Nitrogen cycling and relationships between ammonia oxidizers and denitrifiers in a clay-loam soil.

This study investigated the effect of municipal solid waste (MSW) compost (0, 50, and 100 t/ha) on N cycling and the microorganisms involved in it, in a clay-loam soil. After a release of nitrates (NO3(-)-N) in the first 6 days after compost incorporation, soil NO3(-)-N content remained constant in all the treatments until day 62, suggesting N immobilization induced by the soil used in this study. Then, soil NO3(-)-N content increased in all treatments and especially in the highest compost dose, providing evidence that immobilization effect has been at least partially relieved. amoA gene copies of ammonia-oxidizing archaea (AOA) and bacteria (AOB) followed the overall pattern of soil NO3(-)-N content; however, no differences were found in amoA gene copies among treatments, except in the last sampling, an effect attributed to the slight differences in the potential nitrification rate among them. Ammonia oxidizer pattern provided evidence that both groups were involved in ammonia oxidation and changes in their abundance can be used as 'indicator' to predict changes in soil nitrification status. Moreover, the strong correlation between AOA and AOB amoA copies (R(2) = 0.94) and the high slope (13) of the curve suggest that AOA had probably an important role on ammonia oxidation. Denitrifying genes (nirS, nirK, nosZ) also followed the general pattern of soil NO3(-)-N, and they were strongly correlated with both groups of ammonia oxidizers, and particularly AOA, suggesting strong interrelationships among them. Losses of N through denitrification, as they were estimated by total nitrogen, were inversely related to soil NO3(-)-N content. Similar to ammonia oxidizers, denitrifying gene copies did not differ among compost treatments an effect that could be probably explained by the low availability of organic-C in the MSW compost and hence the competition with aerobic heterotrophs.

Bioconversion of oleuropein to hydroxytyrosol by lactic acid bacteria.

The aim of this work is to study the conversion of oleuropein-a polyphenol present in olives and olive oil by-products-into hydroxytyrosol, a polyphenol with antioxidant and antibacterial properties. The hydrolysis reaction is performed by lactic acid bacteria. Six bacterial strains (Lactobacillus plantarum 6907, Lactobacillus paracasei 9192, Lactobacillus casei, Bifidobacterium lactis BO, Enterococcus faecium 32, Lactobacillus LAFTI 10) were tested under aerobic and anaerobic conditions. The oleuropein degradation and hydroxytyrosol formation were monitored by HPLC. Results showed that oleuropein could be successfully converted into hydroxytyrosol. The most effective strain was Lactobacillus plantarum 6907, with a reaction yield of hydroxytyrosol of about 30 %. Different reaction mechanisms were observed for different microorganisms; a different yield was observed for Lactobacillus paracasei 9192 under aerobic or anaerobic conditions and an intermediate metabolite (oleuropein aglycone) was detected for Lactobacillus paracasei 9192 and Lactobacillus plantarum 6907 only. This study could have significant applications, as this reaction can be used to increase the value of olive oil by-products and/or to improve the taste of unripe olives.

Use of green roofs for greywater treatment: Role of substrate, depth, plants, and recirculation.

This work focuses on the use of green roof as a modified shallow vertical flow constructed wetland for greywater treatment in buildings. Different design parameters such as substrate (perlite or vermiculite), substrate depth (15 cm or 25 cm), and plant species (Geranium zonale, Polygala myrtifolia or Atriplex halimus) were tested to determine optimum selection. In addition, the application of a 40% recirculation rate was applied during last month of the experiment to quantify the efficiency of pollutants removal. The experiment was conducted for a period of 12 months under typical Mediterranean climatic conditions in Lesvos island, Greece. Results showed that green roofs planted with Atriplex halimus and filled with 20 cm of vermiculite had the best COD (91%), BOD (91%), TSS (93%) and turbidity (93%) average removal efficiencies. In contrast, significant lower removals were observed when the substrate depth was decreased to 10 cm (60-75%). Green roof vegetation had significant impact on TN removal as the average TN concentration decreased from 6.5 ± 1.8 mg/L in the effluent of unplanted systems to 4.9 ± 2.7 mg/L in the effluent of green roofs planted with Atriplex halimus. The recirculation of a portion of the effluent in the influent had as a result a significant improvement of turbidity, organic matter and (especially) nitrogen removal. For example, BOD removal in green roofs planted with Atriplex halimus and filled with 20 cm of perlite increased from 76% to 92%, while TN removal in green roofs planted with the same plants and filled with 20 cm of vermiculite increased from 56% to 87%. Overall, the operation of green roofs as modified vertical unsaturated constructed wetlands seems a sustainable nature-based solution for greywater treatment and reuse in urban areas.

