Immobilization of phenol degrader pseudomonas sp in repeated batch culture using bioceramic and sponge as support materials

The performance of two types of inert support, namely bioceramic and sponge to immobilize a locally isolated phenol degrader Pseudomonas sp. in a packed column was investigated in repeated batch culture. Prior to this, our study indicated that immobilization had doubled the tolerance limit of the cells towards phenol from 1000 ppm (in the suspended culture), to 2000 ppm. For the same volume, the bioceramic managed to trap bacterial cells 1.8 times greater than the sponge did. As a result, it was able to remove 100% of 1000 ppm 600–ml phenol fed at a rate of 2.5 ml/min within 24 hours, and the phenol removal capacity was sustained in the next six consecutive batches. Cells entrapped in sponge however, managed to remove around 90% phenol in five batches. Despite lower performance, at large scales, the use of sponge for cell entrapment offers some merits such as lightness, and easily available at cheaper cost

EMPLOYMENT OF MICROBES ISOLATED FROM RESIDENTIAL WASTEWATER TO DEGRADE CHLOROBENZENE

Abstrak: Keupayaan konsortium mikrob daripada air sisa kediaman dalam penguraian klorobenzena (CB) telah dikaji. Konsortium ini terlebih dahulu didedahkan pada kepekatan CB yang tinggi (0.2 mg/l) selama tujuh bulan. Hasilnya, dua spesies yang paling dominan berjaya dipencilkan, iaitu 'Koloni Kuning' (YC), dan 'Koloni Putih' (WC). Sebagai perbandingan, gabungan YC and WC memberikan kadar penguraian spesifik CB, Q s (7.12 x 10 -6 g CB terurai/g biojisim per jam) tiga kali ganda lebih tinggi berbanding hasil gabungan bagi individu WC dan YC. Ini membuktikan wujud kesan sinergistik bagi YC dan WC dalam penguraian CB. Keputusan daripada kultur selanjar menunjukkan penambahan sumber nitrogen telah meningkatkan kadar pencairan kritikal, D c daripada 0.08 jam -1 kepada 0.11 jam -1 . Ini membuktikan penghadan nitrogen dalam penguraian CB tidak boleh diabaikan. Keputusan juga menunjukkan penguraian CB tidak berlaku di bawah kepekatan 0.6 µg/l, di mana CB tidak dapat dikesan oleh mikrob di bawah tahap ambang ini. Hasil kajian ini boleh dijadikan panduan dalam menganggar parameter-parameter untuk kerja-kerja perskalaan pada masa akan datang atau percubaan di tapak sebenar. Kata kunci: Konsortium Mikrob, Air Sisa Kediaman, Penguraian Klorobenzena, Mod Kultur Selanjar Abstract: The performance of microbial consortia from residential wastewater to degrade chlorobenzene (CB) was investigated. The consortia were firstly exposed to high CB concentration (i.e. 0.2 mg/l) for seven months. As a result, two most dominant survivors, denoted as 'Yellow Colony' (YC) and 'White Colony' (WC) were isolated. In a comparison study, the mixture of WC and YC yielded three times greater maximum CB specific degradation rate, Q s (7.12 x 10 -6 g CB degraded/g cell per hour) than the individual WC and YC did, combined. This clarified that there was a synergistic effect of YC and WC on CB degradation. Result in a continuous culture indicated that nitrogen-enriched feed (yeast extract) has improved the critical dilution rate, D c from 0.08 hour -1 to 0.11 hour -1 . This proved that the nitrogen limitation could not be ignored. Our result also indicated that no degradation was witnessed below 0.6 µg/l, where CB was almost undetectable by microbes below this threshold level. Outcomes of this study have provided useful parameter estimates for future up scaling works, or on site trial

High performance phenol degrading microorganisms isolated from wastewater and oil-contaminated soil

