Bioconversion of green macroalgae Chaetomorpha antennina for microbial growth and production of 3-hydroxypropionic acid from glycerol using engineered Escherichia coli

Abstract

The concept of seaweed biorefinery has attracted considerable research interest recently due to its nutrient-rich and lignin-free characteristics. In this study, the hydrolysate rich in simple sugars derived from the acid hydrolysis of green alga Chaetomorpha antennina was evaluated for its suitability as a carbon source for the growth of microbial systems. The alga was first pretreated with a range of concentrations of H₂SO₄ or HCl (100–600 mM), followed by thermal hydrolysis with differential time exposure (20–90 min) at constant temperature (121 °C) and pressure (15 psi). The highest concentration of simple reducing sugar release, 18.94 ± 0.78 g L⁻¹ was observed at 400 mM of HCl, with 60 min exposure time, while 18.89 ± 0.69 g L⁻¹ was obtained with 300 mM of H₂SO₄ for 75 min treatment. The differences in the exposure time played an important role in the determination of the energy requirements for the conversion process. HCl-assisted conversion required less energy (720 kJ/kg total solids) than H₂SO₄-based conversion (900 kJ/kg total solids) while using 10% (w/v) of biomass. Further, the seaweed hydrolysate obtained through HCl treatment resulted in superior growth characteristics with various Gram-positive and Gram-negative bacterial isolates. Additionally, the production of 3-hydroxypropionic acid (3-HP) from glycerol was validated at flask scale with seaweed lysate as a carbon source for recombinant E. coli W in a shaker incubator at 37 °C and 200 rpm. The metabolically engineered E. coli W produced a higher level of 3-HP (43 ± 2.09 mM) when grown with seaweed lysate supplemented medium than glucose (35 ± 1.62 mM).

Introduction

In recent years, biorefineries have been emerging as a sustainable alternative to petroleum-based refineries for the production of biofuels and biochemicals¹. These biorefineries are designed to be self-sustaining and environmentally friendly and are developed to meet the energy and material needs by converting biomass into various value-added products such as biofuels, biomaterials, and biochemicals. These biorefineries are classified as first, second, third, and fourth generation based on the feedstocks used². Due to their primary usage for food and feed, first-generation feedstocks are unable to meet the global demand as a sustainable feedstock. Second-generation feedstocks serve as an effective alternative to the first generation of food crops, primarily due to their non-edible nature and higher availability³. However, the higher operational costs and the formation of inhibitors are the major challenges for the utilization of lignocellulosic feedstock⁴.

On the other hand, as a third-generation feedstock, seaweeds are rich in nutrients and contains no lignin, making them attractive for the fermentation of biofuels and biochemicals. As seaweeds grow in coastal regions, they do not compete for land and water resources, unlike first and second-generation feedstocks. They can photosynthetically assimilate dissolved inorganic carbon, mainly CO₂ and bicarbonates in seawater into energy in the form of polysaccharides in the cell, which serves as feedstock⁵. The presence of 30–40% of dry biomass of seaweeds is composed of carbohydrates, which strengthens their potential utilization in biorefinery applications, particularly for the production of biofuels and biochemicals⁶. Feedstocks for fourth-generation biofuels or biochemicals represent an advancement over third-generation feedstock, involving the use of genetically modified algae, for the production of biofuels and biochemicals⁷. However, only a limited number of studies have explored for genetically modified macroalgae system still faces several challenges like genetic stability, large-scale cultivation, regulatory constrains which further requires scientific and technological advancements^8–10. It has been reported that India has a coastline length of 11,098 km, containing 865 seaweed species, in which Tamil Nadu alone accounts for about 282 species¹¹. In this rich diversity, red and brown algae holds greater economic significance for their demand for marine phycocolloids such as agars, carrageenans and alginates¹². Therefore, the utilization of red and brown seaweed biomass as fermentation feedstock may not be viable.

On the other hand, green seaweeds remain largely unexplored and offer a greater potential as a fermentation feedstock. Various species of green marine macroalgae, including Ulva, Caulerpa and Chaetomorpha have been reported as fermentation feedstock for the production of biofuels such as methane^13,14, bio-hydrogen^15–17, ethanol¹⁸ and biochemicals such as lactic acid^19,20. While the majority of research on the green seaweeds has focused on biofuels, only a limited number of studies have explored for the production of biochemicals. Among these green seaweeds, Chaetomorpha antennina, commonly found on the rocks in the intertidal zone hardly been utilized for economic gains. In the absence of any literature evidence of its use in edible or phycocolloidal products, this represents a significant opportunity to conduct value addition research for this alga²¹. Owing to its availability throughout the year and underutilized nature, C. antennina was selected as a fermentation feedstock¹⁷. While large-scale cultivation or quantitative estimation of C. antennina is not available, its natural availability and absence of lignin, makes it a sustainable feedstock for microbial fermentation. Utilizing such an underutilized resource supports circular bioeconomy strategies by converting minimally explored marine biomass into value-added biochemicals^13,14,22.

