Cellulosic Production Research Articles

Production of Kombucha SCOBY Cellulose by Using Tea and Pumpkin Peel Waste as Fermentation Substrates: A Comparison Study

A market research survey by Technavio has estimated that the global market size of kombucha is projected to grow by USD 3.56 billion from 2021 to 2026 with a Compound annual growth rate (CAGR) value of 19.36%. Kombucha can generate new pellicle Symbiotic Culture of Bacteria and Yeast (SCOBY) that produces biopolymer of cellulose. The production of cellulose from fermentation of kombucha SCOBY has received great attention from scientists. The main objective of this preliminary study was to produce kombucha SCOBY cellulose (KSC) from tea and pumpkin peel waste as fermentation substrates. The carbohydrate and protein contents of both tea and pumpkin peel waste were determined by proximate analysis. The physicochemical properties of kombucha SCOBY cellulose (KSC) were studied using two spectroscopic techniques, namely Fourier Transform Infrared (FTIR) and X-Ray Diffraction (XRD). FTIR spectra revealed the existence of functional groups in KSC. Meanwhile, XRD spectra presented the crystallinity by diffraction peak and crystal lattice of KSC. Overall, results from FTIR and XRD analyses suggest that both kombucha SCOBY cellulose obtained from tea and pumpkin peel fermentation substrates possess similar physicochemical properties to that of commercial cellulose. In conclusion, the application of tea and pumpkin peel waste as fermentation substrates in production of kombucha SCOBY cellulose is feasible.

Integrated production of bacterial cellulose and biogas from agro-industrial wastewater: technical feasibility of implementing a biorefinery concept

Integrated production of bacterial cellulose and biogas from agro-industrial wastewater: technical feasibility of implementing a biorefinery concept

Production and characterization of bacterial cellulose by Rhizobium sp. isolated from bean root

Bacterial cellulose (BC) is a natural polymer renowned for its unique physicochemical and mechanical attributes, including notable water-holding capacity, crystallinity, and a pristine fiber network structure. While BC has broad applications spanning agriculture, industry, and medicine, its industrial utilization is hindered by production costs and yield limitations. In this study, Rhizobium sp. was isolated from bean roots and systematically assessed for BC synthesis under optimal conditions, with a comparative analysis against BC produced by Komagataeibacter hansenii. The study revealed that Rhizobium sp. exhibited optimal BC synthesis when supplied with a 1.5% glucose carbon source and a 0.15% yeast extract nitrogen source. Under static conditions at 30 °C and pH 6.5, the most favorable conditions for growth and BC production (2.5 g/L) were identified. Modifications were introduced using nisin to enhance BC properties, and the resulting BC-nisin composites were comprehensively characterized through various techniques, including FE-SEM, FTIR, porosity, swelling, filtration, and antibacterial activity assessments. The results demonstrated that BC produced by Rhizobium sp. displayed properties comparable to K. hansenii-produced BC. Furthermore, the BC-nisin composites exhibited remarkable inhibitory activity against Escherichia coli and Pseudomonas aeruginosa. This study contributes valuable insights into BC’s production, modification, and characterization utilizing Rhizobium sp., highlighting the exceptional properties that render it efficacious across diverse applications.

GROUPING OF CLONES OF 4-YEAR-OLD Eucalyptus spp. FOR PULP AND PAPER

As in other countries, In Brazil, new genetic materials of Eucalyptus spp. and their hybrids are multiplied through cloning. These materials, currently in experimental trials, must undergo several stages to select the best ones for pulp and paper production. Therefore, new studies on wood quality are essential. Therefore, this study aimed to group 11 clones of Eucalyptus spp. wood, from a clonal plantation in the municipality of Palmital, São Paulo State, for the production of paper and cellulose. For this purpose, four trees of each clone of 4-year-old Eucalyptus spp. were collected. From each tree, a log of 1 m in length was taken from the base of the tree, for the study of the characterization of the basic density and cellular dimensions of the wood. The results showed that there were significant differences between clones for basic density, fiber length, vessel element length and fiber wall thickness. The Runkel ratio, wall fraction and stiffness coefficient did not show significant differences between the different genotypes. From the results obtained, we can conclude that clones can be differentiated only by basic density, fiber length, vessel element length and fiber wall thickness. The Runkel index, flexibility coefficient and wall fraction of Eucalyptus spp. were more efficient to group the clones into two groups.

