Browsing by Author "Govinden, Roshini."

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Characterization and analysis of keratinous material from waste chicken feathers as protein ingredient for animal feed.
(2022) Kekana, Lizzy Mpho.; Sithole, Bishop Bruce.; Govinden, Roshini.
Keratin is one of the most abundant proteins, which is derived from wool, feathers, nails, hair, and other sources. Chicken feathers are a well-known keratin waste by-product, produced in large quantities by poultry slaughterhouses. Their disposal is expensive, and includes incineration of the waste thus contributing to greenhouse gases; or disposal in landfills, also leading to environmental pollution or they can be recycled into low-quality feeds for animals. Research is done worldwide for the beneficiation of waste chicken feathers into commercial products; these include cosmetics, pharmaceutical products, and biomedical products, and it is also useful in the production of animal feed. The focus of this research was to characterize and analyze keratinous hydrolysates formed from waste chicken feathers using enzymatic and chemical hydrolysis for their suitable applications in different industries. The novelty of this project is based on looking at analytical techniques of the keratinous hydrolysate produced from newly formed keratinolytic microorganisms and newly optimized chemical methods from the waste chicken feathers. Different fungal and bacterial strains were tested for the degradation of waste chicken feathers. The quality and quantity of the hydrolysate formed were determined by using a combination of analytical techniques, where the characterization is done via proximate and ultimate analysis. We used Fourier Transform Infrared Spectroscopy (FTIR), which showed the presence of the keratinous structure, which is known to have high protein content. Thermogravimetric Analysis (TGA), showed that a thermally stable hydrolysates were obtained, which is known to be formed by the hydrophobic hydrolysate, which is best for animal feed. CHNS analysis showed evidence that we have high protein content in the hydrolysate. Bradford assay revealed different quantities of the hydrolysate while Sodium Dodecyl Sulphate–Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), showed mostly medium to low molecular weight, due to the presence of amino acids and small peptide chain. A low Ash Content was obtained which means a cleaner fraction of keratin. The hydrolysate formed from the enzymatic hydrolysis contains a mixture of amino acids and peptides. These peptides and essential amino acids formed are known to play a special role in various biological activities. The hydrolysates formed from different degradation methods were also compared, focusing on the qualities and quantities formed from enzymatic and chemical hydrolysis. While looking at all the characterization techniques, enzymatic was the best and suitable for animal feed due to the obtained keratin structure, which is more soluble, contains high protein content, has low molecular weights, and has a cleaner fraction of keratin. Future work will be based on obtaining a peptide chain using Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS), then testing the hydrolysates for bioactivities.
Cloning of the endomannanase from Scopulariopsis candida LMK008 and evaluation of its effect on the digestibility on animal feed.
(2012) Gareeb, Ashant Pravin.; Govinden, Roshini.; Setati, Mathebata Evodia.
Present within the biodiverse hypersaline environment are a wide variety of halotolerant filamentous fungi. Many of these phytopathogens are capable of hydrolysing plant cell wall polysaccharides such as hemicellulose which are comprised of mannans and heteromannans which are polymers of the mannose sugars. Endoacting hydrolytic enzymes such as endo-β-1,4-mannanases are secreted into the extracellular environment and are involved in the catalysis of the random hydrolysis of β-1,4-mannosidic linkages within the backbone of mannan, galactomannan, glucomannan, and galactoglucomannan. Poultry are monogastric animals that are unable to efficiently digest high-fibre and mannan rich feeds such as soybean meals and this results in decreased or depressed animal performance. The use of feeds supplemented with β-mannanases has been shown to enhance the feeding value of mannan-based meals. In the current study, the degradation of β-mannan polysaccharides present in poultry feed by halotolerant Scopulariopsis candida LMK008 β-mannanase was investigated. SDS-PAGE, Native-PAGE in conjunction with zymogram analysis was used to assess the molecular weight of the endomannanases. At least three isozymes were detected: two of 56 kDa (pI 3.5 and 6.7) and one of 28 kDa. Anion exchange chromatography was used to purify the 28 kDa isozyme. Three mannan-based substrates, viz., locust bean gum, guar gum and soybean flour, were used to evaluate the hydrolysis capability of the crude as well as the purified β-mannanase via the release of reducing sugars and was detected using the DNS assay. The β-mannanase exhibited low activity with pure guar gum but high activity with locust bean gum galactomannan and soybean flour mannan. The hydrolysis activities of the crude and purified enzyme were then tested further on mannan-based soybean meals. In general it was found that more reducing sugars were released from the grower feed than the starter and layer feeds. Another common hydrolysis pattern observed in all feed types was that after prolonged incubation of 24 h there was a decrease in the amount of reducing sugars released which could be attributed to the presence of naturally-occurring microorganisms in the feed sample which metabolised the simple sugars resulting from the enzymatic hydrolysis of the mannan components in the feed samples. This was confirmed by standard plate count assays. The results obtained are encouraging and the purified β-mannanase could be applied as an industrial feed additive within the animal feed industry, however, further testing of the enzyme in situ is needed in order to prove its applicability. The cloning of the endomannanase has to date proven unsuccessful despite numerous techniques being employed and further research is also needed to accomplish this task.