Four bacteria, two each isolated from wastewater treatment and oil-contaminated soil showed great potential as phenol degraders. RWC-Cr1, the isolate from wastewater was not only demonstrating the highest specific phenol degradation rate at all tested phenol concentrations, but also not affected by the highest phenol concentration employed, namely 1000 mg/l. After a serial transfer of all four isolates into a series of increasing phenol level, all but RWC-Cr1 demonstrated significant improvement on degradation ability. ISC-Tra which was isolated from oil-contaminated soil exhibited greatest improvement (e.g. 750% at 600 mg/l phenol) after adaptation in phenol. Thus, the ISC-Tra was the most acclimatizable isolate while in contrary RWC-Cr1 was not significantly influenced by acclimatization. Outcomes of this study offer a useful guideline in evaluating potential phenol degraders from the environment

Microbial consortia from residential wastewater for bioremediation of phenol in a chemostat

The study aimed at evaluating the potential of employing microbial consortia from residential wastewater to degrade phenol, a common industrial pollutant. The consortia was subjected to high shock loads and different levels of phenol in continuous cultures. In the shock load study with 1000 ppm phenol in a 1.5 litre working volume chemostat, the culture with prior acclimatization to phenol demonstrated 17 times higher affinity towards phenol (i.e. Ks = 173 ppm). This resulted in double growth rate (µmax=Dc=0.04 h-1) compared to non-acclimatized culture. However, the levels of phenol reduction in both cases were almost identical, approximately 60%. This indicated that the performance of different types of bacteria present in the non-acclimatized culture which was comparable to the acclimatized culture although with less number of microbe types. The effects of two loading concentrations, namely 200 ppm and 1000 ppm were examined at constant dilution rate of 0.02 h-1. The feeding of 200 ppm exhibited 97% of phenol reduction in comparison to only 57% of 1000 ppm. This result reconfirmed the inhibitory effect of phenol reduction at concentration above 500 ppm as previously determined in batch culture. The study has successfully established key operating parameters and provided a useful guideline for future up-scaling studies and site trials

Scale-Up Of Stirred And Aerated BioengineeringTM Bioreactor Based On Constant Mass Transfer Coefficient

The Scale-Up Studies Based On The Constant Oxygen Transfer Coefficient, Kla From 16 L To 150 L Of Aerated And Agitated Bioreactor Were Performed. By Employing The Static Gassing Out Techniques, The Kla Values Were Calculated At Different Sets Of Impeller Speeds And Air Flow Rates Performed In Various Viscosities And Temperatures In The 16 L And 150 L Bioengineeringtm Stirred Bioreactor. Empirical Correlation Was Employed To Correlate And Investigate The Dependence Of Kla On Specific Power Input And Superficial Air Velocity. In Maintaining A Constant Kla Upon Scale-Up From 16 L To 150 L, The Specific Power Input And The Superficial Air Velocity Cannot Be Maintained, Thus An Adjustment Has To Be Done. From The Experimental Results At 150 L, It Was Discovered That The Specific Power Input From 0.0001 To 4.2 Kw/M3 And Superficial Air Velocity Within The Range Of 9 × 10-4 M/S To 7 × 10-3 M/S Employed Gave A Comparable Kla Values Achieved At 16 L Scale. Hence, The Calculated Scaling-Up Factor For Impeller Speed And Air Flow Rate Were 0.28 And 3.1, Respectively. The Comparable Results Obtained From Both Scales Confirmed That The Scale-Up Protocol Developed Works

The kinetic of dilute-acid hydrolysis of agrowaste for the production of reducing sugar

There is a growing incentive for us to identify alternative and ideally renewable energy sources. Among the sources, grains, plant matters (biomass) and their wastes are commonly been used for biofuel production due to their sustainability. Oil palm empty fruit bunch (EFB) is a type of lignocellulosic waste from palm oil mills. Fermentable sugars mainly the glucose from EFB can be further fermented for the production of bioethanol. This study aims to investigate the effect of the key parameters during dilute acid pretreatment and enzymatic treatment for the hydrolysis of EFB to produce optimised yield of glucose. Three parameters for the dilute acid pretreatment, namely the reaction temperature, acid concentration and reaction time and two parameters for the enzymatic treatment, namely the substrate concentration and treatment time were investigated to optimise the yield of glucose. Batch reactions were carried out under different combination of operational conditions as proposed by the experimental design produced by the RSM (response surface methodology). RSM was used to optimise both the pretreatment and enzymatic processes in order to obtain the highest glucose yield. An optimised glucose yield of 53.96 % was obtained at the operating condition of 130 °C pretreatment temperature, 6 %w/w sulphuric acid concentration, 37 min of pretreatment time and 96 hours of enzymatic treatment using 6 %w/v of substrate concentration. The optimised yield has also been validated through experiment work

