988 resultados para Acartia sp., copepodites, production of carbon


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This study describes differences in plankton community structure and in chemical and physical gradients between the offshore West Greenland Current system and inland regions close to the Greenland Ice Sheet during the post-bloom in Godthabsfjorden (64° N, 51° W). The offshore region had pronounced vertical mixing, with centric diatoms and Phaeocystis spp. dominating the phytoplankton, chlorophyll (chl) a (0.3 to 3.9 µg/l) was evenly distributed and nutrients were depleted in the upper 50 m. Ciliates and heterotrophic dinoflagellates constituted equal parts of the protozooplankton biomass. Copepod biomass was dominated by Calanus spp. Primary production, copepod production and the vertical flux were high offshore. The water column was stratified in the fjord, causing chl a to be concentrated in a thin sub-surface layer. Nutrients were depleted above the pycnocline, and Thalassiosira spp. dominated the phytoplankton assemblage close to the ice sheet. Dinoflagellates dominated the protozooplankton biomass, whereas copepod biomass was low and was dominated by Pseudocalanus spp. and Metridia longa. Primary production was low in the outer part of the fjord but considerably higher in the inner parts of the fjord. Copepod production was exceeded by protozooplankton production in the fjord. The results of both physical/chemical factors and biological parameters suggest separation of offshore and fjord systems.

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Megabenthos plays a major role in the overall energy flow on Arctic shelves, but information on megabenthic secondary production on large spatial scales is scarce. Here, we estimated for the first time megabenthic secondary production for the entire Barents Sea shelf by applying a species-based empirical model to an extensive dataset from the joint Norwegian? Russian ecosystem survey. Spatial patterns and relationships were analyzed within a GIS. The environmental drivers behind the observed production pattern were identified by applying an ordinary least squares regression model. Geographically weighted regression (GWR) was used to examine the varying relationship of secondary production and the environment on a shelfwide scale. Significantly higher megabenthic secondary production was found in the northeastern, seasonally ice-covered regions of the Barents Sea than in the permanently ice-free southwest. The environmental parameters that significantly relate to the observed pattern are bottom temperature and salinity, sea ice cover, new primary production, trawling pressure, and bottom current speed. The GWR proved to be a versatile tool for analyzing the regionally varying relationships of benthic secondary production and its environmental drivers (R² = 0.73). The observed pattern indicates tight pelagic? benthic coupling in the realm of the productive marginal ice zone. Ongoing decrease of winter sea ice extent and the associated poleward movement of the seasonal ice edge point towards a distinct decline of benthic secondary production in the northeastern Barents Sea in the future.

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Synthesis of Polyhydroxyalkanoates (PHAs) by Pseudomonas mendocina, using different vegetable oils such as, coconut oil, groundnut oil, corn oil and olive oil, as the sole carbon source was investigated for the first time. The PHA yield obtained was compared with that obtained during the production of PHAs using sodium octanoate as the sole carbon source. The fermentation profiles at shaken flask and bioreactor levels revealed that vegetable oils supported the growth of Pseudomonas mendocina and PHA accumulation in this organism. Moreover, when vegetable oil (coconut oil) was used as the sole carbon source, fermentation profiles showed better growth and polymer production as compared to conditions when sodium octanoate was used as the carbon source. In addition, comparison of PHA accumulation at shaken flask and fermenter level confirmed the higher PHA yield at shaken flask level production. The highest cell mass found using sodium octanoate was 1.8 g/L, whereas cell mass as high as 5.1 g/L was observed when coconut oil was used as the feedstock at flask level production. Moreover, the maximum PHA yield of 60.5% dry cell weight (dcw) was achieved at shaken flask level using coconut oil as compared to the PHA yield of 35.1% dcw obtained using sodium octanoate as the sole carbon source. Characterisations of the chemical, physical, mechanical, surface and biocompatibility properties of the polymers produced have been carried out by performing different analyses as described in the second chapter of this study. Chemical analysis using GC and FTIR investigations showed medium chain length (MCL) PHA production in all conditions. GC-MS analysis revealed a unique terpolymer production, containing 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid when coconut oil, groundnut oil, olive oil, and corn oil were used as the carbon source. Whereas production of the homopolymer containing 3-hydroxyoctanoic acid was observed when sodium octanoate was used as the carbon source. MCL-PHAs produced in this study using sodium octanoate, coconut oil, and olive oil exhibited melting transitions, indicating that each of the PHA was crystalline or semi-crystalline polymer. In contrast, the thermal properties of PHAs produced from groundnut and corn oils showed no melting transition, indicating that they were completely amorphous or semi-crystalline, which was also confirmed by the X-Ray Diffraction (XRD) results obtained in this study. Mechanical analysis of the polymers produced showed higher stiffness of the polymer produced from coconut oil than the polymer from sodium octanoate. Surface characterisation of the polymers using Scanning Electron Microscopy (SEM) revealed a rough surface topography and surface contact angle measurement revealed their hydrophobic nature. Moreover, to investigate the potential applicability of the produced polymers as the scaffold materials for dental pulp regeneration, multipotent human Mesenchymal stem cells (hMSCs) were cultured onto the polymer films. Results indicated that these polymers are not cytotoxic towards the hMSCs and could support their attachment and proliferation. Highest cell growth was observed on the polymer samples produced from corn oil, followed by the polymer produced using coconut oil. In conclusion, this work established, for the first time, that vegetable oils are a good economical source of carbon for production of MCL-PHA copolymers effectively by Pseudomonas mendocina. Moreover, biocompatibility studies suggest that the produced polymers may have potential for dental tissue engineering application.