Pilot-scale comparison of constructed wetlands operated under high hydraulic loading rates and attached biofilm reactors for domestic wastewater treatment.

Four different pilot-scale treatment units were constructed to compare the feasibility of treating domestic wastewater in the City of Heraklio, Crete, Greece: (a) a free water surface (FWS) wetland system, (b) a horizontal subsurface flow (HSF) wetland system, (c) a rotating biological contactor (RBC), and (d) a packed bed filter (PBF). All units operated in parallel at various hydraulic loading rates (HLR) ranging from 50% to 175% of designed operating HLR. The study was conducted during an 8 month period and showed that COD removal efficiency of HSF was comparable (>75%) to that of RBC and PBF, whereas that of the FWS system was only 57%. Average nutrient removal efficiencies for FWS, HSF, RBC and PBF were 6%, 21%, 40% and 43%, respectively for total nitrogen and 21%, 39%, 41% and 42%, respectively for total phosphorus. Removals of total coliforms were lowest in FWS and PBF (1.3 log units) and higher in HSF and RBC (2.3 to 2.6 log units). HSF showed slightly lower but comparable effluent quality to that of RBC and PBF systems, but the construction cost and energy requirements for this system are significantly lower. Overall the final decision for the best non-conventional wastewater treatment system depends on the construction and operation cost, the area demand and the required quality of effluent.

Combining electrokinetic transport and bioremediation for enhanced removal of crude oil from contaminated marine sediments: Results of a long-term, mesocosm-scale experiment.

Marine sediments represent an important sink of harmful petroleum hydrocarbons after an accidental oil spill. Electrobioremediation techniques, which combine electrokinetic transport and biodegradation processes, represent an emerging technological platform for a sustainable remediation of contaminated sediments. Here, we describe the results of a long-term mesocosm-scale electrobioremediation experiment for the treatment of marine sediments contaminated by crude oil. A dimensionally stable anode and a stainless-steel mesh cathode were employed to drive seawater electrolysis at a fixed current density of 11 A/m2. This approach allowed establishing conditions conducive to contaminants biodegradation, as confirmed by the enrichment of Alcanivorax borkumensis cells harboring the alkB-gene and other aerobic hydrocarbonoclastic bacteria. Oil chemistry analyses indicated that aromatic hydrocarbons were primarily removed from the sediment via electroosmosis and low molecular weight alkanes (nC6 to nC10) via biodegradation.

Constructed wetlands treating highway runoff in the central Mediterranean region.

Two free water surface (FWS) and two subsurface flow (SSF) pilot-size constructed wetlands treating highway runoff (HRO) were monitored over a period of two years (September 2005-August 2007). One FWS and one SSF were designed with a hydraulic retention time (HRT) of 12h, named FWS12 and SSF12, respectively, with each one capable of treating a maximum HRO of 12.6 m(3) d(-1). The other couple, named FWS24 and SSF24, respectively, was designed with an HRT of 24h, with each receiving a maximum HRO of 6.3 m(3) d(-1). The influent flowed from a highway section with a total surface 2752 m(2) on the island of Crete, Greece, in the heart of the South-Central Mediterranean region. Influent and effluent were monitored for COD, TSS, total N (TN), NO(3)(-) and total P (TP) concentrations. Furthermore, removal efficiencies were examined for heavy metals (Cu, Ni, Pb, Zn) for both years, while polycyclic aromatic compounds (PAHs) were examined for the period between September 2006 and August 2007. The influent had a two-year average COD value of 101 mg l(-1), whereas the mean values for TSS, TN, N-NO(3)(-) and TP were 203, 4.30, 1.25 and 4.17 mg l(-1), respectively. For Cu, Ni, Pb and Zn the respective two-year mean influent concentrations were 56, 114, 49 and 250 microg l(-1). Mean concentration of total PAHs in runoff (summation operator PAHs, 16 compounds) were 12.01 microg l(-1). The performance among the four beds was not significantly different according to ANOVA analysis followed by Tukey test (at p<0.05) for almost all the above physicochemical parameters, suggesting that all systems performed in a similar way. All studied systems, achieved a mean of two-year removal efficiencies of 47% for COD, 89% for TSS, 49% for TN, 58% for N-NO(3)(-), 60% for TP, 47% for Cu, 23% for Ni, 33% for Pb, 61% for Zn and 59% for summation operator PAHs (16 compounds).

