Red macroalgae Biorefinery to platform chemicals: high-titer of levulinic acid and lactic acid : 홍조류 바이오리파이너리를 활용한 고농도 레불린산 및 젖산 생산

다국어 초록 (Multilingual Abstract)

Red macroalgae have emerged as particularly significant in biorefineries as sustainable and environmentally friendly feedstocks. Owing to their rapid growth rates and high carbohydrate content, they represent a promising biomass source that does not compete with food crops or require arable land. This study focuses on producing levulinic acid (LA), 5-hydroxymethyl furfural (5-HMF), and lactic acid, key high-value platform chemicals, from the red macroalgae species. The production pathway of LA begins with the dehydration of sugars to form 5-HMF, followed by the rehydration of 5-HMF to form LA. In this process, 5-HMF functions as a key intermediate that links these compounds within an integrated biorefinery framework. Accordingly, this study adopted two complementary strategies: (i) producing high-titer LA through process optimization, and (ii) generating 5-HMF and LA by utilizing lactic acid derived from microbial fermentation. The first strategy for high-titer LA production involved the optimization of agar—the primary polysaccharide of red macroalgae—using sulfuric acid as a cost-effective conventional catalyst. Under these optimized conditions, an LA titer of 49.8 g/L was achieved, representing one of the highest values reported from seaweed-derived biomass via homogeneous acid catalysis. The second strategy for 5-HMF production utilized galactose from agar hydrolysate to produce lactic acid via yeast fermentation. The resulting lactic acid and unconsumed 3,6-anhydro-L-galactose (AHG) were then converted into 5-HMF. This approach presents a novel valorization pathway that synergistically links microbial fermentation with chemical synthesis, a design intended to maximize the utilization of all carbohydrates within the biomass. Overall, this study demonstrates an efficient and integrated strategy for converting marine biomass into valuable platform chemicals, thereby contributing to the advancement of sustainable biorefinery technologies.

다국어 초록 (Multilingual Abstract)

Levulinic acid (LA) is a versatile bio-based platform chemical with wide-ranging applications in biofuels, solvents, and biodegradable materials. To establish an efficient LA production strategy, red macroalgae were selected as the biomass feedstock due to their high carbohydrate content—particularly galactan, which is rich in 3,6-anhydro-L-galactose (AHG), a sugar that readily converts to LA under acidic conditions. In this study, a response surface methodology (RSM) based on a Box–Behnken design was used to optimize LA production from agar, the primary polysaccharide in red algae. The optimized reaction conditions (173°C, 67.4 min, 8% H2SO4) were successfully applied to two species of red macroalgae, Gracilaria vermiculophylla and Gelidium elegans, resulting in high LA yields of 52.9% and 48.4%, respectively. Moreover, high-solids loading up to 25% yielded an LA titer of 49.8 g/L—the highest reported to date from biomass using a single-step acid-catalyzed process. These findings highlight a practical and scalable approach for LA production from marine biomass, underscoring its potential in sustainable biorefinery applications.

다국어 초록 (Multilingual Abstract)

Lactic acid serves as a key bio-based platform chemical with critical applications in biodegradable plastics like polylactic acid (PLA) and various industrial sectors. Red macroalgae were selected as an ideal sustainable feedstock for efficient lactic acid production, owing to their abundant carbohydrates, fast growth rates, and non-competition with food agriculture. Herein, an integrated biological-chemical process was developed to valorize agar hydrolysate using the engineered Saccharomyces cerevisiae strain BK02, enabling high-titer lactic acid production coupled with autocatalytic upgrading of residual 3,6-anhydro-L-galactose (AHG) to 5-hydroxymethylfurfural (5-HMF) and levulinic acid. In flask-scale experiments, 30 g/L agar hydrolysate achieved the highest lactic acid titers and productivity while minimizing inhibition from higher loadings. These conditions were scaled up in a pH-stat controlled (5.5-6.0) bioreactor (30°C, 200 rpm, 1.0 VVM aeration, fed-batch operation), achieving 53.8 g/L lactic acid over 144 h. The ensuing chemical conversion confirmed lactic acid in fermentation broths (10-30 g/L initial substrate) as an effective autocatalyst under microwave-assisted thermolysis (180°C, 1 h), transforming residual AHG (2.9-8.0 g/L) to 1.5-6.8 g/L 5-HMF and 0.9-2.9 g/L levulinic acid catalysts. This cascade process optimizes carbon partitioning in red macroalgal agar—directing galactose to lactic acid and AHG to furanics—thereby enhancing biorefinery viability for marine biomass-derived platform chemicals.