3-hydroxypropionic acid (3-HP; C₃H₆O₃, MW-90.08) is an economically important industrial chemical that has been listed as one of the top 12 platform chemicals²³. The global market value for 3-HP has been estimated to be more than 10 billion USD per year with an increasing demand of 3.6 million tons per year²⁴. Due to the involvement of toxic and carcinogenic precursors, chemical synthesis was not found to be viable. As an alternative, the production of 3-HP from biobased methods using recombinant microorganisms has become a more renewable and sustainable alternative to chemical synthesis²⁵. 3-HP is valued as a versatile industrial chemical due to the presence of carboxyl (-COOH) and hydroxyl (-OH) reactive groups, which enable it to undergo diverse chemical transformations. 3-HP can undergo redox reactions to be converted into high-value derivatives such as acrylic acid, 1,3-propanediol (1,3-PDO), malonic acid, acrylamide, acrylonitrile, propiolactone and other specialty chemicals. Acrylic acid, a 3-HP derivative was valued to be 12 billion USD in 2020 which has been projected to be increase around 19.2 billion USD by 2030²⁴. Due to its biodegradability and biocompatibility, 3-HP can be polymerized to form poly-3-hydroxypropionate (P-3HP), which is a widely studied alternative to petrochemical plastics. Although biological routes offer a sustainable approach for the production of 3-HP from glycerol, several challenges limit the yield and titre. While using glycerol as a sole source of carbon for the growth and production, the yield of 3-HP decreases significantly. In addition, redox imbalance caused by glycerol is relatively higher than other simple sugars such as glucose, which results in a drastic downtrend in 3-HP production. To address this challenge, a co-substrate strategy was adopted to separate growth and production. By deleting glycerol assimilating genes glpK and gldA, the utilization of glycerol for microbial growth (oxidative pathway) was restricted. The developed strain will utilize glucose or seaweed-derived sugars as a co-substrate for cell growth, while glycerol will be used as a carbon source for the synthesis of 3-HP.

The aim of this study is to develop a process to convert the biomass of green seaweed, C. antennina, to simple fermentable sugars by means of acid-thermal hydrolysis. Also, validate seaweed lysate (sugars) as carbon source for cell growth in metabolically engineered E. coli in this study, in addition to the production of 3-HP from glycerol.

Materials and methods

Seaweed collection and preparation

The location, availability and genus of the seaweed were based on the previous investigations¹⁵. The biomass was collected from a single coastal location (13°11′13.7″ N 80°19′ 01.5″ E) Ennore, Tamil Nadu, India, during March 2024. Dense mats of Chaetomorpha antennina biomass was observed covering the stone revetments at the selected location. Samples were collected from a single spot to avoid spatial variability. Samples of healthy and mature thalli have been collected and preserved separately in a bag with clean seawater containing 6% (v/v) formaldehyde solution for species authentication. The species was morphologically identified and authenticated by Prof. B. Babu, Algal Taxonomist, Department of Botany, Madras Christian College, Chennai, India. A voucher specimen was deposited at the MCC—Herbarium collection under the voucher number VTU 2307. Further, the harvested C. antennina biomass was washed thoroughly with seawater to remove the sand and transported to the laboratory. Other undesirable materials, such as shells and barnacles, were manually removed. It was then shade-dried to remove the excess moisture. The dried biomass was again washed thoroughly with fresh water to remove the intracellular or ionically bound salts. Finally, the biomass was dried again and stored in an airtight container under room conditions for further research. The collection and use of seaweed material complies with all relevant institutional, national and international legislations and guidelines. The samples were collected following standard guidelines. No permits or permissions were required for collection at the sample site, as the alga is not protected and is classified as a weed species.

Biochemical composition

The proximate analysis for the collected Chaetomorpha biomass was conducted as per methods recommended in the Association of Official Analytical Chemists (AOAC, 2022) followed by the pre-processing steps. For the determination of biochemical composition, all the experiments were conducted in biological triplicate and the results are presented as mean ± standard deviation. For each experiment, three sub-samples of dried C. antennina biomass were taken from the collected material. Each sub-sample was separately processed, hydrolyzed, and analyzed to obtain the results. Technical replicates were also performed during the assays to ensure analytical precision.

Analysis for moisture

Shade-dried biomass samples of about 5 g were placed in a pre-dried crucible and subjected to oven drying for 5 h at 105 °C. The difference between the initial weight and the dry weight of the biomass samples was used to determine the moisture content²⁶.

Analysis of carbohydrates

Samples of 100 mg were weighed, and 2 ml of 2.5 M HCl was added and hydrolyzed for 2 h at 95 °C. The hydrolyzed samples are then cooled to ambient temperature and neutralized with Sodium carbonate until the effervescence ceases. Then the mixture was made up to 100 mL using distilled water. After centrifuging the solution at 3000 rpm for 10 min, the supernatants were analyzed for total carbohydrates using the Phenol–Sulphuric acid²⁷.

Analysis of protein

The estimation of protein was determined using the Kjeldahl method. In this process, 0.5 g of Chaetomorpha samples were mixed with a catalytic solution containing K₂HPO₄ and CuSO₄ in a 1:5 ratio and digested using concentrated H₂SO₄ until the change in colour from brown to green. The digested samples were neutralized using 40% NaOH, followed by distillation against boric acid solution. Subsequently, the ammonium borate solution obtained was titrated against 0.1 N HCl until the pink colour endpoint²⁸. The protein content was determined by multiplying the concentration of nitrogen by the conversion factor of 5, which is specific for macroalgae.

Analysis of ash

For the analysis of ash, a crucible was pre-heated to 550 °C in a furnace to remove the surface-bound impurities. Then, 5 g of biomass samples were weighed in the crucibles and heated to 550 °C overnight. After reaching the room temperature, the crucibles were again weighed, and the percentage of ash was determined based on the weight difference²⁹.

Analysis of crude fiber

For the analysis of fiber, 5 g of the samples were mixed with 1.2% of 200 mL H₂SO₄ and boiled for 30 min. The H₂SO₄ was then removed, and the acid-digested biomass was treated with 1.2% NaOH and boiled again. The NaOH-digested sample solutions were filtered using an oven-dried, pre-weighed filter paper and dried in an oven for 3 h. After drying, the crude fiber content was estimated using the gravimetric method³⁰.