Single stage treatment of saline wastewater with marine bacterial-microalgae consortia in a fixed-bed photobioreactor.

Currently, the treatment of aquaculture-origin effluents is mainly performed through land-based recirculating aquaculture systems (RAS). In this study, we evaluate and introduce a novel immobilized/packed bed bioreactor which uses a synthetic textile as the support medium. A marine microbial consortium was developed on the textile by its inoculation with the microalgae Picochlorum sp. The bioreactor was tested with variable loadings of C and N and showed outstanding performance approaching removal rates up to 95% within a few hours (4-5h) of operation. Pyrosequencing analysis revealed a novel microbial consortium consisting mainly of chitrinomycetes, Pseudomonas sp. and the absence of ß-proteobacteria, which is the Class encompassing autotrophic nitrifiers. Quantitative polymerase chain reaction further confirmed these findings suggesting heterotrophic nitrification and aerobic denitrification as the principal mechanisms of N-removal from the bioreactor. Overall our findings reveal the potential of the AdvanTex System for the treatment of marine aquaculture effluents-COD reduction and N-removal, in a single stage.

Allochthonous bioaugmentation in ex situ treatment of crude oil-polluted sediments in the presence of an effective degrading indigenous microbiome.

Oil-polluted sediment bioremediation depends on both physicochemical and biological parameters, but the effect of the latter cannot be evaluated without the optimization of the former. We aimed in optimizing the physicochemical parameters related to biodegradation by applying an ex-situ landfarming set-up combined with biostimulation to oil-polluted sediment, in order to determine the added effect of bioaugmentation by four allochthonous oil-degrading bacterial consortia in relation to the degradation efficiency of the indigenous community. We monitored hydrocarbon degradation, sediment ecotoxicity and hydrolytic activity, bacterial population sizes and bacterial community dynamics, characterizing the dominant taxa through time and at each treatment. We observed no significant differences in total degradation, but increased ecotoxicity between the different treatments receiving both biostimulation and bioaugmentation and the biostimulated-only control. Moreover, the added allochthonous bacteria quickly perished and were rarely detected, their addition inducing minimal shifts in community structure although it altered the distribution of the residual hydrocarbons in two treatments. Therefore, we concluded that biodegradation was mostly performed by the autochthonous populations while bioaugmentation, in contrast to biostimulation, did not enhance the remediation process. Our results indicate that when environmental conditions are optimized, the indigenous microbiome at a polluted site will likely outperform any allochthonous consortium.

Optimization of the physiochemical parameters for the culture of Bombyx mori insect cells used in recombinant protein production.

Silkworm (Bombyx mori, Bm5) cell growth at different environmental conditions was systematically studied in controlled bioreactors. The physiochemical parameters, which affect the growth of Bm5 cells, were defined and optimized. The growth rate was maximal at a medium osmolality of about 370 mosm kg-1; and more than 90% of the maximum growth rate was achieved with osmolality between 350 and 385 mosm kg-1. The optimum pH for Bm5 cell growth was from 6.10 to 6.30. Moreover, the effect of dissolved oxygen concentrations (higher than 20% air saturation) on growth was minimal. However, the maximum cell density decreased markedly with dissolved oxygen tensions less than 20%. The shear protection additive Pluronic F-68 had a very important effect on the growth of Bm5 cells in bioreactors with an optimum concentration of 0.2% (w/v). Compared to the original culture condition, the optimized conditions resulted in a twofold increase in cell yield and a reduced population doubling time from 48 to 36 h. Furthermore, the yield of a recombinant protein (CAT) was increased substantially in the optimized cultures.

Cell cycle dynamics of microcarrier cultures.

In anchorage-dependent cell microcarrier cultures knowledge of the cell's growth kinetics is necessary in order to design and successfully operate bioreactors, particularly on a large scale. However, in addition to growth kinetics, an understanding of the physiological state of the culture is also important. In this paper the cell cycle progression of Vero and MRC-5 microcarrier cultures have been observed utilizing a flow cytometer. Flow cytometry analysis enabled the differentiation of the various phases of the cell cycle as the culture moved from initial inoculation to the stationary, or confluent stage. Not only was the flow cytometer able to distinguish contact inhibited cells from noncontact inhibited cells, but the measured fraction of contact inhibition cells were found to be in agreement with fractions predicted from a previously developed cellular automation model for microcarrier cultures. Further, the data from the stationary phase was used to quantify the death rate in microcarrier cultures.