Genetic manipulation of saccharomyces cerevisiae for improved ethanol production from d-xylose.
(1999) Govinden, Roshini.
No abstract available.
Heterologous expression and application of lipolytic enzymes for mitigation of lipophilic compounds in eucalyptus species.
(2017) Ramnath, Lucretia.; Govinden, Roshini.; Sithole, Bishop Bruce.
Abstract available in PDF file.
Location and partial purification of phosphate dependent glutaminase from rat small intestine.
(2014) Manzini, Christie Zandile.; Masola, Bubuya.; Govinden, Roshini.
Phosphate dependent glutaminase (PDG) is a key enzyme in intestinal energy metabolism and hence has an impact on nutrient absorption and nutritional status of the whole animal. PDG is known to be a mitochondrial enzyme but its sub-mitochondrial location is still controversial. Due to its instability, PDG has never been purified from the small intestine. The sub-mitochondrial localization of PDG was investigated by tracking the release of PDG and that of marker enzymes for sub-mitochondrial compartments following fractionation of mitochondria using digitonin. The dependence of PDG activity on the membrane phospholipids was investigated using phospholipase A2 treatment while the orientation of the enzyme in mitochondria was probed using sulphydryl inhibitors Mersalyl (Mers) and N-ethylmaleimide (NEM). PDG was partially purified after solubilizing PDG using different methods including lyophilization combined with digitonin fractionation and sonication in the presence of a stabilizing buffer. Solubilized proteins were separated by gel filtration chromatography. SDS-PAGE and Western blotting were conducted on fractions collected to show protein profiles and location of PDG bands. Release of PDG was intermediate between that of cytochrome c oxidase and matrix enzymes. Phospholipase A₂ treatment exhibited a time dependent loss of PDG activity. In intact mitochondria Mers inhibited 97% and NEM inhibited 64% of PDG activity at 1 mM concentration; with a more pronounced effect when combined with sonication. Pre-incubation of mitochondria in a stabilizing buffer before solubilization activated PDG 37-fold. Partial purification was achieved after using Sephacryl S-300 HR. Coomassie stained SDS-PAGE confirmed the partial purification of PDG with bands on Western blot observed to be 63-65 kDa, 50 kDa and 42 kDa. In conclusion, the results suggest that intestinal PDG is localized in two sub-mitochondrial fractions with each displaying a different form: PDG with a molecular weight of 50 kDa being localized in the mitochondria matrix and a 63-65 kDa PDG being bound to the mitochondria inner membrane. The membrane bound PDG requires the presence of phospholipids to retain its activity.
Molecular and biochemical characterisation of ethanolic D-xylose fermenting Pichia stipitis, Candida shehatae and their fusants.
(1994) Govinden, Roshini.; Pillay, Basil Joseph.; Pillay, D.
No abstract available.
Partial purification and characterisation of Phialophora alba xylanases and its application to pretreated sugarcane bagasse.
(2013) Mosina, Leticia Ntsoaki.; Govinden, Roshini.; Masola, Bubuya.
