Enhanced production of lactic acid from pretreated rice straw using co-cultivation of Bacillus licheniformis and Bacillus sonorenesis.

Lactic acid (LA) production from sugar mixture derived from lignocellulosic rice straw employing co- culture system of thermotolerant and inhibitor tolerant Bacillus licheniformis DGB and Bacillus sonorenesis DGS15 was carried out. In minimal media, both the strains of Bacillus DGB and DGS15 worked together by efficiently utilising glucose and xylose respectively. Response Surface Methodology (RSM) was used for optimisation of pretreatment of rice straw to achieve maximum yield of 50.852 g/L total reducing sugar (TRS) from 100 gm of rice straw biomass. Pretreatment of rice straw resulted in its delignification, as confirmed by FTIR spectroscopy, since the peak at 1668 cm-1 disappeared due to removal of lignin and scanning electron microscopy (SEM) revealed disruption in structural and morphological features. Crystallinity index (CrI) of treated rice straw increased by 15.54% in comparison to native biomass. DGB and DGS15 individually yielded 0.64 g/g and 0.82 g/g lactic acid respectively, where as their co-cultivation led to effective utilisation of both glucose and xylose within 15 h (70%) and complete utilisation in 48 h, producing 49.75 g/L LA with a yield of 0.98 g/g and productivity of 1.036 g/L/h, and resulting in reduction in fermentation time. Separate hydrolysis of rice straw and co-fermentation (SHCF) of hydrolysates by Bacillus spp. enhanced the production of lactic acid, can circumvent challenges in biorefining of lignocellulosic biomass.

Bioprospecting Microalgae from Sewage Water: Assessment of Biochemicals for Biomass Utilization.

Microalgal species from sewage treatment plant were identified by 18S rRNA sequencing and were explored for total lipids, carbohydrate, and protein contents, to serve as a potential candidate for biorefinery. Seven unicellular microalgae were identified as Chlorella sorokiniana, Dictyosphaerium sp., Graesiella emersonii belonging to Chlorellaceae and Scenedesmus sp., Desmodesmus sp., Tetranephris brasiliensis, and Coelastrella sp. belonging to Scenedesmaceae family. Biochemical assessment of all isolates revealed total lipid content from 17.49 ± 1.41 to 47.35 ± 0.61% w/w, total carbohydrate content from 12.82 ± 0.19 to 64.29 ± 0.63% w/w, and total protein content from 8.55 ± 0.19 to 16.65 ± 0.20% w/w. FAME analysis of extracted lipid was found to be rich in Hexadecane (C16:0), Tetradecane (C17:0), Octadecane (C18:0), Eicosane (C20:0), Tetracosane (C24:0), Pentacosane (C25:0) fatty acids, the presence of which makes excellent candidate for biodiesel. Being rich in lipid, microalgae Chlorella sorokiniana, Coelastrella sp., and Scenedesmus sp. have high potential for biofuels. Due to the presence of high protein content, Scenedesmus sp. and Chlorella sorokiniana can serve as food or feed supplement, whereas the high carbohydrate content of Dictyosphaerium sp., Coelastrella sp., and Scenedesmus sp. makes them an ideal candidate for fermentative production of alcohol and organic acids. Chlorella sp. and Scenedesmus sp., being dominant microalgae across all seasons, demonstrate remarkable resilience for their cultivation in sewage water and utilization of biomass in biorefineries.

Ecotoxicity of as-synthesised copper nanoparticles on soil bacteria.

Release of metallic nanoparticles in soil poses a serious threat to the ecosystem as they can affect the soil properties and impose toxicity on soil microbes that are involved in the biogeochemical cycling. In this work, in vitro ecotoxicity of as-synthesised copper nanoparticles (CuNPs) on Bacillus subtilis (MTCC No. 441) and Pseudomonas fluorescens (MTCC No. 1749), which are commonly present in soil was investigated. Three sets of colloidal CuNPs with identical physical properties were synthesised by chemical reduction method with per batch yield of 0.2, 0.3 and 0.4 gm. Toxicity of CuNPs against these soil bacteria was investigated by MIC (minimum inhibitory concentration), MBC (minimum bactericidal concentration), cytoplasmic leakage and ROS (reactive oxygen species) assay. MIC of CuNPs were in the range of 35-60 µg/ml and 35-55 µg/ml for B. subtilis and P. fluorescens respectively, while their MBC ranged from 40-70 µg/ml and 40-60 µg/ml respectively. MIC and MBC tests reveal that Gram-negative P. fluorescens was more sensitive to CuNPs as compared to Gram positive B. subtilis mainly due to the differences in their cell wall structure and composition. CuNPs with smaller hydrodynamic size (11.34 nm) were highly toxic as revealed by MIC, MBC tests, cytoplasmic leakage and ROS assays, which may be due to the higher active surface area of CuNPs and greater membrane penetration. Leakage of cytoplasmic components and generation of extra-cellular oxidative stress by reactive oxygen species (ROS) causes cell death. The present study realizes in gauging the negative impact of inadvertent release of nanoparticles in the environment, however, in situ experiments to know its overall impact on soil health and soil microflora can help in finding solution to combat ecotoxicity of nanoparticles.