Analysis of lipids

Determination of lipids was done using the method of Blight and Dyer with slight modifications as suggested for the green seaweed Ulva sp.,^18,31,32. Powdered Chaetomorpha samples of 5 g were mixed with 25 mL of methanol: Chloroform (2:1 v/v) mixture and left undisturbed for an hour. The seaweed-solvent mixtures were subjected to ultrasonication for 15 min followed by 15–20 min of incubation at room temperature. The mixture was then centrifuged at 10,000 rpm for 20 min, and the supernatant was collected in a separate tube. Again, the solvent-extracted biomass was resuspended in 25 mL of solvent mixture for residual lipid extraction following the same methodology. To the extracted supernatants, an equal volume of distilled water was added and vortexed. Then the mixture was allowed to stand overnight at 4 °C without disturbance for the phase separation. Then the bottom and interphase layers were separated carefully and dried at oven for 24 h at 60 °C. After drying, the lipid content was determined by weight difference.

Biomass pretreatment

For the acid thermal hydrolysis (ATH) pretreatment of C. antennina, the optimum conditions for maximum sugar release were determined by fixing one condition as constant and varying the other conditions. Two acids, H₂SO₄ and HCl, were used to determine the efficient acid for hydrolyzing the C. antennina biomass. ATH was carried out at 121℃ temperature and 15 psi pressure for all the conditions. Initially, for the ATH process, the biomass concentration 10% (w/v), representing a solid-to-liquid ratio of 1:10 (i.e., 10 g of dry seaweed in 100 ml of dilute acid) was fixed with varying concentrations of acids (100—600 mM) and varying duration for thermal hydrolysis (20—90 min). From the above conditions, the acid concentration, and thermal hydrolysis (TH) time at which the maximum sugar release obtained were determined. By fixing those optimal conditions as constant, the biomass concentration was further optimized by varying the biomass loading in 5% increments from 5 to 15% (w/v).

Acid Thermal hydrolysis efficiency was calculated using the formula,³³.

1

where Ep is the efficiency of pretreatment (%), ΔSp is the difference in reducing sugar concentration (g L⁻¹) before and after the thermal acid hydrolysis, and TC is the total carbohydrate concentration (g L⁻¹) of seaweed.

As the formulation strategy for the ATH process implies varying TH time and biomass concentration, it is necessary to calculate the amount of energy required per unit of biomass for the conversion. Specific Energy can be calculated using the formula,³⁴.

2

where Power = watts, Time = TH duration (min), volume of sample = litres, Total solids = kg L⁻¹.

All the experiments were carried out in triplicate, and the values are expressed as mean ± standard deviation. Based on the obtained optimal conditions, the C. antennina biomass was ATH processed to yield the maximum sugars. Finally, the ATH-processed seaweed slurry was filtered and neutralized using calcium carbonate, followed by centrifuging at 13,000 rpm for 15 min. The clear lysate obtained in this process was used as a carbon source in modified minimal media for the growth in the production process of 3-HP.

Development of E. coli W mutant strain

An in-frame tagged deletion strategy was employed to generate E. coli W ∆glpK ∆gldA. The deletion process involved the individual amplification of a 500 bp upstream region (fragment A) and a 500 bp downstream (fragment B) region of the target gene. Then, the fragments A and B were fused by overlap PCR and subsequently inserted into the cloning vector. It was further sub-cloned into the pKOV plasmid between BamHI and NotI restriction sites and transformed into the host E. coli W. The target genes glpK and gldA were deleted sequentially from the chromosomal DNA of the E. coli W, through homologous recombination process³⁵. Deletion screening was done through PCR, and successful deletion was further confirmed by PCR amplification and sequencing³⁶.

Cloning of kgsadh and dhaB genes for 3-HP production

All the genes of the 3-HP pathway were cloned and expressed in two different plasmids (Table 1). The genes dhaB and gdrAB encoding glycerol dehydratase (Gene bank #YP 001,337,149.1, YP 001,337,150.1, YP 001,337,151.1) and glycerol dehydratase reactivase, respectively (Gene bank #YP 001,337,148.1), were overexpressed using the tac promoter in the modified plasmid pDK7(p15A). For the overexpression of kgsadh gene (Gene bank #AB241137), a modified pUC19 plasmid was used³⁷. LB (Lysogen Broth) medium was used for routine manipulation of genes, expression of proteins and strain maintenance. Kanamycin (Km^r) at 50 mg L^-1 was used for the maintenance of pUC19/kgsadh, and chloramphenicol (Cm^r) at 25 mg L^-1 was used for pDK7 (p15A)/dhaB123, gdrAB. Standard recombinant DNA procedures were used for gene cloning, plasmid isolation and electroporation³⁵.

Table 1.

Bacterial strains and plasmids used in this study.