Xylan is the major component of hemicellulose and its degradation can be achieved through the hydrolytic action of microbial xylanases. Xylanases have an array of applications one being bioethanol production. The lack of thermophilic xylanases has prompted the search for new enzymes with increased thermostability. Previous work on the crude enzyme of Phialophora alba has demonstrated optimal activity (39 U/μg) at a pH of 4 and two temperature optima of 50°C and 90°C. These desirable properties highlighted the need for further research on the purified enzyme. In the present study P. alba was identified as a thermophilc Ascomycete that forms conidia and chlamydospores during the asexual and sexual stages of its life cycle, respectively. The various isozymes present in the crude enzyme extract were subsequently detected by zymogram analysis. Up to six xylanase isozymes ranging from 90-210 kDa in size were detected. The crude enzyme was subsequently purified by precipitation and ion exchange chromatography (IEX). Protein precipitation methods, desalting methods, IEX resins, elution buffers and NaCl gradients were optimized. The 31-70% ammonium sulphate precipitate had the highest levels of xylanase activity. Separation of proteins with the anion exchanger, HiTrap Q sepharose fast flow column and a linear gradient of 0-2.5 M NaCl in phosphate buffer (50 mM, pH 7) yielded a partially pure xylanase isozyme with molecular weight of 210 kDa. A final yield of 1.4% and purification fold 10.6 was obtained after ion exchange chromatography. The specific activity of the xylanase was 21 IU/μg. At optimum pH (pH 4) and temperature (50°C) a combined xylanase activity of 32 IU.ml⁻¹ was detected. The partially pure xylanase was stable from pH 4-6 with 86% of xylanase activity retained for 90 minutes. Thermostability was observed from 40-70°C with 95% of activity retained for 90 minutes at optimum temperature. The ability of the partially pure xylanase and crude enzyme to hydrolyze untreated and pretreated (alkali and temperature/pressure) sugarcane bagasse was tested at a constant enzyme loading rate of 15 IU/g. Overall, maximum hydrolysis was achieved with the alkali pretreatment and saccharification with the crude enzyme: approximately, 2.4 g/ml of reducing sugars were liberated over a 48 hours. The partially pure xylanase liberated a maximum amount of 2.3 g/ml reducing sugars after 48 hours. The results obtained highlight the desirable characteristics of the partially pure enzyme and its applicability to bioethanol production.
Polyhydroxyalkanoate production by Bacillus thuringiensis: an aspect of biorefining pulp and paper mill sludge.
(2021) Singh, Sarisha.; Govinden, Roshini.; Sithole, Bishop Bruce.; Lekha, Prabashni.; Permaul, Kugenthiren.
The substantial success of plastic as a material is owed to its unparalleled designs with unique properties and proved versatility in an extensive range of applications. Unfortunately, the reliance on single-use plastic commodities consequently results in the incorrect disposal and accumulation of this waste at staggering rates in our environment and landfill sites. In this regard, there is a vested interest in replacing petrochemical plastics with natural, biodegradable plastics (bioplastics). Of the many natural polymers available, microbially synthesized polyhydroxyalkanoates (PHAs) have gained popularity. Eco-friendly PHA-based bioplastics are characteristically as robust and as durable as their oil-based equivalents. Pulp and paper mill sludge (PPMS) is another solid waste stream that is predominantly disposed of via landfilling. The environmentally hazardous gases and leachate emitted from PPMS together with limited landfill space availability and the implementation of strict waste management legislation may not make landfilling practicable in the future. However, this carbohydrate-rich biomass has favorable traits that make it applicable as a feedstock for microbial biomass and PHA production. Hence, in the interest of addressing the issues mentioned above, this study aimed to beneficiate PPMS into PHAs by applying it as the sole feedstock for microbial cell proliferation and subsequent PHA production. Presently, to the best of the author’s knowledge, there are no reports on PHA production as a route for valorization of PPMS from South African pulp and paper mills. Thus, the novelty of the present study is marked by the unique ways of incorporating PPMS as a low-cost substrate as well as the various fermentative strategies navigated to enhance both microbial cell biomass and PHA productivity. In the present study, it was established that Bacillus thuringiensis had promising PHA-producing capability. The strain synthesized a copolymer and terpolymer using untreated (raw) neutral semi-sulphite chemical pulping and cardboard recycling mill (NSSC-CR) and prehydrolysis kraft and kraft pulping mill (PHKK) PPMS in a consolidated bioprocessing fermentation. A separate hydrolysis and fermentation strategy was pursued whereby a glucose-rich hydrolyzate was obtained from enzymolysis of PPMS and subsequently utilized in a cyclic fed-batch fermentation (CFBF) strategy to obtained enhanced yields of cell