratory size has little influence on the recovery yield and purity. After optimization, the yield, purity and productivity are found to be about 82.6%, 96.2% and 1.16 g LA/(g-resin day), respectively.
    Drowning-out crystallization by ethanol with subsequent batch distillation was employed to separate lactic acid from fermentation broth. Lime and ethanol were added to fermentation broth in order to convert soluble lactic acid to an insoluble calcium lactate form. The recovery yield of lactic acid by re-precipitation was 87.1% with the purity of 99.3%.
    This work highlights the improvement of lactic acid production using SSF from paper sludge. In collusion, modifying the microorganism and integrated bioprocess combining SSF with recovery process can be important step toward improving lactic acid production for commercial applications.
Research result report
  • Abstract
  • Biotechnology is often defined as a clean technology, which produces valuable products from renewable resources. Cellulose waste from the pulp and paper industries is a fine biomass resource, from which valuable products such as ethanol and organic acids, can be fermented. Production of lactic acid from paper sludge was carried out using complex biological processes.
    The effects of water soluble materials in paper sludge (sludge for short) on cellulase and beta-glucosidase activities and the effects of pre-drying of sludge and initial glucose concentrations on the enzymatic sludge hydrolysis were studied. The optimization of enzyme system for hydrolysis of the sludge for production of glucose was also made. Water soluble materials in the sludge showed stimulatory effect on carboxymethyl cellulose (CMC) activity, inhibitory effect on filter paper (FP) activity and no effect on avicelase and beta-glucosidase activities. Production of lactic acid from paper sludge was carried out using simultaneous saccharification and fermentation (SSF). The SSF process design was based upon the experimental data obtained from cellulose hydrolysis and fermentation. The SSF process was employed to avoid excessively dense solution, when the sludge content in the feed is higher than 15%, which is one of the several benefits of SSF. The final product concentration by SSF was observed to be limited by the cellulose content in the system, which can probably be resolved by the intermittent feeding of paper sludge. The SSF of paper sludge via a fed-batch mode, with intermittent feeding, produced lactic acid at 162g/l, with an yield of 74% and a productivity of 1.4g/lh. The modified bioreactor had improved lactic acid production performance after removing indigestible solid materials from the upper compartment, increasing paper sludges feed. Modeling and simulation for simultaneous saccharification and fermentation (SSF) process in bioconversion of paper sludge to lactic acid was carried out. SSF process combined the enzymatic hydrolysis of paper sludge into glucose and the fermentation of glucose into lactic acid in one reactor. A mathematical modeling for cellulose hydrolysis was developed, based on the proposed mechanism of cellulase adsorption deactivation. Another model for simple lactic acid fermentation was also made. A whole mathematical model for SSF was developed by combining the above two models for cellulose hydrolysis and lactic acid fermentation.
    This paper first reports the purification results of lactic acid from the fermentation broth with paper sludge as a cellulosic feedstock using weak anion exchanger Amberlite IRA-92. Some factors such as flow rate, sample volume loaded, pH, and column were systematically examined to improve the purity, yield and productivity in lactic acid purification. Results indicate that in purification process the increase of pH of the fermentation broth ranging from 5.0 to 6.0 can significantly enhance the recovery yield and purity but apparently reduce the productivity. In addition, the scale-up of purification process in laboratory size has little influence on the recovery yield and purity. After optimization, the yield, purity and productivity are found to be about 82.6%, 96.2% and 1.16 g LA/(g-resin day), respectively.
    Drowning-out crystallization by ethanol with subsequent batch distillation was employed to separate lactic acid from fermentation broth. Lime and ethanol were added to fermentation broth in order to convert soluble lactic acid to an insoluble calcium lactate form. The recovery yield of lactic acid by re-precipitation was 87.1% with the purity of 99.3%.
    This work highlights the improvement of lactic acid production using SSF from paper sludge. In collusion, modifying the microorganism and integrated bioprocess combining SSF with recovery process can be important step toward improving lactic acid production for commercial applications.
  • Research result and Utilization method
  • 동시당화 발효공정에서 glucose로부터 L. rhamosus를 이용하여 젖산을 생산했을 경우와 제지 슬러지로부터 L. KLB #58를 이용하여 젖산을 생산했을 때 약 3배의 경제적 가치가 있었다. 결과를 토대로 보면, 현재까지 동시당화 발효공정에서는 효소인 β-glucosidase를 이용하여 젖산을 생산하는 경우에는 경제성이 없을 것으로 보인다. 즉, 미생물내에 β-glucosidase이 존재하며, 높은 생산성을 보이는 균주를 가지고 생산을 한다면 경제적으로 젖산을 생산할 수 있을 것이다. 본 연구에서 탐색한 Lactobacillus KLB58을 이용하여 젖산 생산을 진행할 경우 높은 경제성이 있는 것을 확인하였다. 이의 경제성 비교를 위해서, 현재 poly-lactate를 생산 중인 미국 Cargill-Dow사가 이용하는 기질인 starch와 경제적 평가도 이루어져야 할 것이다.
  • Index terms
  • Simutaneous saccharification and fermentation, paper sluge, cellulose, cellulase, lactic acid
  • List of digital content of this reports
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