Thermal induction and overexpression of a foreign protein in 'Escherichia Coli' culture

Engineering studies were performed on foreign protein production by a recombinant Escherichia coli, which overexpresses intracellular Ecoli P-galactosidase from the plasmid vector pBRG 401 under transcriptional regulation of the ?pL promoter and cL857 protein repressor. Both genetic/physiological and environmental/bioprocess factors that influenced the performance and the kinetics of protein expression and growth in complex medium were investigated. A novel approach in describing protein expression in the thermal induction system in an E.coli culture was proposed. The disappearance of protein expression during induction was primarily due to plasmid instability. The instability was non-segregational, irreversible, and strongly temperature-dependent. The loss of resistance towards the selective pressure was in parallel with the decline of protein expression, despite the fact that both events were regulated by separate promoters. The absence of inclusion bodies implied that the overexpressed proteins were completely soluble. The occurrence of intraceliular proteolysis was not evident during the induction. However, proteolysis was observed even in the presence of a respiratory inhibitor at extended incubation time if glucose was present. This proved that the additional metabolic energy needed for the protein degradation was minimal. Although oxygen concentration was not a limiting factor in the culture, the increased secretion of acetic acid following protein overexpression signified a shift of metabolism from oxidative to fermentative pathway. This resulted in a feedback repression in the Kreb's cycle, with consequently lowering the metabolic overload due to reduced protein expression on the plasmid. The accumulated acetic acid and the acidic pH lowered both growth and protein expression. However, in the absence of acetic acid, the growth seemed unaffected by pH. The exclusion of glucose during the induction phase in complex medium was beneficial. The average specific enzyme activity decreased from 95.3 U/mg in the absence of glucose to 20.5 U/mg when glucose was present at an induction temperature of 42 OC. Complex-substrate concentrations also affected plasmid stability, and hence determined protein expression levels. One may conclude that the inactivation of plasmid was not due to temperature per se, but rather due to the negative effect of a strong promoter and protein expression on the plasmid. The average specific productivity decreased as the induction time elapsed. The plateau in productivity marked the complete inactivation of plasmid, and hence the end of protein expression. The decline of the total viable cell counts during the induction was mainly due to the metabolic load. When the culture was totally freed from the productive population, the total viable cell count started to incline. The increase of biomass from the unproductive cells resulted in the decline of the total specific protein activity in the culture. The time of the incline coincided with the time where all the resistant (productive) cells completely disappeared from the culture. A novel semi-empirical model was developed to describe the kinetics of protein expression, growth, and plasmid stability. Good agreement between the predicted and experimental results, in both instantaneous and ramped induction studies, demonstrated the applicability of the model. A novel approach to predicting the induction temperature operating range was established and experimentally proven between 37.3 OC and 42 OC in this study. The ultimate specific P-galactosidase activity ranged from 66 U/mg to 149 U/mg at induction temperature of 42 OC and 38 OC respectively. This approach also provided an accurate means of estimating the average plasmid number per cell at a given time and temperature during the induction. The dependence of parameters on the temperature was determined using an Arrhenius relationship. The temperature strongly affected the plasmid decay constant (h) and the number of activated plasmids per cell (n), while the specific growth rate (U) was relatively independent. This suggested that the protein expression and growth were uncoupled. In addition, the maximum biomass concentration (Xmax), the proteolysis rate (Kprot), and lysis rate (Klys) were also barely influenced by temperature