Juncus rigidus high biomass and cellulose productivity under wastewater salinity stress - A paradigm shift to the valorization of RO reject water

The use of water purifiers is intensively catching up and disposing of reverse osmosis reject water is of great concern. Reject water management using conventional methods is costly and harmful to the environment. To address this issue, the present study aims to utilize reverse osmosis reject wastewater using an eco-friendly approach. Juncus rigidus was treated with reject wastewater containing different salinity levels. Wastewater-treated plant dry biomass increased with increasing reject water salinity, and 625.3 g dry biomass recovered in treatment-B (~18,520 ppm). However, ~23,220 ppm wastewater salinity was lethal to the plants. The cellulose was extracted by alkali hydrolysis. The cellulose content in the wastewater-treated biomass was significantly higher in Treatment-B compared to both the control and Treatment-A (~12,744 ppm). The water salinity enhanced the cellulose (26.49 %) production in J. rigidus. Cellulose purity was confirmed using spectroscopic and thermogravimetric means. XRD shows highest crystallinity Index (77.29) with a d-spacing of 4.7 Å and 5.7 nm crystallite size in treatment-B. FTIR results reveal well-defined relevant peaks for OH, CH, CO, CH2, C-O-C, CO groups in treatment-B cellulose. Salinity impacts carboxyl groups in treatment B cellulose with a sharper and intense peak at 1644 cm−1 responsible for water absorption. Treatment-B exhibits higher thermal stability due to increased crystallinity. DSC shows endothermic depolymerization of cellulose with distinct peaks for different treatments. Morphological traits got better with increasing salinity with no adverse effect on cellulose. Salinity moderately affected the water absorption capacity of cellulose. All cellulose samples were devoid of gram-negative bacteria known by microbial test. This pioneering work underscores the plant's remarkable capacity not only to accomplish the circular economy by the valorization of wastewater obtained from various water purifiers for Juncus cultivation for cellulose production for diverse applications but also to generate income from wastewater.

Enhancing cellulose fiber properties from chromolaena odorata and anana comosus through novel pulping chemical mixtures

The production of cellulose with exceptional properties has led to the modification of chemical pulping methods and the development of newer chemical pulping methods. This study developed mixtures of sodium hydroxide, ethanol, and anthraquinone as new pulping chemicals to produce alpha-cellulose from pineapple leaves and Saim weed stems. The addition of anthraquinone was noticed to influence the pulp yield positively and the FTIR of all the cellulose has the characteristic cellulose bands. Calculated Total crystallinity index (TCI), (Lateral order index) LOI., and Hydrogen bond intensity (HBI) from the FTIR presented PLSHAEC (68.38 %) and SWSHQEC (67.74 %) having high crystallinity. From the XRD, the 2? by all the materials is attributed to cellulose I diffraction, and the crystallinity index aligned with FTIR determined crystallinity. The determined particle average diameter using ImageJ software showed that PLSHQEC 3.00 had the smallest value, followed by 5.17 for PLSHQC, 5.32 for SWSHQEC, and 6.03 for SWSHQEC. From the thermal analysis, the onset of the degradation of all the cellulose occurred at different temperatures: 247.10? (PLSHQC), 253.38? (PLSHQEC), 240.20? (SWSHQC) and 242.58? (SWSHQEC). The increase in yield, higher crystallinity index, and smaller fiber diameter of the cellulose demonstrated anthraquinone as a better additive to produce quality cellulose that would undergo easy chemical modification.