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The hydrothermal liquefaction(HTL) of algal biomass is a promising route to viable second generation biofuels. In this investigation HTL was assessed for the valorisation of algae used in the remediation of acid mine drainage (AMD). Initially the HTL process was evaluated using Arthrospira platensis (Spirulina) with additional metal sulphates to simulate metal remediation. Optimised conditions were then used to process a natural algal community (predominantly Chlamydomonas sp.) cultivated under two scenarios: high uptake and low uptake of metals from AMD. High metal concentrations appear to catalyse the conversion to bio-oil, and do not significantly affect the heteroatom content or higher heating value of the bio-oil produced. The associated metals were found to partition almost exclusively into the solid residue, favourable for potential metal recovery. High metal loadings also caused partitioning of phosphates from the aqueous phase to the solid phase, potentially compromising attempts to recycle process water as a growth supplement. HTL was therefore found to be a suitable method of processing algae used in AMD remediation, producing a crude oil suitable for upgrading into hydrocarbon fuels, an aqueous and gas stream suitable for supplementing the algal growth and the partitioning of most contaminant metals to the solid residue where they would be readily amenable for recovery and/or disposal.

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The hydrothermal liquefaction(HTL) of algal biomass is a promising route to viable second generation biofuels. In this investigation HTL was assessed for the valorisation of algae used in the remediation of acid mine drainage (AMD). Initially the HTL process was evaluated using Arthrospira platensis (Spirulina) with additional metal sulphates to simulate metal remediation. Optimised conditions were then used to process a natural algal community (predominantly Chlamydomonas sp.) cultivated under two scenarios: high uptake and low uptake of metals from AMD. High metal concentrations appear to catalyse the conversion to bio-oil, and do not significantly affect the heteroatom content or higher heating value of the bio-oil produced. The associated metals were found to partition almost exclusively into the solid residue, favourable for potential metal recovery. High metal loadings also caused partitioning of phosphates from the aqueous phase to the solid phase, potentially compromising attempts to recycle process water as a growth supplement. HTL was therefore found to be a suitable method of processing algae used in AMD remediation, producing a crude oil suitable for upgrading into hydrocarbon fuels, an aqueous and gas stream suitable for supplementing the algal growth and the partitioning of most contaminant metals to the solid residue where they would be readily amenable for recovery and/or disposal.