biomass and PHAs. Response surface methodology (RSM) was first implemented to optimize the conditions for enzymatic saccharification of de-ashed PHKK PPMS. The optimized variables were; pH 4.89; 51°C; hydrolysis time 22.9 h; 30 U/g β-glucosidase and 60 U/g cellulase; and 6.4% of dried de-ashed PPMS fiber resulting in a hydrolyzate comprising of 48.27% glucose. Thereafter, CFBF was pursued where the glucose-rich hydrolyzate was employed as the sole carbon source for cell proliferation and PHA production. The statistically optimized fermentation conditions to obtain high cell density biomass (OD600 of 2.42) were; 8.77 g L-1 yeast extract; 66.63% hydrolyzate (v/v); a fermentation pH of 7.18; and an incubation time of 27.22 h. The CFBF comprised of three cycles and after the third cyclic event, maximum cell biomass (20.99 g L-1) and PHA concentration (14.28 g L-1) were achieved. This cyclic strategy yielded an almost 3-fold increase in biomass concentration and a 4-fold increase in PHA concentration compared with batch fermentation. The properties of the synthesized PHAs were similar to commercial polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) and also displayed slightly higher thermostability and lower crystallinity compared with commercial PHB and PHBV. This is the first report detailing the proof of concept of using PPMS from South African pulp and paper making mills for cell biomass and PHA production by B. thuringiensis. In addition, this study reports on the practicality and novelty of utilizing PPMS either in its raw, untreated state or as enzymatically saccharified glucose-rich hydrolyzate as cheap substrates applicable for both cell biomass and PHA production using different fermentation strategies. Finally, to the best of our knowledge, this is also the first report that has successfully applied B. thuringiensis in a CFBF strategy coupled with glucose-rich hydrolyzate as the sole carbon source for the production of high cell density biomass and enhanced PHA production. From this study, it is intended that innovative insights and prospective solutions to valorizing pulp and paper mill sludge are provided, whilst simultaneously generating a value-added product.
Screening of fungal isolates for endoglucanase activity and cloning of a thermostable endoglucanase from phialophora alba.
(2016) Isaac, Ashley.; Govinden, Roshini.
Microbes are fast becoming the solution to the energy crisis. Alternative fuels that can be produced by microbes include bioethanol, biodiesel, methane and biohydrogen. This study aimed to identify and characterize a thermostable endoglucanase, a carbohydrate active enzyme which would be part of a cocktail of enzymes used to breakdown cellulose to liberate glucose. The glucose in turn can be fermented to produce bioethanol. Twenty seven fungal isolates were screened for endoglucanase activity on agar plates supplemented with 1% carboxymethyl cellulose. Zones of hydrolysis were visualized by Congo red staining. Ten isolates were selected based on the diameters of hydrolytic zones (presenting high endoglucanase activity) for time course analysis for optimal production of endoglucanase. Isolate D9 (Aspergillus fumigatus) was found to be the highest producer and time course analysis revealed that the highest production is achieved on Day 9 after inoculation. The crude enzyme extract was produced in bulk and precipitated in 40 - 60% ammonium sulphate in order to remove unwanted proteins and to concentrate the endoglucanases. However, following dialysis, more than 80% proteins and endoglucanase activity was lost. Other avenues such as IEF fractionation and freeze drying were explored to achieve partial purification and concentration, respectively, but these proved fruitless. Thus the focus of the study shifted to cloning, heterologously expressing and characterizing an endoglucanase from Phialophora alba, a fungus known to produce lignocellulosic enzymes. Whole genome sequencing was performed and open reading frames (ORFs) were identified using the CLC Main Workbench. These ORFs were analyzed using the CAZymes Analysis Toolkit to identify carbohydrate active enzyme genes. A single endoglucanase gene (GH5) was identified and selected for the duration of the study. Primer sets GH5_ORF_F; GH5_ORF_R and Exp_GH5_ORF_F; Exp_GH5_ORF_R, were specifically designed for cloning into plasmids pTZ57R/T and pPIC9, respectively. The recombinant plasmid pPIC9_13_Eg_GH5 was successfully integrated into Pichia pastoris GS115 genome. The heterologous enzyme was produced; however, no endoglucanase activity was detected. The gene was expressed in Escherichia coli BL21 (DE3), however, no endoglucanase activity was detected in this instance as well. Bioinformatic analysis of the CAZome of P. alba shows a preference for xylan and chitin over cellulose. P. alba appears to be a poor cellulose degrader possessing only a single endoglucanase gene, however, it does possess auxillary activity enzymes enzymes that may also be involved in the breakdown on cellulose. This therefore provides insight into why this enzyme could be inactive as well as sheds light on the plant polysaccharide degradative abilities of this fungus.