Application of solvent microextraction to the analysis of nitroaromatic explosives in water samples.

The application of solvent microextraction to the analysis of nitroaromatic explosives is presented. Extraction of 11 nitroaromatics was achieved by suspending 1 microl of organic solvent to the tip of a microsyringe in a stirred aqueous solution. Parameters such as extraction solvent, stirring rate, salt concentration and sampling time were studied and optimized. The limits of detection using bench-top quadrupole mass spectrometry and short extraction times (15 min) were found to be between 0.08 and 1.3 microg/l and the relative standard deviations ranged between 4.3 and 9.8%. Although precision and accuracy of quantification of the method are still needed, solvent microextraction proved to be a fast, simple and inexpensive tool for preconcentration and matrix isolation of nitroaromatics on a microscale.

Solid-phase microextraction versus single-drop microextraction for the analysis of nitroaromatic explosives in water samples.

This paper compares solid-phase microextraction (SPME) with a recently developed extraction method called single-drop microextraction (SDME) for the analysis of nitroaromatic explosives in water samples. The two techniques are examined in terms of procedure, chromatographic analysis and method performance. All practical considerations for both techniques are also reviewed. SPME requires dedicated apparatus and is relatively expensive, as the fiber's lifetime is limited. However, it has the advantages over SDME that it can be easily used for headspace analysis and has lower detection limits for all the target analytes. SDME requires more elaborate manual operations, thus affecting linearity and precision.

Optimum infection conditions for recombinant protein production in insect cell (Bm5) suspension culture.

The baculovirus/insect cell expression system is an efficient and practical method for the production of many active therapeutic proteins on a large scale. The advantages of suspension cultures have been demonstrated with the study of a baculovirus/insect cell (BmNPV/Bm5) expression system for the production of recombinant chloramphenicol acetyltransferase (CAT), a model heterologous protein. Key infection parameters such as infection time and multiplicity of infection were examined systematically for the maximization of protein production. Furthermore, emphasis was placed on the development of possible medium replenishment strategies, which were necessary to achieve higher volumetric protein production from the infection of high-density cell cultures without sacrificing specific protein productivity. The highest protein production was achieved with the infection of suspended cells in the mid to late exponential growth phase.

Evaluation of autochthonous bioaugmentation and biostimulation during microcosm-simulated oil spills.

Oil spills are treated as a widespread problem that poses a great threat to any ecosystem. Following first response actions, bioremediation has emerged as the best strategy for combating oil spills and can be enhanced by the following two complementary approaches: bioaugmentation and biostimulation. Bioaugmentation is one of the most controversial issues of bioremediation. Studies that compare the relative performance of bioaugmentation and biostimulation suggest that nutrient addition alone has a greater effect on oil biodegradation than the addition of microbial products because the survival and degradation ability of microbes introduced to a contaminated site are highly dependent on environmental conditions. Microbial populations grown in rich media under laboratory conditions become stressed when exposed to field conditions in which nutrient concentrations are substantially lower. There is increasing evidence that the best approach to overcoming these barriers is the use of microorganisms from the polluted area, an approach proposed as autochthonous bioaugmentation (ABA) and defined as a bioaugmentation technology that exclusively uses microorganisms indigenous to the sites (soil, sand, and water) slated for decontamination. In this work, we examined the effectiveness of strategies combining autochthonous bioaugmentation with biostimulation for successful remediation of polluted marine environments. Seawater was collected from a pristine area (Agios Onoufrios Beach, Chania) and was placed in a bioreactor with 1% v/v crude oil to facilitate the adaptation of the indigenous microorganism population. The pre-adapted consortium and the indigenous population were tested in combination with inorganic or lipophilic nutrients in the presence (or absence) of biosurfactants (rhamnolipids) during 90-day long experiments. Chemical analysis (gas chromatography-mass spectrometry) of petroleum hydrocarbons confirmed the results of previous work demonstrating that the biodegradation processes were enhanced by the addition of lipophilic fertilizers (uric acid and lecithin) in combination with biosurfactants (rhamnolipids), resulting in increased removal of petroleum hydrocarbons as well as reduction of the lag phase within 15 days of treatment. Considering this outcome and examining the results, the use of biostimulation additives in combination with naturally pre-adapted hydrocarbon-degrading consortia (bioaugmentation) has proved to be an effective treatment and is a promising strategy that could be applied specifically when an oil spill approaches near a shore line and an immediate hydrocarbon degradation effort is needed.