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Carbon materials are found versatile and applicable in wide range of applications. During the recent years research of carbon materials has focussed on the search of environmentally friendly, sustainable, renewable and low-cost starting material sources as well as simple cost-efficient synthesis techniques. As an alternative synthesis technique in the production of carbon materials hydrothermal carbonization (HTC) has shown a great potential. Depending on the application HTC can be performed as such or as a pretreatment technique. This technique allows synthesis of carbon materials i.e. hydrochars in closed vessel in the presence of water and self-generated pressure at relatively low temperatures (180-250 ˚C). As in many applications well developed porosity and heteroatom distribution are in a key role. Therefore in this study different techniques e.g. varying feedstock, templating and post-treatment in order to introduce these properties to the hydrochars structure were performed. Simple monosaccharides i.e. fructose or glucose and more complex compounds such as cellulose and sludge were performed as starting materials. Addition of secondary precursor e.g. thiophenecarboxaldehyde and ovalbumin was successfully exploited in order to alter heteroatom content. It was shown that well-developed porosity (SBET 550 m2/g) can be achieved via one-pot approach (i.e. exploitation of salt mixture) without conventionally used post-carbonization step. Nitrogen-enriched hydrochars indicated significant Pb(II) and Cr(VI) removal efficiency of 240 mg/g and 68 mg/g respectively. Sulphur addition into carbon network was not found to have enhancing effect on the adsorption of methylene blue or change acidity of the carbon material. However, these hydrochars were found to remove 99.9 % methylene blue and adsorption efficiency of these hydrochars remained over 90 % even after regeneration. In addition to water treatment application N-rich high temperature treated carbon materials were proven applicable as electrocatalyst and electrocatalyst support. Hydrothermal carbonization was shown to be workable technique for the production of carbon materials with variable physico-chemical properties and therefore hydrochars could be applied in several different applications e.g. as alternative low-cost adsorbent for pollutant removal from water.

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Carbon and nitrogen loading to streams and rivers contributes to eutrophication as well as greenhouse gas (GHG) production in streams, rivers and estuaries. My dissertation consists of three research chapters, which examine interactions and potential trade-offs between water quality and greenhouse gas production in urban streams of the Chesapeake Bay watershed. My first research project focused on drivers of carbon export and quality in an urbanized river. I found that watershed carbon sources (soils and leaves) contributed more than in-stream production to overall carbon export, but that periods of high in-stream productivity were important over seasonal and daily timescales. My second research chapter examined the influence of urban storm-water and sanitary infrastructure on dissolved and gaseous carbon and nitrogen concentrations in headwater streams. Gases (CO2, CH4, and N2O) were consistently super-saturated throughout the course of a year. N2O concentrations in streams draining septic systems were within the high range of previously published values. Total dissolved nitrogen concentration was positively correlated with CO2 and N2O and negatively correlated with CH4. My third research chapter examined a long-term (15-year) record of GHG emissions from soils in rural forests, urban forest, and urban lawns in Baltimore, MD. CO2, CH4, and N2O emissions showed positive correlations with temperature at each site. Lawns were a net source of CH4 + N2O, whereas forests were net sinks. Gross CO2 fluxes were also highest in lawns, in part due to elevated growing-season temperatures. While land cover influences GHG emissions from soils, the overall role of land cover on this flux is very small (< 0.5%) compared with gases released from anthropogenic sources, according to a recent GHG budget of the Baltimore metropolitan area, where this study took place.

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The dual problems of sustaining the fast growth of human society and preserving the environment for future generations urge us to shift our focus from exploiting fossil oils to researching and developing more affordable, reliable and clean energy sources. Human beings had a long history that depended on meeting our energy demands with plant biomass, and the modern biorefinery technologies realize the effective conversion of biomass to production of transportation fuels, bulk and fine chemicals so to alleviate our reliance on fossil fuel resources of declining supply. With the aim of replacing as much non-renewable carbon from fossil oils with renewable carbon from biomass as possible, innovative R&D activities must strive to enhance the current biorefinery process and secure our energy future. Much of my Ph.D. research effort is centered on the study of electrocatalytic conversion of biomass-derived compounds to produce value-added chemicals, biofuels and electrical energy on model electrocatalysts in AEM/PEM-based continuous flow electrolysis cell and fuel cell reactors. High electricity generation performance was obtained when glycerol or crude glycerol was employed as fuels in AEMFCs. The study on selective electrocatalytic oxidation of glycerol shows an electrode potential-regulated product distribution where tartronate and mesoxalate can be selectively produced with electrode potential switch. This finding then led to the development of AEMFCs with selective production of valuable tartronate or mesoxalate with high selectivity and yield and cogeneration of electricity. Reaction mechanisms of electrocatalytic oxidation of ethylene glycol and 1,2-propanediol were further elucidated by means of an on-line sample collection technique and DFT modeling. Besides electro-oxidation of biorenewable alcohols to chemicals and electricity, electrocatalytic reduction of keto acids (e.g. levulinic acid) was also studied for upgrading biomass-based feedstock to biofuels while achieving renewable electricity storage. Meanwhile, ORR that is often coupled in AEMFCs on the cathode was investigated on non-PGM electrocatalyst with comparable activity to commercial Pt/C. The electro-biorefinery process could be coupled with traditional biorefinery operation and will play a significant role in our energy and chemical landscape.