Screening, purification and characterisation of anti-pseudomonas aeruginosa compounds produced by endophytic fungi from Kigelia Africana.
(2018) Mamokoena, Kuali.; Govinden, Roshini.; Moodley, R.
The emergence of new diseases and drug resistant pathogens coupled with the side effects presented by conventional or synthetic drug use calls for the discovery of new antibiotics and chemotherapeutics. Pseudomonas aeruginosa is a multifaceted Gram-negative opportunistic pathogen which is responsible for ten percent of all hospital infections. This, therefore, directed the search for novel bioactive molecules with new targets from previously understudied sources. Plants have bioactive compounds that have been used for traditional healthcare for thousands of years. In the interest of plant preservation, the focus has shifted to include the plant microbiome; interestingly, not only were the plants themselves producing the bioactive metabolites but also their associated microbiome. The focus of this study was to screen and determine the optimum time to produce anti-Pseudomonas aeruginosa metabolites, to purify the compounds of interest and characterise them. Forty-five endophytic fungi were grown in solid substrate fermentation on rice to produce extracts of varying ages (one week to four weeks). Thin layer chromatography (TLC) coupled with bio-autography revealed that the anti-P. aeruginosa compound was produced after three weeks. Average zones of inhibition of 40.33, 20.33 and 22 mm were obtained using the Kirby-Bauer disc diffusion assay. All rows A, B, C and D show very strong activity as the MICs of the extracts were 156.5 μg/mL, 39.06 μg/mL, 78.73 μg/mL and 19.53 μg/mL for T1, T2, T3 and T4, respectively. Thin layer chromatography was conducted, and optimum separation was observed using hexane: ethyl acetate (60:40, v/v). Fractionation was carried out using a silica gel column with six different ratios of a hexane: ethyl acetate solvent system. The first round of purification resulted in twenty-seven fractions with five fractions having similar TLC profiles. These fractions were combined and subjected iii to a second round of purification that gave three fractions. One fraction was observed to have good anti-P. aeruginosa activity and acceptable purity levels after nuclear magnetic resonance spectroscopy. Compound one, a dilactone (3a,10b-dimethyl-1,2,3,3a,5a,7,10b,10c-octahydro-5,8-dioxa-acephenanthrylene-4,9-dione, molecular formula C16H18O4) was isolated as a white solid from the extract of the fungus Neofusicoccum luteum. This compound was previously isolated from the fungus Oidiodedron griseum. The relative configuration of the compound was confirmed by X-ray crystallography. Although the isolated compound is not novel, its ability to inhibit the growth of P. aeruginosa is new. This suggests that known compounds need to be screened across a wide range of pathogens and organisms to determine potential activity.
Seasonal variation of microflora and their effects on the quality of wood chips intended for pulping.
(2013) Govender, Lucretia.; Bush, T.; Govinden, Roshini.