Recycling kaolin from paper waste and assessment of its application for paper coating

In the cellulose industry, the paper coating process plays an essential role in reducing manufacturing costs while increasing the quality of the products. Given that cellulose products are being used increasingly, the ability to recycle and reuse these materials might be highly beneficial in this industry. Thus, kaolin—one of the most commonly utilized ingredients in the cellulose industry that has a major impact on the final quality—was recycled from paper waste for this study. Then, to confirm the successful recycling process, the particle distribution, morphology, and chemical composition of recycled kaolin were analyzed by DLS, SEM/EDS, FTIR, XRF, and XRD. The related results verified the recycled kaolin's high purity and crystallinity. Subsequently, coatings with thicknesses of 30, 60, 90, and 120 μm were applied to paper using a suspension of recycled kaolin. Their mechanical properties (burst strength and tensile strength), permeability (air passage, the amount of which indicates the porosity of the paper structure), wettability (the contact angle can reveal the wetting ability of the paper), and water absorbance (the Cobb test) were then assessed. The pertinent findings demonstrated that applying a coating considerably improves paper quality. So, a coating with a thickness of 30 µm is superior to uncoated paper. In addition, among all the coating thicknesses, the treatment with the best mechanical strength, permeability, contact angle, and Cob value values had a coating thickness of 120 µm. The corresponding values for the tensile index, burst index, air passage amount, and contact angle are 70 kNm/kgr, 3.61kPa m2/gr, 680 ml/min, and 46֯ ±2. Consequently, the 120 µm-thickness treatment was determined to be the most effective one in this investigation.

RESOURCE-SAVING WHEAT STRAW PROCESSING TECHNOLOGY

The wheat straw delignification condition influence in the medium “acetic acid-water-hydrogen peroxide-citric acid catalyst” to the yield and cellulose product composition was studied. It was established that the fibrous semi-finished product output and the residual lignin content are decrease with increasing temperature and delignification duration. The oxidative wheat straw delignification process was described by a first-order kinetic equation, and it was characterized by low activation energy values of 16.7 kJ mol-1. The oxidized cellulose sample’s structure was studied using IR spectroscopy.

Production and Characterization of Bacterial Cellulose Synthesized by Komagataeibacter sp. Isolated from Rotten Coconut Pulp

Bacterial cellulose is a naturally occurring polysaccharide that is produced by acetic acid bacteria. It has become increasingly popular due to its numerous biotechnological applications. Bacterial cellulose is free from contaminants that are often associated with plant cellulose. This study aims at isolation of bacterial cellulose producing microbial strains from rotten fruits. The selected bacterial cellulose producer was identified using biochemical tests, including biochemical, carbohydrate fermentation, morphology, gram staining and 16S rRNA sequence analysis. Furthermore, several different media were evaluated for higher production of cellulose. The produced cellulose sheets were washed, purified and characterized using various analytical techniques, including FTIR, XRD and SEM analysis. A potential high bacterial cellulose yielding microbial strain was isolated from coconut pulp waste and identified as Komagataeibacter saccharivorans strain BC-C1 using 16S rRNA sequencing. Effect of media components on bacterial cellulose production was studied. The isolate yielded 0.52 g/100 mL with Hestrin-Schramm (HS) culture media (designated as M1) and 1.10 g/100 mL in M5 media. The physico-chemical characterization demonstrated that produced bacterial cellulose sheets show characteristics IR bands and three-dimensional fibrillar interconnected network structure. The study revealed that isolated Komagataeibacter saccharivorans strain BC-C1 can be utilized for large scale production of bacterial cellulose.