Enhanced ex situ bioremediation of crude oil contaminated beach sand by supplementation with nutrients and rhamnolipids.

Mediterranean coastal regions are particularly exposed to oil pollution due to extensive industrialization, urbanization and transport of crude and refined oil to and from refineries. Bioremediation of contaminated beach sand through landfarming is both simple and cost-effective to implement compared to other treatment technologies. The purpose of the present study was to investigate the effect of alternative nutrients on biodegradation of crude oil contaminated beach sand in an effort to reduce the time required for bioremediation employing only indigenous hydrocarbon degraders. A natural sandy soil was collected from Agios Onoufrios beach (Chania, Greece) and was contaminated with weathered crude oil. The indigenous microbial population in the contaminated sand was tested alone (control treatment) or in combination with inorganic nutrients (KNO3 and K2HPO4) to investigate their effects on oil biodegradation rates. In addition, the ability of biosurfactants (rhamnolipids), in the presence of organic nutrients (uric acid and lecithin), to further stimulate biodegradation was investigated in laboratory microcosms over a 45-day period. Biodegradation was tracked by GC/MS analysis of aliphatic and polycyclic aromatic hydrocarbons components and the measured concentrations were corrected for abiotic removal by hopane normalizations. It was found that the saturated fraction of the residual oil is degraded more extensively than the aromatic fraction and the bacterial growth after an incubation period of approximately 3 weeks was much greater from the bacterial growth in the control. The results show that the treatments with inorganic or organic nutrients are equally effective over almost 30 days where C12-C35n-alkanes were degraded more than 97% and polyaromatic hydrocarbons with two or three rings were degraded more than 95% within 45 days. The results clearly show that the addition of nutrients to contaminated beach sand significantly enhanced the activity of indigenous microorganisms, as well as the removal of total recoverable petroleum hydrocarbons (TRPH) over a 45-day study period.

Measurements of particulate matter concentrations at a landfill site (Crete, Greece).

Large amounts of solid waste are disposed in landfills and the potential of particulate matter (PM) emissions into the atmosphere is significant. Particulate matter emissions in landfills are the result of resuspension from the disposed waste and other activities such as mechanical recycling and composting, waste unloading and sorting, the process of coating residues and waste transport by trucks. Measurements of ambient levels of inhalable particulate matter (PM(10)) were performed in a landfill site located at Chania (Crete, Greece). Elevated PM(10) concentrations were measured in the landfill site during several landfill operations. It was observed that the meteorological conditions (mainly wind velocity and temperature) influence considerably the PM(10) concentrations. Comparison between the PM(10) concentrations at the landfill and at a PM(10) background site indicates the influence of the landfill activities on local concentrations at the landfill. No correlation was observed between the measurements at the landfill and the background sites. Finally, specific preventing measures are proposed to control the PM concentrations in landfills.

Toxicity bioassays in core sediments from the Bay of Santander, northern Spain.

The use of Vibrio fischeri as luminescence bacteria is particularly effective in evaluating contaminated sediment. In this study, the ecotoxicity of five core sediments from the Bay of Santander, northern Spain, utilising V. fischeri as marine bacterium, was carried out. Different toxicity assay procedures were applied in order to study the influence of the mobility and bioavailability of the pollutants. Basic Solid Phase Test (BSPT) in whole sediment and acute toxicity test, using pore water and three leaching test procedures as liquid extracts, were applied. In addition, the study of the influence of the pH value on the toxicity results of the leaching tests was conducted. The obtained results show toxicity units (TU50) values in BSPT test ranging from 0.42 to 39.06 with a decrease with depth as general trend and TU50 values from 0.010 to 0.389 in the liquid extracts, where TU50 is calculated as the inverse of EC50 (%). The obtained data show the historical toxicity trends of the Bay of Santander and provides a technical database for the management of contaminated sediments. Moreover, these results showed evidence that each sediment test procedure provided independent and complementary ecotoxicological responses useful for a sediment classification. In order to analyse the correlations between chemical parameters (both organic and inorganic) and the toxicity results, the self-organising map (SOM) neural network and regression equations were applied. Satisfactory correlations (R=0.93) between chemical concentrations of sum of five heavy metals and 16 PAHs and BSPT toxicity were obtained.