Fabrication of chitosan-alginate nanospheres for controlled release of cartap hydrochloride.

Insecticide cartap hydrochloride (C) was fabricated as nanospheres by a two-step method of ionic gelification and polyelectrolyte complexation of alginate (ALG) and chitosan (CS) to undermine its adverse effects on environment. Nanospheres were characterized by field emission scanning electron microscope, Fourier transform infrared spectra and x-ray diffraction. The size of cartap hydrochloride entrapped chitosan alginate nanospheres (C-CS-ALG nps) was in range of 107.58-173.07 nm. Cartap hydrochloride nanospheres showed encapsulation efficiency of 76.19% and were stable for 30 d at ambient temperature. Release of cartap from nanospheres fitted best with first order linear kinetics followed by Hixson and Higuchi model suggesting super case II transport release. With the application of such control release nanoformulations, it is possible to reduce the frequency of field application of insecticide due to its slow release to the target organism, which is economical as well as environmentally safe.

Cartap hydrochloride induced stress response in Anabaena variabilis ARM 441.

Cartap hydrochloride is a moderately hazardous nereistoxin analogue insecticide that is predominantly applied in paddy fields of India, at a recommended dose of 10 µg ml-1 to kill chewing and sucking insect pests of rice crop. Toxicity of cartap hydrochloride was studied on non-target free-living nitrogen fixing cyanobacterium Anabaena variabilis ARM 441 commonly used as algal biofertilizer in rice cultivation. Anabaena sp. could tolerate commercial grade insecticide up to 30 µg ml-1. However, at the recommended dose of 10 µg ml-1, it caused reduction in algal growth, total nitrogen and heterocyst frequency by 47.28, 24.29 and 17.72% respectively, as well as photosynthetic pigments under pure culture conditions. Scanning electron micrographs revealed cell rupture and breakage in filaments due to cartap exposure with the formation of akinetes. Cartap hydrochloride induced stress, since level of superoxide dismutase, peroxidase and catalase were increased by 108.57, 187.5 and 117% respectively. Generation of superoxide radicals and hydrogen peroxide were also increased by 152.48 and 34% respectively. Lipid peroxidation was increased by 31.03%, whereas there was decline in ascorbate content by 48.45%, however the glutathione content was increased by 128.57%. Increase in osmolytes such as proline from 8.6 to 32.8% and sucrose from 61.22 to 90.13% indicates their possible role in overcoming cartap induced oxidative stress and can be helpful in assessing its detrimental effect on Anabaena variabilis ARM 441, since cyanobacterial biofertilizers are purposely used in paddy fields as nitrogen contributors.

Chitosan/PVA silver nanocomposite for butachlor removal: Fabrication, characterization, adsorption mechanism and isotherms.

Silver nanoparticle (AgNP) incorporated chitosan-Polyvinyl alcohol (Ch/PVA) hydrogel nanocomposite was fabricated by repeated freeze-thaw treatment using glutaraldehyde as crosslinker for removal of herbicide butachlor from aqueous solution. Ch/PVA hydrogel provided a matrix for in-situ immobilization of AgNPs and were characterized by various physicochemical techniques. AgNPs of size 5-20 nm possessed crystalline structure, led to increase in thermal stability and surface area after incorporation into Ch/PVA hydrogel. Ch/PVA hydrogel nanocomposite showed maximum adsorption of butachlor (86.55 %) at 30 °C and pH 3.0, while Ch/PVA-Ag showed a slight increase in adsorption of butachlor following pseudo-second order kinetics. Langmuir and Freundlich models with different error functions (R2, R2adj, RSME, χ2 and RSS), confirmed monolayer adsorption of butachlor.