Strains and plasmids	Description	Sources	References
Strains
Bacillus pacificus NAA2	Cell growth study	Isolated strain	38
Escherichia coli W	Cell growth study	(ATCC 9637)	35
Klebsiella quasipneumonia NAA4	Cell growth study	Isolated strain	39
Lactococcus lactis	Cell growth study	Isolated strain	–
Pseudomonas aeruginosa	Cell growth study	(ATCC 15,442)	40
E. coli W ΔglpK ΔgldA_ pUC19/kgsadh, pDK7(p15A)/dhaB123, gdrAB	3-HP production	Present study	–
Plasmids
pUC19/kgsadh	kgsadh in pUC19; Kmr		37
pDK7(p15A)/dhaB123, gdrAB	dhaB123, gdrAB in pDK7 plasmid with p15A; Cmr		37

Culture conditions for microbial cell growth study

Strains and plasmids used in this study are given in Table 1. The media used for cell growth study consists of the following components in one litre: NaCl 1.0 g, NH₄Cl 0.5 g, MgSO₄.7H₂O 0.25 g, KH₂PO₄ 4 g, Yeast Extract 0.5 g³⁵ and HEPES buffer (pH 7.0) 100 mM. Specific volume of green seaweed lysate (GSL) was filtered using Whatman Grade 1 filter paper with vacuum-assisted filtration to remove any suspended particles. The resulting clear lysate was then supplemented to the culture media at 10 g L⁻¹ concentration of reducing sugar. A comparative study was performed using culture media supplemented with glucose at a concentration of 10 g L⁻¹. All the strains were cultivated in a 250 mL conical flask with 50 mL of culture media. The flasks were sealed with cotton plugs, ensuring sterile and microaerobic conditions. The cultures were incubated at 37 °C in a rotary shaker incubator at 200 rpm. Samples were withdrawn at regular intervals for the measurement of cell growth at OD₆₀₀.

Culture condition for 3-HP production study

The engineered strain E. coli W ΔglpK ΔgldA-pUC19/kgsadh, pDK7(p15A)/dhaB123, gdrAB (EcWGP), was evaluated in a shaker incubator for 3-HP production potential using glycerol (100 mM) as primary substrate, and green seaweed lysate (GSL) as a co-substrate for the cell growth. A comparative evaluation was performed with glucose as co-substrate. Fermentation under both co-substrate conditions (GSL and glucose) were initiated with 0.1 OD₆₀₀ of overnight-grown cells. 0.1 mM of IPTG was added to the shake flask cultures once the 0.6 OD₆₀₀ was reached. 2 µM of coenzyme B₁₂ was added during 3, 6, 9 and 12 h of cultivation. Samples were withdrawn at regular intervals, and the metabolites were analyzed using HPLC.

Analytical methods

Filaments of the seaweed at different stages were viewed and photographed using (Dino-Lite AM4515) Digital Microscope under 200 × magnification. A single beam UV spectrophotometer [Lynx 2373] was used to measure the cell growth using a cuvette with 10 mm pathlength at OD₆₀₀. The Dinitro Salicylic Acid (DNS) method was used to determine the concentration of reducing sugars produced during the Acid-Thermal Hydrolysis (ATH) process. Briefly, seaweed lysate was mixed and incubated with DNS reagent, followed by the absorbance of the developed colour was measured at OD₅₄₀. A standard curve was generated using varying concentrations of glucose, and the OD values obtained from the ATH-processed samples were quantified using a linear regression equation based on the standard curve.

The concentration of substrates and metabolites was quantified using High Performance Liquid Chromatography (HPLC) equipped with Refractive Index Detector (RID) and Photodiode Array (PDA) detectors (Shimadzu, Japan). Briefly, the culture supernatant was filtered appropriately using a 0.22-micron filter. For the chromatographic analysis, 2.5 mM H₂SO₄ was employed as a mobile phase, with a flow rate of 0.6 mL/min. The separation was carried out through a Phenomenex Rezex 300 × 7.8 mm column accompanied by a guard column (35 × 7.8 mm), both maintained at a constant temperature of 50 °C.

Results

Identification and pre-processing of collected seaweed

During collection, dense mats of seaweed thalli were firmly attached to rocks and found emerging from the holdfast (Fig. 1a)⁴¹. The collected seaweed was characterized by a tufted filamentous thallus measuring approximately 9–14 cm in length, with a branched holdfast (Fig. 1b). The filaments are stout, thick, cylindrical and unbranched. Based on its morphological traits, the collected green marine macroalga was identified as C. antennina (Bory) Kützing 1847, following standard identification manuals. Generally, green macroalgae should not be washed immediately with fresh water after harvesting. The drastic drop in external salinity disrupts the osmotic balance across their cell membranes which leads to water influx into cells. This imbalance causes swelling which destabilizes the cell membrane and can result in leakage of intracellular components⁴². To avoid such osmotic shock, the biomass was dried and then washed ensuring structural stability and nutritional integrity of the collected seaweed.

Fig. 1.

a Sample collection site—growth of green seaweed on intertidal rocks of the shore, b Thallus of the collected green seaweed fresh biomass, c Compositional analysis of the green seaweed C. antennina.

Compositional analysis of C. antennina

The proximate analysis C. antennina revealed a composition of carbohydrates, proteins, crude fiber, ash and lipids with varying concentrations (Fig. 1c). Briefly, C. antennina contains substantial levels of 42.8 ± 1.71% carbohydrates, 22.33 ± 1.11% protein, 11.01 ± 0.55% crude fiber, 23 ± 0.92% ash content and 0.87 ± 0.04% of lipid content of its total biomass.

Optimization of feedstock conditions for acid thermal hydrolysis

In this study, the efficient conversion of seaweed biomass into reducing sugars was achieved using two strong acids, HCl and H₂SO₄. The effect of various parameters such as concentration of acids, the duration of thermal hydrolysis and the concentration of biomass was also evaluated in this study. The maximum concentration of fermentable sugars obtained from the biomass of C. antennina after the ATH process by varying the conditions are shown in Fig. 2. H₂SO₄ treatment resulted 18.89 ± 0.69 g L⁻¹ of reducing sugars at 75 min of TH time (Fig. 2a), whereas the highest concentration of reducing sugars, 18.94 ± 0.78 g L⁻¹ was achieved with 400 mM of HCl with 60 min of thermal hydrolysis time (Fig. 2b). The effect of ATH on C. antennina biomass before and after treatment was illustrated in Fig. 3a, b, c.