Physico-chemical characterization of indoor/outdoor particulate matter in two residential houses in Oslo, Norway: measurements overview and physical properties--URBAN-AEROSOL Project.

Indoor/outdoor measurements have been performed in the Oslo metropolitan area during summer and winter periods (2002-2003) at two different residential houses. The objective of the measurement study was to characterize, physically and chemically, the particulate matter (PM) and gaseous pollutants associated with actual human exposure in the selected places, and their indoor/outdoor relationship. In this paper, we focus on the PM measurements and examine the relationship between the indoor and outdoor PM concentrations taking into account the ventilation rate, indoor sources and meteorological conditions. The indoor/outdoor measurements indicate the important contribution of the outdoor air to the indoor air quality and the influence of specific indoor sources such as smoking and cooking to the concentration of PM inside houses. However, no specific correlation was found between the indoor/outdoor concentration ratio and the meteorological parameters. This study provides information on the physical characteristics and the relationship of indoor to outdoor concentration of particulate matter in residential houses. Moreover, the parameters that influence this relationship are discussed. The results presented here are specific to the sampled houses and conditions used and provide data on the actual human exposure characteristics which occur in the spatial and temporal scales of the present study.

Bacterial community dynamics during in-situ bioremediation of petroleum waste sludge in landfarming sites.

In-situ bioremediation of petroleum waste sludge in landfarming sites of Motor Oil Hellas (petroleum refinery) was studied by monitoring the changes of the petroleum composition of the waste sludge, as well as the changes in the structure of the microbial community, for a time period of 14 months. The analyses indicated an enhanced degradation of the petroleum hydrocarbons in the landfarming areas. A depletion of n-alkanes of approximately 75-100% was obtained. Marked changes of the microbial communities of the landfarms occurred concomitantly with the degradation of the petroleum hydrocarbons. The results obtained from terminal restriction fragment length polymorphism (T-RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rRNA genes demonstrated that bacteria originating from the refinery waste sludge and newly selected bacteria dominated the soil bacterial community during the period of the highest degradation activity. However, the diversity of the microbial community was decreased with increased degradation of the petroleum hydrocarbons contained in the landfarms. T-RFLP fingerprints of bacteria of the genera Enterobacter and Ochrobactrum were detected in the landfarmed soil over the entire treatment period of 14 months. In contrast, the genus Alcaligenes appeared in significant numbers only within the 10 month old landfarmed soil. Genes encoding catechol 2,3-dioxygenase (subfamily I.2.A) were detected only in DNA of the untreated refinery waste sludge. However, none of the genes known to encode the enzymes alkane hydroxylase AlkB, catechol 2,3-dioxygenase (subfamily I.2.A) and naphthalene dioxygenase nahAc could be detected in DNA of the landfarmed soils.

Effect of endogenous proteins on growth and antibody productivity in hybridoma batch cultures.

It has been shown that some B-cell hybridomas secrete autocrine factors in vitro which can influence cell metabolic processes. Rather than screen specifically for suspected cytokines, that may or may not affect our cell line, we have examined the lumped effects of intracellular and secreted factors on cell proliferation and monoclonal productivity in hybridoma batch cultures. Firstly, supplements of total soluble intracellular proteins combined with other intracellular metabolites were found to both decrease the specific growth rate and increase the antibody production rate at higher concentrations in batch culture. This is an important consideration in high cell density cultures, such as perfusion systems, where a reduction of growth by the presence of intracellular factors may be compensated by an increase in MAb production. In addition, flow cytometry data revealed that the average cell cycle G1 phase fraction was unaffected by the variation in the maximum specific growth rates during the exponential growth phase, caused by the addition of intracellular factors; this suggests that higher MAb productivity at lower growth rates are not a result of cell arrest in the G1 phase. Secondly, secreted extracellular proteins larger than 10,000 Daltons, which were concentrated from spent culture supernatant, were shown to have no significant effect on growth and specific MAb productivity when supplemented to batch culture at levels twice that encountered late in normal batch culture. This indicates that endogenous secreted cytokines, if at all present, do not play a major autocrine role for this cell line.