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This thesis attempts to find the least-cost strategy to reduce CO2 emission by replacing coal by other energy sources for electricity generation in the context of the proposed EPA’s regulation on CO2 emissions from existing coal-fired power plants. An ARIMA model is built to forecast coal consumption for electricity generation and its CO2 emissions in Michigan from 2016 to 2020. CO2 emission reduction costs are calculated under three emission reduction scenarios- reduction to 17%, 30% and 50% below the 2005 emission level. The impacts of Production Tax Credit (PTC) and the intermittency of renewable energy are also discussed. The results indicate that in most cases natural gas will be the best alternative to coal for electricity generation to realize CO2 reduction goals; if the PTC for wind power will continue after 2015, a natural gas and wind combination approach could be the best strategy based on the least-cost criterion.

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Hardboard processing wastewater was evaluated as a feedstock in a bio refinery co-located with the hardboard facility for the production of fuel grade ethanol. A thorough characterization was conducted on the wastewater and the composition changes of which during the process in the bio refinery were tracked. It was determined that the wastewater had a low solid content (1.4%), and hemicellulose was the main component in the solid, accounting for up to 70%. Acid pretreatment alone can hydrolyze the majority of the hemicellulose as well as oligomers, and over 50% of the monomer sugars generated were xylose. The percentage of lignin remained in the liquid increased after acid pretreatment. The characterization results showed that hardboard processing wastewater is a feasible feedstock for the production of ethanol. The optimum conditions to hydrolyze hemicellulose into fermentable sugars were evaluated with a two-stage experiment, which includes acid pretreatment and enzymatic hydrolysis. The experimental data were fitted into second order regression models and Response Surface Methodology (RSM) was employed. The results of the experiment showed that for this type of feedstock enzymatic hydrolysis is not that necessary. In order to reach a comparatively high total sugar concentration (over 45g/l) and low furfural concentration (less than 0.5g/l), the optimum conditions were reached when acid concentration was between 1.41 to 1.81%, and reaction time was 48 to 76 minutes. The two products produced from the bio refinery were compared with traditional products, petroleum gasoline and traditional potassium acetate, in the perspective of sustainability, with greenhouse gas (GHG) emission as an indicator. Three allocation methods, system expansion, mass allocation and market value allocation methods were employed in this assessment. It was determined that the life cycle GHG emissions of ethanol were -27.1, 20.8 and 16 g CO2 eq/MJ, respectively, in the three allocation methods, whereas that of petroleum gasoline is 90 g CO2 eq/MJ. The life cycle GHG emissions of potassium acetate in mass allocation and market value allocation method were 555.7 and 716.0 g CO2 eq/kg, whereas that of traditional potassium acetate is 1020 g CO2/kg.