Oxidative Stress Induced Cytotoxicity of Colloidal Copper Nanoparticles on RAW 264.7 Macrophage Cell Line.

Copper is an essential trace mineral that plays an important role in various physiological processes of human body and also possesses excellent antimicrobial properties, however its high dose results in the formation of free-radicals, which can induce cytotoxicity through chromosomal and DNA damage. Therefore, cytotoxicity of colloidal copper nanoparticles (CuNPs) on murine macrophage cell line (RAW 264.7) was studied to understand the correlation between the cytotoxicity and the nanoparticle yield. Three identical sets of CuNPs with similar physical properties having hydrodynamic particle size of 11-14 nm were prepared by chemical reduction method with target yield of 0.2 g, 0.3 g and 0.4 g. CuNPs exhibited dose-dependent (0.001-100 µg/mL) cytotoxicity due to the mitochondrial damage as indicated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay. Oxidative stress induced by reactive oxygen species (ROS) in RAW 246.7 macrophage cell lines exposed to CuNPs was the primary cause of observed cytotoxicity in all CuNPs test samples. Morphological changes in cells also indicated strong dose-dependent oxidative damage by CuNPs. IC50 (half maximal inhibitory concentration) values of CuNPs were independent of nanoparticle yield. This suggests that per batch variation in CuNPs yield from 0.2 g to 0.4 g had no negative correlation with their toxicity that makes CuNPs a potential candidate for further development of nanotherapeutics and anticancer drugs.

Biodegradation of Butachlor by Bacillus altitudinis and Identification of Metabolites.

Butachlor is a chloroacetamide pre-emergence herbicide, with a half-life of 1.6 to 29 days. It is a suspected carcinogen, genotoxin, neurotoxin and persists in the environment having toxic effect on living systems. Butachlor degrading bacterial strain A16 was isolated from coal tar contaminated soil, which showed 99.38% similarity with Bacillus altitudinis 41KF2bT as revealed by 16S rRNA analysis. B. altitudinis strain A16 utilised butachlor as a sole source of carbon and degraded 90% of 50 mg L-1 butachlor in 5 days at a rate constant and half-life (t1/2) of 0.02 h-1 and 34.65 h, respectively, following the first-order reaction kinetics. Five metabolites (N-(butoxymethyl)-N-(2-chloroethyl)-2,6-diethylaniline, (N-(butoxymethyl)-2-chloro-N-(2-ethylphenyl) acetamide, N-(butoxymethyl)-2,6-diethyl-N-propylaniline, 2-chloro-N-(2,6-diethylphenyl) acetamide and 2,6-diethylaniline) were produced during the breakdown of butachlor by B. altitudinis A16 as identified by GC-MS analysis, which are further mineralized to carbon dioxide and water. A metabolic pathway is proposed and compared with other bacteria. The findings have immense beneficial application since such microbes can be used on large scale for faster soil bioremediation and minimizing negative impact of pesticide butachlor on health and environment.

Algae-based metallic nanoparticles: Synthesis, characterization and applications.

Nanomaterials (NMs) tailored via conventional physicochemical routes play havoc with the environment that has led to the evolution of competent green routes for the actualization of a circular economy on an industrial-scale. Algae belonging to the class Cyanophyceae, Chlorophyceae, Phaeophyceae and Rhodophyceae have been harnessed as nano-machineries through intracellular and extracellular synthesis of gold (Au), silver (Ag) and several other metallic nanoparticles. Algae are an appealing platform for the production of diverse NMs, primarily due to the presence of bioactive compounds such as pigments and antioxidants in their cell extracts that act as biocompatible reductants. Chlorella spp. and Sargassum spp. have been extensively explored for the synthesis of nanoparticles having antimicrobial properties, which can potentially substitute conventional antibiotics. Characterization of nanoparticles (NPs) synthesised from algae has been done using advanced spectroscopic, diffractographic and microscopic techniques such as UV-Vis FT-IR, DLS, XPS, XRD, SEM, TEM, AFM, HR-TEM, and EDAX. The present paper reviews the information available on algae-mediated biosynthesis of various NPs, their characterization and applications in different domains.

Potential pancreatic lipase inhibitory activity of an endophytic Penicillium species.