Application of beneficial microorganisms on agriculture

Food crisis has increasingly become a main issue discussed worldwide. In addition with the recent fuel price increase and the depletion of oil resources, there is a call for bio-fuel as an alternative, which further competes with food production in the agricultural sector. A sustainable agriculture development is needed to mitigate these issues. The ultimate goal of sustainable agriculture according to the National Research Council of USA is to develop farming systems that are productive, profitable, energyconserving, environmentally-sound, conserving of natural resources, and that ensure food safety and quality. Natural farming may provide a good alternative to solve the issues. The concepts and practices of natural farming make use of a consortium of beneficial microorganisms to improve soil health have originated from Han Kyu Cho and the Korean Natural Farming Association (KNFA) more than 30 years ago. However, this knowledge has only come into the English language in the last one to two decades. The practice is very similar to the technology of effective microorganisms (EM) originated by Professor Teruo Higa, University of the Ryukyus, Okinawa, Japan [1, 2, 6]. EM consists of predominant populations of lactic acid bacteria and yeasts, and smaller numbers of photosynthetic bacteria, actinomycetes and other types of organisms that can be applied as microbial inoculants to increase the microbial diversity of soils and plants. EM shifts the soil microbiological equilibrium in ways that can improve soil quality, soil health, and hence the growth, yield, and quality of the crops. Indeed, soil quality is the key to a sustainable agriculture [6]. Beneficial microorganisms and indigenous microorganisms (IMO) generally refer to a large group of naturally occurring and often unknown or ill-defined microorganisms that interact favorably in soils and with plants to render beneficial effects which are sometimes difficult to predict. While EM usually denotes specific mixed cultures of known, beneficial microorganisms that are being used effectively as microbial inoculants that could exist naturally in soil or added as microbial inoculants to soil where they can improve soil quality, enhance crop production and create a more sustainable agriculture and environment [6]. Upon inoculation of beneficial microorganisms to the soil, they can function as biocontrol agents by controlling or suppressing soil-borne plant pathogens through their competitive and antagonistic activities. However, the use of mixed cultures in this approach has been criticized because it is difficult to demonstrate conclusively which microorganisms are responsible for the observed effects, how the introduced microorganisms interact with the indigenous species, and how these new associations affect the soil plant environment. Thus, the use of mixed cultures of beneficial micro-organisms as soil inoculants to enhance the growth, health, yield, and quality of crops has not gained widespread acceptance by the agricultural research establishment because conclusive scientific proof is often lacking [6]. Natural farming which relies on IMO is considered a type of organic farming. As opposed to conventional farming, organic farming does not use any agrochemicals as fertilizers or pesticides. Instead bio-fertilizers and traditional composting of animal or plant manures are used to provide nutrients for the plants. Therefore, it is a low-input but sustainable cropping system, enabling the natural roles of micro-organism to maintain soil fertility and bio-control of plant pathogens [3,4]. In Malaysia, application of IMO in natural farming has been actively adopted by farmers in at least three states since 2001 through the Asian Productivity Organization [4]. However, some of the local technologies only depend on farm testing and lack scientific results [5]. Thus, it suggested conducting research to verify efficiency and dosage of the products before they are marketed. For example, germination test and soil fertility test could be conducted and data should be interpreted using statistical analysis. Microbial diversity can also be determined using scientific methods such as fatty acid profiling, DNA microarray and polymerase chain reaction (PCR). Apart of application on soil and plant, EM have also been widely applied on animal farmland to improve the management of environmental issues such as to reduce the stinky smell due to animal waste and the number of flies; and also to promote the production of organic fertilizer from animal and plant waste. Application of EM to the feed and drinks of the chickens has also been reported to improve the overall health of the animal [23]. The following sections described the case studies where EM was applied to provide improved growth and yield of the maize plants; as well as to improve the quality of meat and to lower the mortality among the Japanese quails

Review : overexpression of protein under transcriptional regulation of lambda pL promoter system in Escherichia coli: consequences and bioprocess improvement approaches

The attraction of employing thermal induction system in the production of heterologous protein is mainly due to 1) a higher expressed protein titer in comparison to constitutive system, and 2) a cleaner process since no chemical inducing agent is added. However, problems pertaining to high temperature and protein overexpression such as inclusion bodies formations, metabolic overload on the host, stress-related proteolysis on the expressed protein, and enhanced acetic acid secretion are commonly encountered. Several bioprocess approaches that include temperature upshift schemes, feeding strategies, timing of induction, two-stage culture mode, and stabilization of plasmid had been implemented to minimize some of these problems. The approaches are reviewed in this article