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This research is about producing recombinant Trichoderma reesei endoglucanase Cel7B by using Kluyveromyces lactis, transformed with chromosomally integrated Cel7B cDNA, as a host cell (K. lactis Cel7B). Cel7B is one of the glycoside hydrolyze family of proteins that are produced by T. reesei. Cel7B together with other endoglucanases, exoglucanases, and â-glucosidases hydrolyze cellulose to glucose, which can then be fermented to biofuels or other value-added products. The research objective of this MS project is to examine favorable fermentation conditions for recombinant Cel7B enzyme production and improved activity. Production of enzyme on different types of media was examined, and the activity of the enzyme was measured by using different tools or procedures. The first condition tested for was using different concentrations of galactose as a carbon and energy source; however galactose also acts as a potent promoter of recombinant Cel7B expression in K. lactis Cel7B. The purpose of this method is to determine the relationship between production of enzyme with increasing sugar concentration. The second culture condition test was using different types of media: a complex medium-yeast extract, peptone, galactose (YPGal); a minimal medium-yeast nitrogen base (YNB) with galactose; and a minimal medium with supplement-yeast nitrogen base with casamino acid (YBC), a nitrogen source, with galactose. The third condition was using different types of reactors or fermenters: a small reactor (shake flask) and a larger automated bioreactor (BioFlo 3000 fermenter). The purpose of this method is to determine the quantity of the protein produced by using different environments of production. Different tools to determine the presence and activity of Cel7B enzyme were used. For the presence of enzyme, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used. Secondly, to detect enzyme activity, the carboxymethyl cellulose- 3,5-dinitrosalicylic acid (CMC- DNS) assay was employed. SDS-PAGE showed that the enzyme band was at 67 kDa, which is larger than native Cel7B (52 kDa.), likely due to over glycolylation during post-translational processing in K. lactis. For the different types of media used in our fermentation, recombinant Cel7B was produced from yeast extract peptone galactose (YPGal), and yeast nitrogen base with casamino acid (YBC), but was not produced and no activity was detected from yeast nitrogen base (YNB). This experiment concluded that the Cel7B production requires the amino acid resources as part of fermentation medium. In experiments where recombinant Cel7B net activity was measured at 1% galactose initial concentration in YPGal and YBC media, higher enzyme activity was detected for the complex medium YPGal. Higher activity of recombinant Cel7B was detected for flask culture in 2% galactose compared to 1% galactose for YBC medium. Two bioreactor experiments were conducted under these culture conditions at 30°C, pH 7.0, dissolved oxygen of 50% of saturation, and 250 rpm agitation (variable depending on DO control) K. lactis-Cel7B yeast growth curves were quite reproducible with maximum optical density (O.D) at 600 nm of between 7 and 8 (when factoring dilution of 10:1). Galactose was consumed rapidly during the first 15 hours of bioreactor culture and recombinant Cel7B started to appear in the culture at 10-15 hours and increased thereafter up to a maximum of between 0.9 and 1.6 mg/mL/hr in these experiments. These bioreactor enzyme activity results are much higher than comparable experiments conducted with flask-scale culture (0.5 mg/mL/hr). In order to achieve the highest recombinant Cel7B activity from batch culture of K. lactis-Cel7B, based on this research it is best to use a complex medium, 2% initial galactose concentration, and an automated bioreactor where good control of temperature, pH, and dissolved oxygen can be achieved.

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Abstract: Alcoholic beverages are produced following the fermentation of sugars by yeasts, mainly (but not exclusively) strains of the species, Saccharomyces cerevisiae. The sugary starting materials may emanate from cereal starches (which require enzymatic pre‐hydrolysis) in the case of beers and whiskies, sucrose‐rich plants (molasses or sugar juice from sugarcane) in the case of rums, or from fruits (which do not require pre‐hydrolysis) in the case of wines and brandies. In the presence of sugars, together with other essential nutrients such as amino acids, minerals and vitamins, S. cerevisiae will conduct fermentative metabolism to ethanol and carbon dioxide (as the primary fermentation metabolites) as the cells strive to make energy and regenerate the coenzyme NAD+ under anaerobic conditions. Yeasts will also produce numerous secondary metabolites which act as important beverage flavour congeners, including higher alcohols, esters, carbonyls and sulphur compounds. These are very important in dictating the final flavour and aroma characteristics of beverages such as beer and wine, but also in distilled beverages such as whisky, rum and brandy. Therefore, yeasts are of vital importance in providing the alcohol content and the sensory profiles of beverages. This Introductory Chapter reviews, in general, the growth, physiology and metabolism of S. cerevisiae in alcoholic beverage fermentations.

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O desenvolvimento de materiais de carbono continua a ser, na atualidade, uma das áreas de grande interesse junto das comunidades científica e industrial [1]. Especial atenção é dada à valorização de resíduos da indústria, de baixo valor económico, tentando desta forma resolver problemas de gestão de grandes quantidades de desperdícios [2]. De especial relevo destacamos os resíduos potencialmente perigosos, segundo os dados da FAO – Food and Agriculture Organization das Nações Unidas [3]. O desenvolvimento de novos materiais para a indústria do mobiliário, substitutos da madeira, tem gerado uma enorme diversidade de produtos mas também de resíduos. Os mais comuns no mercado são os materiais compósitos, entre eles o PB – ParticleBoard e o MDF – Medium Density Fibreboard, os quais registam um aumento de consumo na Europa de 1,3% e 4,3%, respetivamente, mesmo em período de crise económica como a que estamos a atravessar [3]. O presente trabalho tem como objetivo o estudo do potencial destes resíduos para a produção de carvões ativados (CA) em formas monolíticas, gerando um produto com um elevado valor acrescentado e com características inovadoras para a posterior aplicação em processos de adsorção.

