Multivariate analysis as a tool for selecting the vine pruning pretreatment towards the highest enzymatic hydrolysis yield

Highlights

• Vine pruning pretreatments were investigated by multivariate statistical analysis.

• The processing removed 68.7% of hemicellulose enabling 95.8% of cellulose recovery.

• It was possible to obtain a material with 75.0% of cellulose and 25.0% of lignin.

• Low levels of acetic acid, furfural, and HMF enable its further bioprocesses.

Abstract

Lignocellulosic materials require pretreatment to remove lignin enabling the enzyme access to the cellulose. This work used multivariate analysis to investigate the acid and alkali pretreatments of vine pruning followed by enzymatic hydrolysis. The best acid pretreatment conditions were H₂SO₄ 1.5%, 120 °C for 30 min, removing 68.7% of hemicellulose, enabling 95.8% of cellulose recovery. However, this treatment was not enough to allow the enzyme hydrolysis. A second step of treatment with NaOH 3.0% at 120 °C without agitation for 60 min led to a material with 75.0% of cellulose and 25.0% of lignin. However, the lowest glucose yield (80.86% and 32.26 g L⁻¹ of glucose) was obtained after the enzyme hydrolysis of this material. The highest glucose yield (98.72% with 35.06 g L⁻¹) was obtained using a pretreated material containing 68.1% of cellulose and 31.9% of lignin obtained after a milder condition (NaOH 2% at 100 °C), thus showing that not all the lignin need to be removed to obtain a high saccharification yield. A less severe pretreatment with no adverse effect on the glucose yield with the advantage of preserving the non-cellulose biomass fractions was effective for vine prune valorization.

Introduction

Some European countries have a long tradition of wine production [1]. In 2015, France, Italy, Spain, Germany, and Portugal were responsible for 70% of worldwide wine production (26 billion liters). Among these countries, Portugal is the fifth largest producer with a production of 705 million liters in that year, according to Instituto da Vinha e Vinho (https://www.ivv.gov.pt/np4/home.html accessed on May 21, 2019). Annually, the vineyard trees are pruned to increase the productivity and the grape quality for the next harvest [2]. In the pruning process, the cut thin branches are often burned [3], which may cause environmental problems related to the lignin combustion [4]. Some studies based on the production of wood-based panels [5]; vine pruning gasification [6]; lactic acid production by fermentation of the hemicellulose sugars [4,7]; biosurfactants production by fermentation of the cellulose sugars [3] were previsouly descibed.

The use of lignocellulosic biomass as a raw material for biofuels and biomaterials production increased [8]. At first, the lignocellulosic biomass was considered a source of glucose for ethanol production by fermentation. However, according to the biorefinery concept, the lignocellulosic biomass is now considered a raw material for many different industrial processes [9]. The industrial application of non-cellulose (hemicellulose and lignin) fractions are also a target in recent studies. Therefore, the use of all fractions of these materials, such as sugars from hemicellulose, cellulose, and lignin, to produce value-added products makes the transformation of biomass economically attractive [9].

The lignocellulosic materials are composed of an aromatic polymer (lignin) and carbohydrates polymers such as cellulose and hemicellulose, which are hydrolyzed to xylose and glucose, respectively [10]. Due to its composition, lignocellulosic materials are highly resistant to degradation. Therefore, the first step of the biomass transformation process is a pretreatment of the residue that results in the chain opening and the lignin and hemicellulose release [11,12]. The pretreatment might also reduce the cellulose crystallinity, increasing the amorphous cellulose fraction, which makes the cellulose available to be converted by enzymes action [13].

Due to the complexity of the lignocellulosic material, there is not a single pretreatment that is equally effective for all of them [14]. Therefore, whenever selecting a pretreatment, it is essential to consider some factors, such as digestibility of the treated material, sugars recovery, the presence of inhibitors, and the energy expenditure [15]. This pretreatment step is crucial for bioprocess development and sustainability because it may comprise 20% of the total costs in the production process [16]. The acid hydrolysis removes the hemicellulose and can solubilize a small fraction of lignin. Nonetheless, most of this polymer remains in the solid fraction, and therefore, another treatment step is necessary to remove the remaining lignin [14]. Afterward, the resultant material can be subjected to enzymatic hydrolysis to release the monomeric sugars (e.g. glucose), used in fermentation processes [10].

Although alkali treatment can be applied to remove lignin and hemicellulose from biomass, it also breaks lignin β-O-4 and α-O-4 bonds, resulting in several phenolic hydroxyl groups. The treatment severity depends on the material composition, and long treatment times are frequently needed. As reported by Lorenci et al. (2020), the alkali pretreatment presents some disadvantages, including high concentration processing media, low cellulose yields, high production of toxic compounds, and generation of polluting residues [17]. Besides, alkali treatment does not allow the use of non-cellulosic fractions (hemicellulose and lignin), which presents a great potential of application in other industries.

On the other hand, biomass processing at mild concentration and temperature conditions, either acid or sequential acid and alkaline, allows the recovery and utilization of the three main fractions of lignocellulosic biomass. The processing is conducted in at least two steps to selectively separate the sugars from hemicellulose, dissolve the lignin and recover less crystalline cellulose for enzyme hydrolysis. The two-step mild acid-alkali pretreatments enhance the biomass enzyme hydrolysis and fermentability. The disadvantages of such processing include additional processing steps; additional research to enhance the whole process and the utilization of biomass constituents [17]. Due to the low yields obtained from some pretreatments [18], in the current work, the effect of acid-alkali two-steps for pretreatment of vine pruning is evaluated.

Therefore, the resultant cellulosic fraction from the solid material was submitted to enzymatic hydrolysis, and the hydrolysis yields were determined since the glucose concentrations obtained from the hydrolysis are relevant to select the most appropriate pretreatment conditions for a given fermentation process. Scanning Electron Microscopy (SEM) allowed to visualize the pretreatment effect on the vine prune biomass and correlate the chemical composition of each portion with the material structure. The SEM images demonstrate that even with residual lignin, the acid-alkali pretreatment was able to open the material structure allowing the enzyme to reach the cellulose portion producing high glucose yields.