Multi-omics revealed the mechanism of feed efficiency in sheep by the combined action of the host and rumen microbiota

This study was conducted to investigate potential regulatory mechanisms of feed efficiency (FE) in sheep by linking rumen microbiota with its host by the multi-omics analysis. One hundred and ninety-eight hybrid female sheep (initial body weight = 30.88 ± 4.57 kg; 4-month-old) were selected as candidate sheep. Each test sheep was fed in an individual pen for 60 days, and the residual feed intake (RFI) was calculated. The ten candidate sheep with the highest RFI were divided into the Low-FE group, and the ten with the lowest RFI were divided into the High-FE group, all selected for sample collection. The RFI, average daily gain and average daily feed intake were highly significantly different between the two experimental groups (P < 0.05). Compared with Low-FE group, the insulin-like growth factor-1 and very low-density lipoprotein in serum and the propionate in rumen significantly increased in High-FE group (P < 0.01), but the acetate:propionate ratio in rumen significantly decreased in High-FE group (P = 0.034). Metagenomics revealed Selenomonas ruminantium, Selenomonas sp. and Faecalibacterium prausnitzii were key bacteria, and increased abundance of the genes encoding the enzymes for cellulose degradation and production of propionate in High-FE group. The results of proteomics and section showed the rumen papilla length (P < 0.001) and expression of carbonic anhydrase and Na+/K+-ATPase were significantly higher in High-FE group (P < 0.05). On the other hand, the acetyl-CoA content significantly increased in the liver of High-FE group (P = 0.002). The relative expression levels of insulin-like growth factor-1 and apolipoprotein A4 genes were significantly up-regulated in the liver of High-FE group (P < 0.01), but relative expression level of monoacylglycerol O-acyltransferase 3 gene was significantly down-regulated (P = 0.037). These findings provide the mechanism by which the collaborative interaction between rumen microbiota fermentation and host uptake and metabolism of fermentation products impacts feed efficiency traits in sheep.

Valorisation of bread wastes via the bacterial cellulose production

AbstractThe short shelf life of bread can be attributed to changes in its textural and sensory properties, a process termed staling, and large amounts of bread residue and waste are generated daily. Because the main component of bread is starch, the use of bread wastes as a substrate for bacterial cellulose (BC) production can significantly contribute to valorisation and reuse of wastes. This study aimed to investigate the BC production potential of various stale breads, convert these wastes into usable forms for food and other industries, and increase their economic value. Stale breads were hydrolyzed with dilute acid, and BC-producing bacteria from Kombucha tea were isolated and identified as Gluconobacter oxydans MG2021 (GO). BCs were produced from bread hydrolysates with GO and Komagataeibacter hansenii GA2016 (KH), and their properties were examined. The results indicated that stale breads represented a good source for BC production, as high BC yields were obtained using GO (8.81%–25.02%) and all BCs had superior properties such as high crystallinity (75.96%–91.39%), thermal stability, liquid holding capacity, and fine fibers (40.16–85.39 nm). This study demonstrated that bread wastes could be used as a low-cost substrate for large-scale BC production, and the abundance of bread wastes demonstrated their potential as a resource for commercial BC producers.

Influence of feedstock selection on cellulosic ethanol production based on densified biomass with calcium hydroxide and regular steam pretreatment

Feedstock selection is a key factor affecting the process of cellulosic ethanol production. Densifying lignocellulosic biomass with chemicals (calcium hydroxide) followed by regular steam autoclave (DLCA(ch)) is an emerging and efficient pretreatment technology. In this study, different crop straws were selected as feedstock in a DLCA(ch) based process for cellulosic ethanol production, and their experimental and economic performances were evaluated. Both single crop straws and mixed crop straws were used and compared as feedstock for DLCA(ch) based biorefinery. Among single crop straws of corn stover, rice straw and wheat straw, wheat straw had the highest ethanol yield and the lowest feedstock cost, with a minimum ethanol selling price of 2.03 $/gal ethanol considering a 10 % internal rate of return and a 2000 tons crop straw per day biorefinery scale. When mixed crop straws were used as the feedstock, both feedstock cost and ethanol production cost were further reduced, and the minimum ethanol selling price was reduced to 2.02 $/gal ethanol. In addition, the use of mixed crop straws has great potential for large scale DLCA(ch) based process for cellulosic ethanol production.