Fig. 2.

Acid Thermal Hydrolysis pretreatment of green seaweed Chaetomorpha antennina by varying concentrations of H₂SO₄ and HCl (mM) and different thermal hydrolysis time (min), a Effect of H₂SO₄ and TH time on reducing sugar concentration, b Effect of HCl and TH time on fermentable sugar concentration. Experiments were done in triplicate (n = 3) and the error bars represent standard deviation (SD).

Fig. 3.

a C. antennina dry biomass during storage, b Acid pretreated biomass before thermal hydrolysis, c Disintegrated seaweed biomass after thermal hydrolysis.

Evaluation of energy and pretreatment efficiency

Energy required for the conversion of biomass into reducing sugars plays a major role in determining the efficiency of the ATH process. HCl pretreated biomass yielded higher sugars at 60 min of thermal hydrolysis (TH) time which required an energy input of 720 kJ/kg total solids, whereas H₂SO₄ pretreated biomass yielded higher sugars at 75 min of thermal hydrolysis time with a higher energy input of 900 kJ/kg total solids as shown in Fig. 4a. Thus, the acid pretreatment conditions, 400 mM of HCl pretreatment along with 60 min of TH time at 121 °C at 15 psi were considered as optimal for the Acid Thermal Hydrolysis process.

Fig. 4.

a Effect of acid concentration H₂SO₄ and HCl on specific energy requirement, b Effect of macroalgal biomass concentration on reducing sugar yield (g L^-1), pretreatment efficiency (Ep%) and specific energy requirement (kJ/kg TS). The error bars represent the standard deviation (SD) and each experiment was performed in triplicate (n = 3).

Under these optimum conditions, the biomass concentration was varied within the range of 5–15% (w/v) to determine the effect of biomass loading on reducing sugar yield. The corresponding pretreatment efficiency (Ep %) and specific energy (kJ/kg TS) were also evaluated (Fig. 4b). At 5% (w/v) of C. antennina biomass, the swelling ratio of the seaweed biomass was lower than the liquid volume resulting 8.88 ± 0.75 g L⁻¹ reducing sugars and a pretreatment efficiency of E_p = 40.13%, with an energy input of 360 kJ/kg TS. Upon increasing the biomass concentration to 10% (w/v), the swelling ratio matched the liquid volume, resulting in a reducing sugar yield of 18.94 ± 0.78 g L⁻¹ and enhanced efficiency of E_p = 44.17%, with a specific energy input of 720 kJ/kg TS. Upon further increasing the biomass concentration to 15% (w/v), the swelling ratio surpassed the liquid level, absorbing the liquid entirely. Though the reducing sugar yield increased slightly to 21.86 ± 0.88 g L⁻¹, this imbalance in solid to liquid ratio—‘thickening effect’ reduced the pretreatment efficiency to 33.30%, with a higher energy demand of 1080 kJ/kg TS. Therefore, 10% (w/v) of biomass concentration, with 400 mM of HCl at 60 min of TH time, was the optimum condition for efficient acid thermal hydrolysis (ATH) of green seaweed C. antennina.

Microbial cell growth study

Five different industrially important organisms comprising two Gram-positive strains (Bacillus pacificus, Lactococcus lactis) and three Gram-negative strains (Escherichia coli W, Klebsiella quasipneumoniae NAA4 and Pseudomonas aeruginosa) were cultivated in a modified minimal medium containing either glucose (Fig. 5a) or green seaweed lysate (GSL) as the sole carbon source (Fig. 5b). The components of the minimal media used in the study have been modified and optimized, where HEPES was used as an alternative buffer and KH₂PO₄ was used as a source of phosphate.

Fig. 5.

Comparison of cell growth performance of two Gram-positive (Bacillus pacificus, Lactococcus lactis) and three Gram-negative (Escherichia coli W, Klebsiella quasipneumoniae NAA4 and Pseudomonas aeruginosa) strains cultivated on two different substrates, a Cell growth profile of five strains in glucose supplemented media, b Cell growth profile of five strains in seaweed lysate supplemented media, c Maximum cell growth of five strains in glucose and seaweed lysate media (OD₆₀₀), d Maximum specific growth rate (µ_max) h⁻¹ of five strains in glucose and seaweed lysate media. Experiments were done in triplicate (n = 3) and the error bars represent the standard deviation (SD).

In glucose-supplemented media, Pseudomonas aeruginosa and E. coli W exhibited the highest cell growth, reaching 5.02 and 4.4 OD_600, respectively. In contrast, K. quasipneumoniae, B. pacificus and L. lactis showed significantly lower growth, with values below 1.0 OD₆₀₀ (Fig. 5 a). On the other hand, all strains supplemented with GSL showed better cell growth. Notably, L. lactis, K. quasipneumoniae, and B. pacificus achieved higher growth of 6.04, 5.15, and 5.86 OD_600, respectively, with a characteristic diauxic growth pattern. This clearly shows the sequential utilization of mixed sugars by the strains present in the seaweed lysate. Interestingly, E. coli W and P. aeruginosa did not exhibit diauxic growth when cultivated in GSL (Fig. 5b). Specifically, P. aeruginosa showed the lowest growth in GSL, with a 3.35 OD₆₀₀. In contrast, E. coli W attained a near similar growth in both glucose and GSL media (Fig. 5c).