Pancreatic lipase (PL) is considered as one of the safest target for diet-induced anti-obesity drug development. Orlistat is the only PL inhibitor approved for anti-obesity treatment till date. In the process of exploration of new PL inhibitors, we have screened culture filtrates of 70 endophytic fungi of medicinal plants using qualitative as well as quantitative in-vitro PL assays. The qualitative assays indicated potential PL inhibition in only three isolates, namely #57 TBBALM, #33 TBBALM and #1 CSSTOT. Only ethyl acetate extracts of the culture filtrates of these isolates exhibited the PL inhibition. #57 TBBLAM ethyl acetate extract of culture filtrate exhibited potential PL inhibition with an IC50 of 3.69 µg/ml which was comparable to the positive control, i.e. Orlistat exhibiting IC50 value of 2.73 µg/ml. Further molecular phylogenetic tools and morphological studies were used to identify the isolate #57 TBBALM as Penicillium species.

Enhanced Cellulase Production from Bacillus subtilis by Optimizing Physical Parameters for Bioethanol Production.

Effect of physical parameters such as initial pH, agitation (rpm), and temperature (°C) for cellulase production from Bacillus subtilis AS3 was investigated. Central composite design of experiments followed by multiple desirability function was applied for the optimization of cellulase activity and cell growth. The effect of the temperature and agitation was found to be significant among the three independent variables. The optimum levels of initial pH, temperature, and agitation for alkaline carboxymethylcellulase (CMCase) production predicted by the model were 7.2, 39°C, and 121 rpm, respectively. The CMCase activity with unoptimized physical parameters and previously optimized medium composition was 0.43 U/mL. The maximum activity (0.56 U/mL) and cell growth (2.01 mg/mL) predicted by the model were in consensus with values (0.57 U/mL, 2.1 mg/mL) obtained using optimized medium and optimal values of physical parameters. After optimization, 33% enhancement in CMCase activity (0.57 U/mL) was recorded. On scale-up of cellulase production process in bioreactor with all the optimized conditions, an activity of 0.75 U/mL was achieved. Consequently, the bacterial cellulase employed for bioethanol production expending (5%, w/v) NaOH-pretreated wild grass with Zymomonas mobilis yielded an utmost ethanol titre of 7.56 g/L and 11.65 g/L at shake flask and bioreactor level, respectively.

Characterization of pyrene utilizing Bacillus spp. from crude oil contaminated soil

Pyrene, a high molecular weight polycyclic aromatic hydrocarbon (PAH), is a priority pollutant present in soil contaminated with crude oil, coal-tar and complex PAHs. Bacterial consortium CON-3 developed from crude oil contaminated soil of Patiala, Punjab (India) cometabolized 50 µg ml-1 pyrene in the presence of glucose (0.5 %; w/v) at 30 °C, as determined by reverse-phase high performance liquid chromatography (HPLC). Bacillus sp. PK-12, Bacillus sp. PK-13 and Bacillus sp. PK-14 from CON-3, identified by 16S rRNA gene sequence analysis, were able to cometabolize 64 %, 55 % and 53 % of pyrene in 35 days, respectively. With the increase in glucose concentration to 1.0 % (w/v) in growth medium isolates PK-12, PK-13 and PK-14 showed 19 - 46 % uptake of 50 µg ml-1 pyrene in 4 days, respectively. Uptake of pyrene was correlated with growth and biosurfactant activity, which is suggestive of the potential role of members of Bacillus genera in pyrene mobilization and its uptake.

Bioethanol production involving recombinant C. thermocellum hydrolytic hemicellulase and fermentative microbes.

The enhancement of the biomass productivity of Escherichia coli cells harbouring the truncated 903 bp gene designated as glycoside hydrolase family 43 (GH43) from Clostridium thermocellum showing hemicellulase activity along with its further use in simultaneous saccharification and fermentation (SSF) process is described. (Phosphoric acid) H(3)PO(4)-acetone treatment and ammonia fibre expansion (AFEX) were the pretreatment strategies employed on the leafy biomass of mango, poplar, neem and asoka among various substrates owing to their high hemicellulose content. GH43 showed optimal activity at a temperature of 50 °C, pH 5.4 with stability over a pH range of 5.0-6.2. A 4-fold escalation in growth of the recombinant E. coli cells was observed when grown using repeated batch strategy in LB medium supplemented with glucose as co-substrate. Candida shehatae utilizing pentose sugars was employed for bioethanol production. AFEX pretreatment proved to be better over acid-acetone technique. The maximum ethanol concentration (1.44 g/L) was achieved for AFEX pretreated mango (1%, w/v) followed by poplar with an ethanol titre (1.32 g/L) in shake flask experiments. A 1.5-fold increase in ethanol titre (2.11 g/L) was achieved with mango (1%, w/v) in a SSF process using a table top 2-L bioreactor with 1 L working volume.

