Bacterial cellulose production using enzymatic hydrolysate of olive pomace

Abstract

Bacterial cellulose is a biopolymer which has the identical chemical and structural properties with plant cellulose. However, it is recently of great scientific interest due to its superior characteristics such as high degree of polymerization, water retention capacity, and biocompatibility, compared to its plant-originated counterpart. Additionally, while plant cellulose is often bound to lignin, pectin and hemicellulose polymers, BC is obtained as a pure product. By virtue of these features, bacterial cellulose has been successfully employed in many applications among which are food, paper, biosensors, electronics, drug delivery systems, cosmetics, and wound healing materials. Along with some bacteria genera such as Acetobacter, Pseudomonas, Rhizobium and Escherichia, this polymer can also be formed by some algae (Valonia, Chaetamorpha) and fungi. It has been considered to procure the microorganisms an evolutionary advantage by protecting them against environmental factors such as UV radiation, desiccation and, contamination. There are several standardized growth media for the production of bacterial cellulose. Nonetheless, high cost of these laboratory-grade ingredients obstructs the large scale use of bacterial cellulose. Heretofore, numerous strategies have been proposed to minimize the medium cost. Molasses, rotten fruit, orange peel can be counted among the alternative media which gave promising results for bacterial cellulose generation. In this study, use of lignocellulosic material, particularly of olive pomace as carbon source for bacterial cellulose production was demonstrated. Lignocellulose, being the most abundant biopolymer in the world, has a great potential to serve as a substitute for fossil fuels. In fact, bioethanol and biofuel generation from lignocellulose by means of microorganims has been widely applied in industry. Similarly, in this work, it was aimed to obtain monomeric sugars from lignocellulose which in turn was used as carbon source for the cellulose producing bacteria- Novacetimonas hansenii. On the other hand, olive pomace (OP) is a by-product of the olive oil industry. Olive pomace is made up of olive pulp, stones, and skin of the fruit. It may pose environmental problems if not conducted properly. Approximately 35 - 45 kg dry olive pomace is obtained from 100 kg olive during oil production. This material is generally sorted and burned for energy production Nevertheless, it has been shown that OP is high in lignocellulosic content and the need for a more efficient way to use this material is apparent. In the first part of the study, two strains were compared according to their cellulose production yields. In addition, several cultivation conditions were performed to determine the most effective method. N. hansenii (ATCC 53582) in static growth condition gave the best results and therefore applied on the next steps. Moreover, three agricultural waste products; meat-bone flour, fish flour, and olive pomace were investigated for their efficiency to function as growth media. While no cellulose was formed with meat-bone flour and fish flour media, little cellulose was obtained in the medium prepared with olive pomace and lactose. After that, several trials with the use of olive pomace as nitrogen source while examining the performance of lactose and glucose as carbon sources were realized. However, elementary analysis revealed that the nitrogen content of olive pomace was not sufficient to supply the growth medium as nitrogen source. Besides, cellulose produced in olive pomace medium had poor mechanical qualities which is not suitable for any further application. On the other hand, olive pomace was shown to possess high organic component with approximately 45 % carbon. Therefore, in the second part of the study, generation of monomeric sugars by degradation of the lignocellulose of olive pomace was aimed. For this, acidic pretreatment and enzymatic hydrolysis were applied respectively. Acidic pretreatment breaks down the complex organization of lignocellulosic material to expose cellulose and hemicellulose for enzymatic degeneration. Subsequent enzymatic hydrolysis generates monomeric sugars which can be used by microorganisms. In this study, olive pomace was pretreated with 1 % phosphoric acid at 170 oC at 8 bar in order to separate cellulose from hemicellulose and lignin, thus unveiling the hydrolysable ends and producing oligosaccharides. Consecutive enzymatic reaction was conducted at 50 oC for 72 h with enzyme:substrate concentrations varying from 1.5 to 30 % (w/w) in static and agitated conditions. The reducing sugar concentration of the liquid part following the acidic pretreatment was determined by glucose hexokinase assay and found to be too low to lead to any microbial growth. Furthermore, reducing sugar concentration of each enzymatic hydrolysate was detected by dinitrosalicylic acid (DNSA) assay. Among varying enzyme:substrate concentrations, 30 % enzyme reaction in static condition resulted in the highest reducing sugar yield with 9.31 g/l. Enzymatic hydrolysates were scanned for the presence of galactose, glucose, mannose, arabinose, xylose, rhamnose, lactose, fructose, maltose and cellobiose by HPLC and distribution of these sugars along different hydrolysates was determined. For each hydrolysate, glucose was found to be the major monosaccharide. Growth media were prepared from selected hydrolysates with the ingredients of Hestrin-Schramm medium, except the carbon source, however, no cellulose formed. Therefore, the hydrolysates were detoxified to eliminate the inhibitory molecules generated in course of pretreatment. Among the methods attempted, Ca(OH)2 treatment was shown to be the most effective. From the media prepared with detoxified hydrolysates, the highest amount of bacterial cellulose production was 0.68 g/l. In addition, Hestrin-Schramm conventional medium and the medium with enzymatic hydrolysates were compared according to the substrate conversion ratio, cellulose production rate and yield. Sugar consumption in the control and test media was also detected. In the third part of the study, bacterial cellulose produced in the alternative medium was characterized with X-ray Diffraction Analysis, Fourier-Transform Infrared Spectroscopy (FTIR), and Scanning Electron Microscopy (SEM). X-ray diffraction demonstrated that the new BC posssesed the typical cellulose I peaks. However, the BC had a small amount of phosphate salts which caused HAP signals on the diffratogram. The FTIR analysis showed that the new bacterial cellulose had the characteristic spectrum of cellulose and no impurities were found. Similarly, with SEM analysis, it was demonstrated that the new material had nano-sized fibrillary structure very similar to the control material.