Based on the maximum specific growth rate calculations shown in Fig. 5d, E. coli W exhibited the highest µ_max of 0.78 h⁻¹ on GSL, followed by P. aeruginosa µ_max: 0.72 h⁻¹ on glucose and L. lactis µ_max : 0.62 h⁻¹ on GSL. In the meanwhile, a µ_max of 0.58 h^-1 was attained with E. coli W while it was grown in glucose-supplemented conventional media. These results demonstrate that seaweed lysate can serve as an effective substrate for the growth of various microorganisms, particularly E. coli W, which exhibits better growth performance than the conventional substrates.

Production of 3-HP from glycerol using glucose and seaweed lysate

Production of 3-HP during the fermentation process involves a two-step enzymatic reaction. Initially, glycerol is converted into (3-HPA) by the enzyme glycerol dehydratase (DhaB) using Vitamin B₁₂ as a coenzyme. In the second step, 3-HPA is converted into 3-HP with the help of NAD⁺-dependent aldehyde dehydrogenase (kgsadh). A major problem in this pathway is that each mole of 3-HP co-produces one mole of NADH, significantly altering the cellular redox balance. The imbalanced redox equivalents significantly affect the overall cellular metabolism and the synthesis of 3-HP as well. The adverse effects of NADH accumulation can be minimized by blocking the glycerol assimilation through the oxidative pathway and by employing a co-substrate. To restrict the glycerol flow towards oxidative pathway/ growth, the glycerol assimilating genes glpK and gldA were deleted from the E. coli genome. Finally, the engineered strain E. coli W ΔglpK ΔgldA pUC19/kgsadh, pDK7(p15A)/dhaB123, gdrAB (EcWGP) was developed (Fig. 6).

Fig. 6.

Development of recombinant E. coli W ΔglpK ΔgldA pUC19/kgsadh, pDK7(p15A)/dhaB123, gdrAB (EcWGP) strain.

The metabolically engineered strain (EcWGP) was employed for 3-HP production using glycerol as primary substrate and glucose as co-substrate (Fig. 7a). In parallel, the potential of GSL to serve as an alternative co-substrate for cell growth was evaluated under similar conditions (Fig. 7b). EcWGP attained a maximum specific growth rate (µ_max) of 0.598 h^-1 and 0.531 h^-1 under GSL and glucose-supplemented media, respectively. Although the µ_max were similar to each other, the overall cell growth achieved at 24 h by the strain in GSL was 54% higher than that of glucose. By consuming 7.09 g L⁻¹ of fermentable reducing sugars, EcWGP in GSL attained a final cell density of 1.54 g CDW g L⁻¹ at 24 h, whereas with glucose supplementation, only 3.93 g/L was consumed, attaining a final density of 1.0 g CDW g L⁻¹. On the other hand, the glycerol consumption by EcWGP co-supplemented with GSL and glucose was around 62 mM and 58 mM, at 24 h respectively. However, the level of 3-HP produced in GSL was 18.6% higher (43 ± 2.09 mM) than glucose (35 ± 1.62 mM).

Fig. 7.

Comparison of time course profile for cell growth, sugar consumption, pH, glycerol consumption, 3-HP and 1,3-PDO production by the recombinant E. coli W ΔglpK ΔgldA pUC19/kgsadh, pDK7(p15A)/dhaB123, gdrAB (EcWGP) strain from glycerol, a Glucose supplementation as a carbon source b green seaweed lysate supplementation as a carbon source. The experiments were done in triplicates (n = 3) and the error bars represent the standard deviation (SD).

Discussion

The proximate compositional profile of C. antennina obtained in this study highlights its carbohydrate and protein-rich nature, with comparatively low lipid content. This trend aligns with previously reported Chaetomorpha species, where carbohydrate levels typically ranged between 14–42% of dry weight. In general, the total carbohydrates in seaweeds (green, brown and red) comprise a significant portion of soluble polysaccharides such as ulvans, alginates, fucoidans, agars and carrageenans, which are categorized as dietary fiber and are solubilized during the carbohydrate estimation process⁴³. In contrast, the insoluble fractions of seaweeds consists of structural cell wall polysaccharides such as cellulose, hemicellulose, xylan and other non-starch polysaccharides that are not completely digestible by HCl and are therefore classified as fibers⁴⁴. The observed fiber levels are within the typical range reported for Chaetomorpha species, 1.7–21.5% of dry weight (Table 2). Likewise, the protein fraction falls within the reported range of 4–22.3% estimated by the Kjeldahl method using a conversion factor of Nx5, as the general conversion factors of Nx6.25 for foods and Nx6.38 for milk are inappropriate due to the presence of pigments and non-proteinaceous substances like nitrate, ammonia and organic nitrates in macroalgae⁴⁵. The observed very low lipid content also corroborates with the usual lipid content in the seaweeds from 0.5 to 8.0%⁴⁶.

Table 2.

Nutritional composition of various Chaetomorpha species.

Macroalgal sp.	Moisture %	Carbohydrates %	Protein %	Ash %	Lipid %	Fiber %	Reference
Chaetomorpha sp.	–	43.0	20.4 ± 0.16	14.0 ± 0.05	0.8 ± 0.03	21.8 ± 0.06	47
Chaetomorpha crassa	33.99 ± 0.38	14.4	7.94 ± 0.16	37.91 ± 0.29	1.05 ± 0.04	–	48
Chaetomorpha sp.	–	42.19	12.63	36.3	8.88	1.7	49
Chaetomorpha clavata	–	24.39	17.80	23.42	6.24	16.52	50
Chaetomorpha linum	3.47	37.43	15.48	40.85	2.25	–	51
Chaetomorpha linum	–	40.71 ± 1.41	4.40 ± 0.09	42.28 ± 0.70	0.57 ± 0.10	12.02 ± 0.13	52
Chaetomorpha antennina	–	42.76 ± 1.710	22.33 ± 1.11	23.00 ± 0.92	0.87 ± 0.04	11.01 ± 0.55	Present study

*Percentage (%)—represents the proportion of each nutrient component in the biomass.