Characterization of pyrene utilizing Bacillus spp. from crude oil contaminated soil.

Pyrene, a high molecular weight polycyclic aromatic hydrocarbon (PAH), is a priority pollutant present in soil contaminated with crude oil, coal-tar and complex PAHs. Bacterial consortium CON-3 developed from crude oil contaminated soil of Patiala, Punjab (India) cometabolized 50 µg ml(-1) pyrene in the presence of glucose (0.5 %; w/v) at 30 °C, as determined by reverse-phase high performance liquid chromatography (HPLC). Bacillus sp. PK-12, Bacillus sp. PK-13 and Bacillus sp. PK-14 from CON-3, identified by 16S rRNA gene sequence analysis, were able to cometabolize 64 %, 55 % and 53 % of pyrene in 35 days, respectively. With the increase in glucose concentration to 1.0 % (w/v) in growth medium isolates PK-12, PK-13 and PK-14 showed 19 - 46 % uptake of 50 µg ml(-1) pyrene in 4 days, respectively. Uptake of pyrene was correlated with growth and biosurfactant activity, which is suggestive of the potential role of members of Bacillus genera in pyrene mobilization and its uptake.

Bioconversion of Agricultural Waste to Ethanol by SSF Using Recombinant Cellulase from Clostridium thermocellum.

The effect of different pretreatment methods, temperature, and enzyme concentration on ethanol production from 8 lignocellulosic agrowaste by simultaneous saccharification and fermentation (SSF) using recombinant cellulase and Saccharomyces cerevisiae were studied. Recombinant cellulase was isolated from E. coli BL21 cells transformed with CtLic26A-Cel5-CBM11 full-length gene from Clostridium thermocellum and produced in both batch and fed-batch processes. The maximum cell OD and specific activity in batch mode were 1.6 and 1.91 U/mg, respectively, whereas in the fed-batch mode, maximum cell OD and specific activity were 3.8 and 3.5 U/mg, respectively, displaying a 2-fold increase. Eight substrates, Syzygium cumini (jamun), Azadirachta indica (neem), Saracens indica (asoka), bambusa dendrocalmus (bamboo), Populas nigra (poplar), Achnatherum hymenoides (wild grass), Eucalyptus marginata (eucalyptus), and Mangifera indica (mango), were subjected to SSF. Of three pretreatments, acid, alkali, and steam explosion, acid pretreatment Syzygium cumini (Jamun) at 30°C gave maximum ethanol yield of 1.42 g/L.

Enhancement of Cellulase Activity from a New Strain of Bacillus subtilis by Medium Optimization and Analysis with Various Cellulosic Substrates.

The cellulase activity of Bacillus subtilis AS3 was enhanced by optimizing the medium composition by statistical methods. The enzyme activity with unoptimised medium with carboxymethylcellulose (CMC) was 0.07 U/mL and that was significantly enhanced by CMC, peptone, and yeast extract using Placket-Burman design. The combined effects of these nutrients on cellulase activity were studied using 2(2) full factorial central composite design. The optimal levels of medium components determined were CMC (1.8%), peptone (0.8%), and yeast extract (0.479%). The maximum enzyme activity predicted by the model was 0.49 U/mL which was in good agreement with the experimental value 0.43 U/mL showing 6-fold increase as compared to unoptimised medium. The enzyme showed multisubstrate specificity, showing significantly higher activity with lichenan and ß-glucan and lower activity with laminarin, hydroxyethylcellulose, and steam exploded bagasse. The optimised medium with lichenan or ß-glucan showed 2.5- or 2.8-fold higher activity, respectively, at same concentration as of CMC.

Biosorption of Pb and Zn by Non-Living Biomass of Spirulina sp.

Removal of heavy metals (Pb(2+), Zn(2+)) from aqueous solution by dried biomass of Spirulina sp. was investigated. Spirulina rapidly adsorbed appreciable amount of lead and zinc from the aqueous solutions within 15 min of initial contact with the metal solution and exhibited high sequestration of lead and zinc at low equilibrium concentrations. The specific adsorption of both Pb(2+) and Zn(2+) increased at low concentration and decreased when biomass concentration exceeded 0.1 g l(-1). The binding of lead followed Freundlich model of kinetics where as zinc supported Langmuir isotherm for adsorption with their r(2) values of 0.9659 and 0.8723 respectively. The adsorption was strongly pH dependent as the maximum lead biosorption occurred at pH 4 and 10 whereas Zn(2+) adsorption was at pH 8 and 10.