Cellulosic rich biomass production with optimized process parameters by using glycerol pretreatment for biofuels applications

In this work, we conduct acidified aqueous glycerol pre-treatment (AAG) on rice husks (RH) and utilize the response surface methodology (RSM) to assess the impact of pre-treatment parameters. The primary objective of this research is to optimize the parameters to maximize the cellulose content within RH. The parameters under consideration encompassed temperature (ranging from 80 to 110 °C), retention time (spanning 15 to 45 min), and biomass loading (varying from 5 to 10 wt. %). To achieve this optimization, we perform the Box-Behnken Design (BBD) within the framework of RSM. Additionally, we scrutinize the interactive effects of these parameters on cellulose content. Our findings unveiled a remarkable increase in cellulose content, escalating from 40 % in untreated RH to an impressive 75 % in pre-treated RH under the optimized conditions of 110 °C for 45 min with a 5.0 wt. % biomass loading. To further evaluate the effectiveness of the pre-treatment process, we conduct scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses, shedding light on alterations in surface morphology and crystallinity of RH. This investigation yields valuable insights, presenting novel opportunities for the efficient conversion of readily available rice husks into high-value products, such as biofuels and composites.

A cellulosomal yeast reaction system of lignin-degrading enzymes for cellulosic ethanol fermentation.

Biofuel production from lignocellulose feedstocks is sustainable and environmentally friendly. However, the lignocellulosic pretreatment could produce fermentation inhibitors causing multiple stresses and low yield. Therefore, the engineering construction of highly resistant microorganisms is greatly significant. In this study, a composite functional chimeric cellulosome equipped with laccase, versatile peroxidase, and lytic polysaccharide monooxygenase was riveted on the surface of Saccharomyces cerevisiae to construct a novel yeast strain YI/LVP for synergistic lignin degradation and cellulosic ethanol production. The assembly of cellulosome was assayed by immunofluorescence microscopy and flow cytometry. During the whole process of fermentation, the maximum ethanol concentration and cellulose conversion of engineering strain YI/LVP reached 8.68g/L and 83.41%, respectively. The results proved the availability of artificial chimeric cellulosome containing lignin-degradation enzymes for cellulosic ethanol production. The purpose of the study was to improve the inhibitor tolerance and fermentation performance of S. cerevisiae through the construction and optimization of a synergistic lignin-degrading enzyme system based on cellulosome.

Whole genome sequencing analysis of Komagataeibacter nataicola reveals its potential in food waste valorisation for cellulose production.

Komagataeibacter nataicola (K. nataicola) is a gram-negative acetic acid bacterium that produces natural bacterial cellulose (BC) as a fermentation product under acidic conditions. The goal of this work was to study the complete genome of K. nataicola and gain insight into the functional genes in K. nataicola that are responsible for BC synthesis in acidic environments. The pure culture of K. nataicola was obtained from yeast-glucose-calcium carbonate (YGC) agar, followed by genomic DNA extraction, and subjected to whole genome sequencing on a Nanopore flongle flow cell. The genome of K. nataicola consists of a 3,767,936bp chromosome with six contigs and 4,557 protein coding sequences. The maximum likelihood phylogenetic tree and average nucleotide identity analysis confirmed that the bacterial isolate was K. nataicola. The gene annotation via RAST server discovered the presence of cellulose synthase, along with three genes associated with lactate utilization and eight genes involved in lactate fermentation that could potentially contribute to the increase in acid concentration during BC synthesis. A more comprehensive genome study of K. nataicola may shed light into biological pathway in BC productivity as well as benefit the analysis of metabolites generated and understanding of biological and chemical interactions in BC production later.