The ash content of various Chaetomorpha species reported to vary between 14 and 42.2% of the dry weight, also aligns with our proximate findings. In general, seaweeds accumulate calcium, potassium, sodium, magnesium, iron, phosphorus, zinc, copper and manganese from seawater, which corresponds to the elevated ash content⁵⁷. Accumulation of higher levels of salts in seaweeds can lower the efficiency of the ATH process, resulting in reduced sugar yield⁵⁸. Moreover, environmental factors such as seasonal variations and geographical patterns also influence the nutritional content of the seaweeds. For instance, it has been reported that the overall nutritional content of the seaweed C. antennina was high during the post-monsoon season, with particularly elevated carbohydrate levels²¹. In our study, carbohydrate levels of 42.7% of dry weight were observed in samples collected during the spring season, consistent with moderate nutritional accumulation. Based on these findings, it is suggested that harvesting of the C. antennina during the post-monsoon season may contain higher carbohydrate levels.

Different pretreatment techniques have been employed for the conversion of green seaweeds into fermentable sugars. The efficiency of sugar release varies with each method, depending on the structural complexity of the algal biomass, choice of chemical or thermal conditions, and the type of end products generated (Table 3). Among the different methods, acid thermal hydrolysis was found to be viable and efficient. In this study, 300 mM H₂SO₄ and 400 mM HCl were found to be optimal for maximum sugar recovery from C. antennina, beyond which a decrease in sugar yield was observed. Additionally, the thermal hydrolysis (TH) duration also plays a vital role in the sugar release. Similar to acid concentrations, the increased TH duration above the optimum led to decreased sugar yield. The decline in sugar yields could be attributed to the degradation of sugars into furfural derivatives at higher acid concentrations and prolonged TH time^59,60.

Table 3.

Different species of green macroalgae used as a feedstock for the fermentation.

Macroalgae	Pretreatment method	Yield	Product	Reference
Chaetomorpha antennina	Chemo-disperser liquefaction	–	Methane	13
Chaetomorpha antennina	Thermo-chemo disperser	–	Methane	53
Chaetomorpha antennina	Sonic disintegration	26% Disintegration efficiency	Biohydrogen	16
Chaetomorpha antennina	Sonic surfactant disintegration	26.9% Disintegration efficiency	Biohydrogen	15
Chaetomorpha antennina	Sonication alkali liquefaction	24.61% Liquefaction efficiency	Biohydrogen	17
Chaetomorpha antennina	Acid-thermal hydrolysis	18.94 ± 0.78 g L−1	3-Hydroxy propionic acid	Present study
Ulva intenstinalis	Hydrothermal hydrolysis enzymatic saccharification	7.3 g L−1 20.1 g L−1	reducing sugars	54
Ulva intestinalis	Acid-thermal hydrolysis enzymatic saccharification	15.1 g L−1 24.20 g L−1	Ethanol Ethanol	33
Ulva reticulata	Acid—H2O2 induced microwave pretreatment	38.5% Liquefaction	Bio hydrogen	55
Ulva prolifera	Acid-Thermal Hydrolysis	47.14 ± 1.36 g L−1	Ethanol	56
Ulva sp.	Mild Acid-Thermal Hydrolysis	25.66 g L−1	Lactic acid	19
Ulva fasciata	Acid-Thermal Hydrolysis	–	Lactic acid	20

During the ATH process, the difference in concentrations of H₂SO₄ and HCl has not shown much impact on the release of reducing sugar. However, the difference in exposure time plays a vital role in determining the energy requirements for the conversion process. Furthermore, our results suggest that HCl is more efficient in maximum sugar release, with minimal energy consumption in a lesser time than compared to H₂SO₄ pretreatment for the C. antennina biomass⁶¹. Various studies have indicated that the optimum biomass concentration for acid thermal hydrolysis depends on the pretreatment conditions and the biomass type. In a study, optimal biomass concentration for drifted marine macroalgae was found to be 15% (w/v) with 2.5% (v/v) of H₂SO_4, yielding a maximum sugar concentration of 18.4 g L^-1, whereas 10% (w/v) of the same biomass has yielded 13.3 g L^-1 of sugars⁶². In terms of biomass type, 10% (w/v) biomass concentration was optimal for Kappaphycus alvarezii⁶³, whereas 2% (w/v) biomass was optimal for G. verrucosa⁶⁴ and 5% (w/v) biomass for Ulva intestinalis and Ulva lactuca⁶¹. Notably, our results indicated that a reduction in pretreatment efficiency with an increase in biomass concentration was due to the swelling and hydration behavior of the biomass.

Carbon/ nitrogen sources play a vital role in cell growth and metabolism of the microorganisms. As seaweeds accumulate higher levels of carbon/ nitrogen along with other various micro and macro elements (Table 2), this study has explored their ability to support the growth of various microbial cell factories. Chaetomorpha sp. are reported to contain various sugars such as arabinose (15–60%), galactose (40–60%), glucose (5–25%), rhamnose (3–20%), xylose (< 5%) and other sugars (< 0.5%)^65,66. In parallel, red seaweeds contain a sugar composition of galactose, 3,6-anhydrogalactose (AHG), glucose, and xylose. Brown seaweeds are composed of glucose, mannose, xylose, fucose, galactose, mannuronic acid and guluronic acids. Sugars such as glucose, galactose, mannose, xylose, arabinose, rhamnose, and fucose are classified as fermentable sugars. Whereas 3,6-anhydrogalactose (AHG), glucuronic acid, iduronic acid, mannuronic acid and guluronic acids are classified as non-fermentable sugars by the microorganisms^67,68. Depending on the extraction methods and the degree of depolymerization, the sugar composition in the lysate varies accordingly⁶⁹. Notably, our ATH pretreated GSL hydrolysate of C. antennina also had a composition of arabinose and galactose. Therefore, the presence of fermentable sugars, lower inhibitor formation and low sulphate content makes Chaetomorpha-derived hydrolysate a good carbon source for microbial fermentation.