국문 초록 (Abstract)

홍조류는 지속가능하고 환경 친화적인 원료로서 바이오리파이너리 분야에서 특히 주목받고 있다. 빠른 성장 속도와 높은 탄수화물 함량으로 인해 식량 작물과 경쟁하지 않으며 경작지 없이도 활용 가능한 유망한 바이오매스 원료이다. 본 연구는 홍조류로부터 고부가가치 플랫폼 화학물질인 레불린산, 5-하이드록시메틸푸르푸랄 (5-HMF), 젖산을 생산하는 데 초점을 맞췄다. 레불린산 생산 경로는 당류의 탈수로 5-HMF를 형성한 후, 5-HMF의 재수화로 레불린산을 생성하는 과정으로 진행되며, 이 과정에서 5-HMF는 바이오리파이너리 체계 내에서 두 화합물을 연결하는 중간체 역할을 한다. 이에 본 연구는 두 전략을 채택하였다: (i) 공정 최적화를 통한 고농도 레불린산 생산, (ii) 미생물 발효 유래 젖산을 활용한 5-HMF 및 레불린산 생성이다. 고농도의 레불린산 생산을 위한 첫 번째 전략은 홍조류의 주성분인 한천을 황산과 같은 경제적인 촉매로 최적화하는 것이다. 최적화를 통해 얻은 조건을 기반으로 해조류 유래 바이오매스에서 기존 문헌 대비 최고 수준인 49.8 g/L을 달성하였다. 5-HMF 및 레불린산 생산을 위한 두 번째 전략은 한천 가수분해물의 갈락토스를 효모 발효로 젖산으로 전환한 후, 생성된 젖산을 촉매로 활용하여 3,6-무수-L-갈락토스 (AHG)를 5-HMF와 레불린산으로 전환하는 것이었다. 이 접근법은 미생물 발효와 화학 합성을 연계하여 바이오매스 내 모든 탄수화물을 최대화하는 새로운 경로를 제시한다. 종합적으로 본 연구는 해양 바이오매스를 귀중한 플랫폼 화학물질로 전환하는 효율적이고 통합된 전략을 제시하며, 지속가능한 바이오리파이너리 기술 발전에 기여한다.

목차 (Table of Contents)

• Chapter Ⅰ 1
• Abstract 2
• 1.1. Introduction 5
• 1.2 Materials and methods 8
• 1.2.1. Materials 8
• 1.2.2. Composition analysis of red macroalgal biomass 8
• 1.2.3. Optimization of LA production from agar using RSM 10
• 1.2.4. Validation with actual biomass and high-solids loading 11
• 1.2.5. Product analysis 12
• 1.3. Results and discussion 13
• 1.3.1. Optimization of LA production from agar using RSM 13
• 1.3.3. Application of the model to actual biomass 25
• 1.3.4. Effect of high-solids loading on LA production 32
• 1.3.5. Mass balance for LA production from red macroalgae 36
• 1.4. Conclusion 39
• 1.5. References 40
• Chapter Ⅱ 46
• Abstract 47
• 2.1. Introduction 50
• 2.2 Materials and methods 53
• 2.2.1. Materials 53
• 2.2.2. Strain construction 53
• 2.2.3. Preparation of agar hydrolysate containing galactose and AHG 55
• 2.2.4. Optimization of lactic acid fermentation in a flask 55
• 2.2.5. Validate Chemical conversion of AHG to 5-HMF and LA with lactic acid autocatalytic 56
• 2.2.6. Optimization for high-titer production of lactic acid in a fermenter 57
• 2.2.7. Validation of lactic acid with agar hydrolysate in a fermenter 57
• 2.2.8. Product analysis 58
• 2.3. Results and discussion 60
• 2.3.1. Optimization of lactic acid fermentation in a flask 60
• 2.3.2. Validate Chemical conversion of AHG to 5-HMF and LA with lactic acid autocatalytic 66
• 2.3.3. For high-titer lactic acid production in a fermenter 69
• 2.3.4. Validation of lactic acid with agar hydrolysate in fermenter 72
• 2.4. Conclusion 75
• 2.5. References 76
• Korean abstract (국문 초록) 80