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The production of activated carbons (ACs) involves two main steps: the carbonization of the carbonaceous of raw materials at temperatures below 1073 K in the absence of oxygen and the activation had realized at the temperature up to 1173 but the most useful temperature at 1073 K. In our study we used the most common industrial and consumer solid waste, namely PET, alone or blended with other synthetic polymer PAN. By mixing the two polymers in different ratios, an improvement of the yield of the AC production was found and some textural properties were enhanced by comparison with the AC prepared using each polymer separately. When all the samples were exposed through the carbonization process with a pyrolysis the mixture of PAN-PET (1:1w/w) yield around 31.9%, between that obtained with PET (16.9%) or PAN (42.6%) separately. The combine activation, with CO2 at 1073 K, allow ACs with a lower burn-off degree isothermally, when compared with those attained with PET or PAN alone, but with similarly chemicals or textural properties. The resultant ACs are microporous in their nature, as the activation time increase, the PET-PAN mixture AC are characterized by a better developed porous structure, when associated with the AC prepared from PAN. The AC prepared from PET-PAN mixture are characterized by basic surface characteristics, with a pHpzc around 10.5, which is an important characteristic for future applications on acidic pollutants removals from liquid or gaseous phase. In this study we had used the FTIR methods to determine the main functional groups in the surface of the activated carbons. The adsorbents prepared from PAN fibres presents an IR spectrum with similar characteristics to those obtained with PET wastes, but with fewer peaks and bands with less intensity, in particular for the PAN-8240 sample. This can be reflected by the stretching and deformation modes of NH bond in the range 3100 – 3300 cm-1 and 1520 – 1650 cm-1, respectively. Also, stretching mode associated to C–N, C=N, can contributed to the profile of IR spectrum around 1170 cm-1, 1585 – 1770 cm-1. And the TGA methods was used to study the loses of the precursors mass according to the excessive of the temperature. The results showed that, there were different decreasing of the mass of each precursors. PAN degradation started at almost 573 K and at 1073 K, PAN preserve more than 40% of the initial mass. PET degradation started at 650 K, but at 1073 K, it has lost 80% of the initial mass. However, the mixture of PET-PAN (1:1w/w) showed a thermogravimetric profile between the two polymers tested individually, with a final mass slightly less than 30%. From a chemical point of view, the carbonisation of PET mainly occurs in one step between 650 and 775 K.

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Increasing environmental awareness has been a significant driving force for innovations and process improvements in different sectors and the field of chemistry is not an outlier. Innovating around industrial chemical processes in line with current environmental responsibilities is however no mean feat. One of such hard to overhaul process is the production of methyl methacrylate (MMA) commonly produced via the acetone cyanohydrin (ACH) process developed back in the 1930s. Different alternatives to the ACH process have emerged over the years and the Alpha Lucite process has been particularly promising with a combined plant capacity of 370,000 metric tonnes in Singapore and Saudi Arabia. This study applied Life Cycle Assessment methodology to conduct a comparative analysis between the ACH and Lucite processes with the aim of ascertaining the effect of applying principles of green chemistry as a process improvement tool on overall environmental impacts. A further comparison was made between the Lucite process and a lab-scale process that is further improvement on the former, also based on green chemistry principles. Results showed that the Lucite process has higher impacts on resource scarcity and ecosystem health whereas the ACH process has higher impacts on human health. On the other hand, compared to the Lucite process the lab-scale process has higher impacts in both the ecosystem and human health categories with lower impacts only in the resource scarcity category. It was observed that the benefits of process improvements with green chemistry principles might not be apparent in some categories due to some limitations of the methodology. Process contribution analysis was also performed and it revealed that the contribution of energy is significant, therefore a sensitivity analysis with different energy scenarios was performed. An uncertainty analysis using Monte Carlo analysis was also performed to validate the consistency of the results in each of the comparisons.