Moreover, due to the presence of multiple elements in the GSL, they tend to interact with the existing inorganic elements supplemented through the minimal media. Such interaction between the media components causes random precipitation and makes the nutrients unavailable for microbial growth. To overcome such limitations, the components of the minimal media used in this study have been optimized accordingly, thereby further supporting the production of value-added biochemicals through biorefinery approach.

Different bacterial species exhibit distinct preferences and metabolic strategies for carbon source utilization. E. coli W preferably utilizes glucose as a carbon source over other sugars⁷⁰. In contrast, P. aeruginosa prioritizes amino acids and organic acids over sugars⁷¹. Klebsiella sp. utilizes glycerol more efficiently⁷², B. pacificus prefers starch and complex sugars⁷³. L. lactis generally utilizes lactose and galactose as its preferred carbon source⁷⁴. In a culture medium containing mixed sugars, initially strains utilize their preferred choice of carbon source, followed by a lag phase to activate the regulating genes for uptake of the other available sugars, and then entering into a second exponential phase^75,76. Similar characteristics were observed in our study with K. quasipneumonia, B. pacificus and L. lactis cultivated in GSL medium. Interestingly, no diauxic growth was observed in E. coli W cultivated in GSL medium containing mixed sugars, exhibiting similar growth characteristics to glucose medium. A similar pattern was already observed for E. coli W while cultivation in a mixed sugars medium containing glucose, lactose, galactose, sucrose, fructose and maltose⁷⁷. This indicates that the E. coli W can effectively overcome carbon catabolite repression under low glucose concentrations by adapting to the mixed sugar environment without any significant lag in their growth⁷⁸.

Among the two different media for validating the 3-HP production potential, the media supplemented with GSL had a better performance, which is attributed to its composition of mixed sugars⁷⁰. The presence of multiple sugars allows the activation of ‘lac’ promoter⁷⁹. In contrast, the media with a glucose-rich environment can hinder the expression of genes under the ‘lac’ operon, potentially limiting overall metabolic flexibility and growth. This observation aligns with earlier studies on carbon catabolite repression in E. coli, where higher glucose levels can repress key enzymes through regulatory networks affecting operons such as ‘lac’⁷⁹.

The disparity observed in 3-HP levels between the glucose and GSL medium can also be correlated to the production of 1,3-PDO as a by-product. Higher glucose concentration tends to lower the enzyme activity of kgsadh, which is regulated under ‘lac’ promoter. Hence, the glycerol flux towards 1,3-PDO was slightly higher in glucose medium. The present study highlights the unique advantage of mixed sugars from GSL in overcoming glucose-mediated repression of ‘lac’ promoter in EcWGP, whereas other studies have focused on glucose as a primary substrate^70,80. The presence of mixed sugars in GSL medium not only improved the 3-HP production but also demonstrates a strategy for optimizing metabolic pathways by leveraging promoter regulation. Furthermore, our study supports the understanding of substrate-mediated gene expression control and suggests practical strategies for improving yields in mixed sugar biorefinery applications. To our knowledge, this is the first report demonstrating the use of seaweed-derived mixed sugars to enhance 3-HP production in EcWGP. These findings open new avenues for seaweed biorefinery scale-up, paving the way for sustainable and efficient industrial biotechnological processes.

Conclusion

This study demonstrated the feasibility of utilizing C. antennina biomass as an alternative source of fermentable reducing sugars through acid thermal hydrolysis process. The highest concentration of reducing sugars, 18.94 ± 0.78 g L⁻¹ was obtained with 400 mM of HCl for 60 min. A notable thermal hydrolysis efficiency of 44.17% with an efficient energy requirement of 720 kJ/kg TS was achieved. Our findings demonstrated that the green seaweed lysate (GSL) effectively supported the growth of various Gram-positive and Gram-negative bacterial strains. Further validation of GSL with metabolically engineered E. coli W (EcWGP) as a growth carbon source showed promising results on cell growth of 1.4 g CDW g L⁻¹ and an increased 3-HP production of 43 ± 2.09 mM, when compared to media supplied with glucose as a co-substrate. Thus, the utilization of C. antennina as a carbon source can serve as an effective alternative to conventional carbon sources for the production of industrially important chemicals through a biorefinery approach.

Author contributions

A. C. E: Writing—Original draft, M. S: Conceptualization, Validation, Investigation, writing—review and editing, N.R.V.G: Visualization, Formal analysis, T. K: Conceptualization, Methodology.

Funding

Open access funding provided by Manipal University Jaipur. The authors gratefully acknowledge the financial support received from Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, under the Seed Fund, Grant No. VTU/Seed Fund/FY 2023–24/019. This support was instrumental in facilitating the successful execution of this research work.

Data availability

The datasets generated during and/ or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing Interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mugesh Sankaranarayanan has patent #Production of 3-hydroxypropionic acid from glycerol using genetically modified Escherichia coli and seaweed lysate #202441019496 pending to Vel Tech Rangarajan Dr Sagunthala R&D Institute of Science and Technology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.