Cellulose and lignin purified from Metroxylon sagu palm fronds by a new technology with 2-methylanthraquinone cooking and peroxymonosulfuric acid bleaching

The demand for high-purity cellulose, optimization of wood utilization, and environmentally friendly processes has increased in dissolving pulp (DP) production. Sago palm fronds (SPF), an abundant agricultural waste in Indonesia, hold great potential as a raw material for cellulose, hemicellulose, and lignin production. This study aimed to explore the production of from SPF by employing a combination of prehydrolysis, soda cooking with 2-methylanthraquinone (MAQ) as a green additive (PHS-MAQ), and totally chlorine-free (TCF) bleaching with peroxymonosulfuric acid (Psa). Furthermore, lignin was recovered from the black liquor of PHS-MAQ. The results showed that prehydrolysis at 150 ºC for 3 h, followed by soda-MAQ cooking at 160 ºC for 1.5 h using 0.03% of MAQ, 23% active alkali (AA), and a five-stage bleaching with oxygen (O), Psa, alkaline extraction with hydrogen peroxide (Ep), Psa, and Ep successfully produced high-purity cellulose as DP, with properties of 94.3% α-cellulose content, 89.9% ISO brightness (SNI ISO 2470–1:2016), 9.1 cP viscosity, and 0.13% ash content. Moreover, the soda-MAQ cooking method exhibited superior delignification compared to prehydrolysis kraft (PHK) and prehydrolysis soda (PHS) processes in a range of kappa numbers of 9.4–22.6 at 17–25% AA. The inclusion of MAQ increased pulp yields by 4.6–4.9% and decreased kappa number by 1.6–3.1 compared to the PHS without additives at similar AA. Lignin was separated from the PHS-MAQ, with yields of 69–77%. This work demonstrated the suitability of SPF processed by PHS-MAQ cooking and Psa bleaching for the preparation of viscose rayon and cellulose derivatives. The lignin recovery could be an attractive biorefinery process in modern pulp mills.

Влияние вида ножевой размалывающей гарнитуры на процесс получения микрокристаллической целлюлозы

The article shows the possibility of intensifying the process of producing microcrystalline cellulose via pregrinding of fibrous semi-finished products before hydrolysis. The technological factors determining the grinding of fibrous materials have been considered. The efficiency of the microcrystalline cellulose production process is influenced by the choice of its production technology. For grinding, a semi-industrial disc mill with a tacking with rectilinear and curvilinear knives has been used. Multiphysics models of the flow of fibrous mass in the grinding zone of a disc mill with different knife tacking patterns have been constructed. The morphological properties of the fiber have been measured on the Morfi Neo automatic fiber analyzer after grinding to 65 °SR. The nature of the change in the properties of bleached sulfate hardwood and softwood cellulose has been analyzed. It has been found that it is identical for all degrees of grinding, but the quantitative characteristics change to the greatest extent when grinding softwood cellulose using a tacking with rectilinear knives: the weighted average length of fibers decreases by 17 %, the width – by 14 %; the content of broken fibers increases by 22 %, the content of fines along the length – by 67 % and the fibrillation index – by 1.9 times. A comparative analysis of the values of the degree of polymerization depending on the knife tacking pattern has been carried out. The conditions for producing microcrystalline cellulose after grinding the fibrous mass in a semi-industrial disc mill, depending on the degree of grinding, have been determined. It has been shown that with an increase in the degree of grinding of the fibrous mass from 15 to 65 °SR, the degree of polymerization of microcrystalline cellulose decreases from 272 to 120, the concentration of hydrochloric acid – from 2.5 to 1.5 N and the duration of hydrolysis – from 120 to 90 min. The developed method for producing microcrystalline cellulose makes it possible to reduce the cost of chemical treatment of the fibrous mass (acid concentration, treatment duration and hydrolysis temperature) by 1.5 times.