Waste water treatment and metal (Pb(2+), Zn(2+)) removal by microalgal based stabilization pond system.

A case study was undertaken for the treatment of domestic wastewater generated at village of Sanghol, Distt. Fatehgarh Sahib, Punjab (India), using a schematic designed algal and duckweed based stabilization pond system, which is discussed here for winter months only (November to March) as there was no growth of duckweeds and only algae dominated the whole system. A proficient increase in pH and dissolved oxygen was observed after the treatment with reduction in chemical oxygen demand and biochemical oxygen demand by 93% and 79% respectively. Chlorella sp. was the dominating algal species in the stabilization pond water during entire period and was studied for its Zn(2+) and Pb(2+) metal removal efficiency. 60-70% removal of Zn(2+) was observed from culture medium containing 5-20 mg L(-1) Zn(2+), which declined to 42% at 50 mg L(-1). A constant decline in cell number from 538 × 10(5) to 8 × 10(5) cells ml(-1) was observed indicating zinc toxicity to Chlorella. Lead was maximally removed by 66.3% from culture medium containing 1 mg L(-1). The lead removal efficiency was 45 50 % at higher 5 to 20 mg L(-1) of external lead concentrations. The increase in cell number indicated no signs of Pb(2+) toxicity up to 20 mg L(-1). The maximum uptake (q(max)) by live Chlorella biomass for both Zn(2+) and Pb(2+) was 34.4 and 41.8 mg/g respectively.

Comparative biosorption of Pb2+ by live algal consortium and immobilized dead biomass from aqueous solution.

The percent removal and uptake of Pb2+ by algal-consortium (CP1) developed from wastewater of biological oxidation pond (Wazirabad, New Delhi, India) was studied under batch conditions with live biomass compared with continuous system using dried biomass immobilized on silica under laboratory conditions. In batch study, algal consortium (CP1) consisting of a mixed culture of Chlorella > Chlamydomonas > Lyngbya sp. was found to remove 17% of Pb2+ after 15 days of incubation from culture media containing 10 mg/L Pb2+, which decreased by increasing metal concentration from 20-50 mg/L. This reduction in removal efficiency was co-related with direct toxic effect of Pb2+ on live consortium as indicated by drastic inhibition in growth. A maximum lead uptake capacity (q(max)) of 33.31 mg/g showed good accumulation potential of live consortium. Bioresin derived from the biomass of dried algal consortium immobilized on silica gel and packed in column exhibited 92.5% removal of Pb2+ with inlet Pb2+ concentration of 33.90 mg/L was brought down to minimum 0.375 mg/L of Pb2+ in outlet stream. The plot of outlet concentration to volume exhibited the typical 'S' shape curve with Pb2+ uptake capacity of 15.95 mg/g. Efficient recovery of 86.16% of Pb2+ was achieved by elution with dilute HCl which indicated multiple usability of immobilized biomass.

Decolorization of Direct Red 28 by mixed bacterial culture in an up-flow immobilized bioreactor.

Aerobic mixed bacterial culture comprised of five isolates (Bacillus vallismortis, B. pumilus, B. cereus, B. subtilis and B. megaterium) identified by 16srDNA analysis was developed from wastewater samples from the aeration tank of an effluent treatment plant of a textile and dyeing industry and evaluated for its ability to decolorize azo dye Direct Red 28 in an up-flow immobilized packed bed bioreactor using marble chips as support matrix. The bioreactor was operated under two parameters: an aeration rate of 0.4 and 0.6 mmol/min at a flow rate of 60, 90 and 120 ml/h, respectively. At a constant aeration rate of 0.4 mmol/min and with flow rates of 60, 90 and 120 ml/h, optimum decolorization of 91, 75 and 72% was observed, while at an aeration rate of 0.6 mmol/min and flow rates of 60, 90 and 120 ml/h, optimum decolorization of 93, 78 and 72% was observed over 10 days. The study concluded that across the two aeration rates and the respective flow rates, the higher aeration rate of 0.6 mmol/min along with a flow rate of 60 ml/h was best suited to decolorize Direct Red 28 in the packed bed bioreactor. Spectral changes of the input and output of the bioreactor by UV-visible spectroscopy indicated decolorization of the dye solution by degradation in addition to the visual observation of the biosorption process.