Introduction

The exploitation of finite fossil fuel resources has given rise to increased price fluctuations and elevated greenhouse gas emissions, contributing mainly to global warming. These drawbacks have escalated the need for alternative, renewable, sustainable, and economically viable energy resources such as carbohydrate-rich biomass to produce bioethanol. Bioethanol production is obtained from the carbohydrate fraction of the biomass, which is extracted and fermented [1–4]. Biomass conversion involves the separation of carbohydrate fraction to simple sugar through pretreatment methods, a vital step in biofuel production [5]. Several attempts have been made towards pretreatment of macroalgae or seaweeds [6–11], including chemical, physical or biological, or combination of these techniques. Pretreatment is carried out to enhance the surface area of the feedstock for the release of the constituent fermentable (reducing) sugars, which depends on the characteristics of chosen feedstock, catalysts, operation parameters, and strength [12–14]. Macroalgal biomasses are composed of a wide range of polysaccharides such as Cellulose, Ulvan, Laminarin, Floridean starch, etc. These polysaccharides are broken down into monosaccharides which serve as raw materials for bioethanol production. Constituents of these monosaccharides vary in the macroalgal biomass, as summarized in Table 1.

Macroalgae with higher moisture (80-85%) content and devoid of lignin polymer is well suited for microbial conversion than combustion or thermochemical conversion [15–17]. Also, the absence of lignin avoids the necessity of employing harsh pretreatment processes required in lignocellulosic biomass [15, 16]. Pretreatment of biomass is carried out for (i) size reduction, (ii) alter or remove structural and compositional impediments before enzymatic hydrolysis. Pretreatments are required to be cost-effective with minimal inhibitor formation while releasing a higher quantum of sugar.

Physico-chemical pretreatment involves liquid catalysts with higher process conditions to treat the feedstock.

Reducing sugar (RS) released using H₂SO₄ from various macroalgal species such as Gracillaria verrucosa (430 mg g^-1 RS, 1.5% H₂SO₄) [24]; Kappaphycus alvarezii (300 mg g^-1 RS, 0.9N H₂SO₄) [25]; Gracilaria verrucosa (7g L^-1 RS, 373mM H₂SO₄) [26]; Laminaria japonica (29.09% RS, 0.06% H₂SO₄) [27]; Kappaphycus alvarezii (81.62 g L^-1 RS, 1% v/v H₂SO₄) [28]; Gelidium amansii (33.7% RS, 3% H₂SO₄ ) [10]; Gracilaria verrucosa (7.47g L^-1 RS, 0.1N H₂SO₄) [29] and Kappaphycus alvarezii (30.5 g L^-1 RS, 0.2M H₂SO₄ ) have been reported [30]. However, drawback of dilute acid (higher concentration >0.9N) pretreatment is the generation of a higher concentration of 5-Hydroxymethyl furfural (HMF) and levulinic acid (LA) (with the degradation of hexose sugars and furfurals from pentose sugar degradation), which acts as inhibitors for microorganisms during the fermentation process by reducing enzymatic and biological activities, breaking down the DNA and inhibiting protein and RNA synthesis [31]. In order to overcome this, enzyme saccharification or biological pretreatment using either cellulase enzyme (of commercial-grade) or enzymes isolated from fungi or bacteria has been tried.

Most common enzymes employed for seaweed hydrolysis in earlier studies are commercial enzymes such as Cellulase, Celluclast 1.5 L, Viscozyme L, Novozyme 188, Termamyl 120 L, β-glucosidase, Multifect, Meicelase, Amyloglucosidase, etc. operated at pH 4.5-5.5 and temperature 35-55^oC, incubation time varies based on the algal feedstock [13, 28, 39, 31–38]. The current study focuses on the evaluation of sugar release from Enteromorpha intestinalis and Ulva lactuca using dilute acid hydrolysis and enzyme (extracted from Vibrio parahaemolyticus) saccharification of the dilute acid pretreated biomass.