The influence of reed nanocellulose on the quality indicators of paper-basis for wallpapers

Improving the production technology of products with improved quality indicators and with less impact on the environment remains an urgent scientific and practical problem of paper industry enterprises. This refers to the technology for the production of paper-bases for wallpaper using environmentally safe chemical additives, in particular nanocellulose (NC), which was obtained by acid hydrolysis from reed cellulose. The production of cellulose was carried out in two stages - extraction from reed stalks with a solution of alkali and an organosolvent method of cooking in a solution of peracetic acid. The obtained organosolvent cellulose contained the remains of lignin and mineral substances, which allows it to be used for the preparation of NC. As a result of the hydrolysis of organosolvent cellulose, a time-stable NC suspension was extracted, the properties of which were investigated by scanning electron microscopy (morphological changes in the structure of reed cellulose-containing materials), atomic force microscopy (determination of topographic characteristics of NC), analysis of electronic absorption spectra (transparency of NC films). It was established that reed NC films had nanoparticles with a cross-sectional size of 5-25 nm, density up to 1.52 g/cm3, transparency up to 81.6 %, tensile strength up to 65 MPa. For the production of castings of paper-bases for wallpaper samples, sulfated pine bleached cellulose and polyester synthetic fiber were used, to the fibrous mass of which were added the following chemical additives: alkyl ketene dimer, reed NC, binder, and optical brightener. It has been shown that the use of nanocellulose at a rate of 0.35 % to 1.0 % of the paper mass leads to a significant improvement in the physical and mechanical quality indicators of the paper base for wallpaper. It was established that the replacement of 50 % of the synthetic chemical auxiliary substance alkyl ketene dimer with nanocellulose or additional application of nanocellulose at a rate of 0.5 g/m2 on the surface of the casting with 0.7 % alkyl ketene dimer allows to obtain paper that meets the requirements of the standard. The obtained results indicate the prospects of using reed nanocellulose for the production of other mass types of paper and cardboard.

Engineering Compositionally Uniform Yeast Whole-Cell Biocatalysts with Maximized Surface Enzyme Density for Cellulosic Biofuel Production.

In recent decades, whole-cell biocatalysis has played an increasingly important role in the food, pharmaceutical, and energy sector. One promising application is the use of ethanologenic yeast displaying minicellulosomes on the cell surface to combine cellulose hydrolysis and fermentation into a single step for consolidated bioprocessing. However, cellulosic ethanol production using existing yeast whole-cell biocatalysts (yWCBs) has not reached industrial feasibility due to their inefficient cellulose hydrolysis. As prior studies have demonstrated enzyme density on the yWCB surface to be one of the most important parameters for enhancing cellulose hydrolysis, we sought to maximize this parameter at both the population and single-cell levels in yWCBs displaying tetrafunctional minicellulosomes. At the population level, enzyme density is limited by the presence of a nondisplay population constituting 25-50% of all cells. In this study, we identified the cause to be plasmid loss and successfully eliminated the nondisplay population to generate compositionally uniform yWCBs. At the single-cell level, we demonstrate that enzyme density is limited by molecular crowding, which hinders minicellulosome assembly. By adjusting the integrated gene copy number, we obtained yWCBs of tunable enzyme display levels. This tunability allowed us to avoid the crowding-limited regime and achieve a maximum enzyme density per cell. As a result, the best strain showed a cellulose-to-ethanol yield of 4.92 g/g, corresponding to 96% of the theoretical maximum and near-complete conversion (∼96%) of the starting cellulose (1% PASC). Our holistic engineering strategy that combines a population and single-cell level approach is broadly applicable to enhance the WCB performance in other biocatalytic cascade schemes.

Current research and advances in the molecular aspect of bacterial cellulose synthesis with special emphasis on the production of bacterial cellulose from waste and its food applications

Current research and advances in the molecular aspect of bacterial cellulose synthesis with special emphasis on the production of bacterial cellulose from waste and its food applications