Eucalyptus, pine and wattle are the predominant exotic wood species used in the production of dissolving pulp in South Africa. On entering the mill, wood is chipped and stored in outdoor piles where it becomes vulnerable to microbial degradation and spontaneous combustion. Major losses of stored chips are due to high temperatures and combustion caused by heat energy released by microbial fermentation. Changes in the chemistry of the wood chips caused by the metabolic activity of indigenous microflora combined with the inherent chemical characteristics of each wood species could have a potential impact on final pulp quality and yield. Therefore the objective of this study was to analyse the microbial (bacteria and fungi) communities present in commercial wood chip piles and correlate this with changes in the chemistry of the wood in summer and winter. The molecular fingerprinting technique of Denaturing Gradient Gel Electrophoresis (DGGE) was optimized for the detection of microbial diversity in commercial wood chips. Wood chips were collected from an industrial wood yard and milled to different specifications. A total of four primer sets with GC-clamps were tested in nested PCR for DGGE analysis. 16S and 18S rRNA genes were amplified using 338f-GC/518r; 933F-GC/1387R (bacteria) and NS26/518R-GC; EF4F/518R-GC (fungi), respectively. Several gel gradients were examined to determine optimal separation of bacterial (40/60%, 35/50%, 30/60%) and fungal (35/50%, 20/45%, 25/50%) PCR-DGGE products. Comparison of the DGGE profiles revealed greater diversity in the milled wood chips amplified using primer sets; 338F-GC/518R (16S) and NS26/518R-GC (18S) with gradients of 30/60% (16S) and 25/50% (18S). Once optimized, this standardized protocol was tested against five samples to assess its applicability to woodyard samples. 16S and 18S DGGE profiles were generated and amplicons excised from gels, re-amplified, sequenced and the microorganism from which the DNA originated was determined. In the second phase a cross-sectional study of wood chip piles from a commercial dissolving pulp mill was conducted with sample collected in summer and winter using the optimized PCR-DGGE technique. Microbial strains were identified after sequencing of 16S and 18S rRNA amplicons separated by DGGE. Chemical characteristics of the wood chips were evaluated by conducting extractive analyses using HPLC. Due to unpredictable combinations of different wood species in commercial wood chip piles, the third phase involved the investigation of individual Eucalyptus species. The microflora indigenous to the two Eucalyptus species (E. dunnii and E. nitens) and a combination of the two were subjected to winter and summer simulations for one month during which samples were tested for wood chemistry properties, microflora and the final samples were used to generate dissolving pulp. Using the PCR-DGGE method eighteen bacterial and twelve fungal species were identified from the five samples collected from the commercial wood chip pile, compared to the ten bacterial and nine fungal isolates which were identified using the culturing technique and standard 16S and 18S rRNA gene sequence analysis. Predominant genera in the optimization phase of this study were Klebsiella spp. (×3), Bacillus spp. (×2), Pantoea spp. (×2), Pseudomonas spp. (×2) and Paecilomyces spp. (×2). Application of the optimized DGGE technique to samples collected from the commercial pulping mill in summer and winter revealed variable profiles indicating a range of bacterial and fungal strains that varied in intensity in the areas and seasons sampled. Seventy nine (45 in summer and 34 in winter) and 29 (20 in summer and 9 in winter) distinct amplicons representing bacteria and fungi, respectively, were visualized. Predominant genera in summer were Pantoea rodasii, Inquilinus limosus, Streptococcus sp., Klebsiella spp., Diversispora sp., Boletaceae sp., Scutellospora sp., and Ophiostoma bicolour. In winter the prevailing genera were Leuconostoc palmae, Streptococcus sp., Bacillus spp., Diversispora sp., Boletaceae sp., and Bullera sp. Lower cellulose levels in summer correlated significantly with high microbial loads and the predominance of Bacillus spp., suggesting that in warm humid environments storage should not exceed 1-2 weeks. No correlations were determined between the decreased hot water levels in winter and microbial activity, however they were correlated to increased exposure of those samples to environmental factors. Chemistry data on the wood chips imparts the quality of the wood which only permitted projection of final pulp quality. This inadequacy was addressed in the third phase which included identification of microbial strains, originating from the individual Eucalyptus species, after sequencing of 16S and 18S rRNA amplicons separated by DGGE. Fungal and bacterial species were also isolated, cultured, identified and screened for lignocellulolytic enzyme activity. Ninety two and 88% of the fungi isolated were capable of producing cellulase and xylanase, respectively. Significant correlations exist between the microflora, seasons (greater diversity and loading in summer) and the chemical and physical properties of wood chips (lower cellulose and viscosity in summer) as well as Eucalyptus species (significantly higher cellulose and viscosity for the combination and E. nitens). Indigenous microflora of each wood species may be one of the contributing factors to poor/good pulp quality, as significant correlations were made between enzyme production of microorganisms and wood chemistry which ultimately has an impact on the final pulp quality and yields. This investigation provides proof of concept that combining wood species with different deterioration rates results in an overall improvement in pulp quality and thus paves the way for a practical and applicable approach to managing quality of chips.