Humic acid inhibition of hydrolysis and methanogenesis with different anaerobic inocula.

There is increasing evidence that humic acid (HA) is hampering the performance of anaerobic digesters treating animal manures and thermally-hydrolysed waste activated sludge. In the present study, HA inhibition and inhibition resilience was examined for hydrolysis (carbohydrate and protein) and acetotrophic methanogenesis with four distinct full-scale anaerobic inocula. The aim was to further understand HA inhibition and to explore potential relationships between microbial factors and inhibition resilience. For two of the four tested inocula, cellulose degradation showed a start-up delay that lengthened as HA concentration increased from 0 to 2 g L-1. This inhibition was reversible because, after the initial delay, subsequent hydrolysis rates and methane yields were not significantly influenced by HA concentration. Cellulose hydrolysis results at HA concentrations below 2 g L-1 support a threshold inhibition mechanism, i.e. HA complexes with hydrolytic enzymes preventing them from binding with cellulose, but once all the HA had been complexed, enzymes subsequently released are free to bind with cellulose. Inocula with higher cellulose hydrolytic activity were less affected by HA inhibition, suggesting a potential link between HA inhibition resilience and microbial activity. However, above 5 gHA L-1, cellulose hydrolysis rates decreased with increasing HA concentration; indicating that the mechanisms of inhibition may change depending on some threshold HA concentration. Protein hydrolysis and acetotrophic methanogenesis were less susceptible to HA inhibition than cellulose hydrolysis, since signs of inhibition were only observed above 5 gHA L-1. Acetotrophic methanogenesis was partially inhibited at 10 gHA L-1 and completely inhibited at 20 gHA L-1. These results further support that HA inhibition is selective towards particular enzymes.

Characterising and modelling free ammonia and ammonium inhibition in anaerobic systems.

Inhibition by ammoniacal nitrogen, consisting of free ammonia (NH3) and ammonium ion (NH4+), has been widely investigated for anaerobic digestion. However, despite the large amount of research on the subject, ammoniacal nitrogen inhibition still threatens many anaerobic digesters. This paper presents (i) a method to reliably characterise ammoniacal nitrogen inhibition and (ii) a robust inhibition modelling approach. Results showed that NH3 and NH4+ inhibition need to be jointly determined, which can only be done by performing inhibition tests at various total ammoniacal nitrogen (TAN) concentrations and pH values. These test conditions were reliably achieved using the salts NH4HCO3 and NH4Cl without pH adjustment, rather than by using NH4Cl with pH adjustment. The use of only salts showed a lower pH change during the inhibition test (∼1.5 days), thereby decreasing the uncertainty in TAN speciation and strengthening the test and model outputs. A threshold inhibition function satisfactorily described (R2 > 0.99) the joint inhibition of NH3 and NH4+ on three distinct inocula, and provided a better description of the inhibition testing results than a non-competitive inhibition function (R2 ∼0.70). The key advantage of the proposed threshold inhibition function is its capacity to identify the inhibition lower limit (concentration where inhibition starts; KImin) and upper limit (concentration where inhibition is complete; KImax). The threshold inhibition function also identifies the 50% inhibition concentration (KI50) at the midpoint of KImin and KImax. Finally, experimental and model results show that at pH 7.3-7.7 and TAN concentrations above 2000 mgN·L-1, both NH3 and NH4+ contribute significantly to overall inhibition.

Deterministic mechanisms define the long-term anaerobic digestion microbiome and its functionality regardless of the initial microbial community.

The impact of the starting inoculum on long-term anaerobic digestion performance, process functionality and microbial community composition remains unclear. To understand the impact of starting inoculum, active microbial communities from four different full-scale anaerobic digesters were each used to inoculate four continuous lab-scale anaerobic digesters, which were operated identically for 295 days. Digesters were operated at 15 days solid retention time, an organic loading rate of 1 g COD Lr-1 d-1 (75:25 - cellulose:casein) and 37 °C. Results showed that long-term process performance, metabolic rates (hydrolytic, acetogenic, and methanogenic) and microbial community are independent of the inoculum source. Digesters process performance converged after 80 days, while metabolic rates and microbial communities converged after 120-145 days. The convergence of the different microbial communities towards a core-community proves that the deterministic factors (process operational conditions) were a stronger driver than the initial microbial community composition. Indeed, the core-community represented 72% of the relative abundance among the four digesters. Moreover, a number of positive correlations were observed between higher metabolic rates and the relative abundance of specific microbial groups. These correlations showed that both substrate consumers and suppliers trigger higher metabolic rates, expanding the knowledge of the nexus between microorganisms and functionality. Overall, these results support that deterministic factors control microbial communities in bioreactors independently of the inoculum source. Hence, it seems plausible that a desired microbial composition and functionality can be achieved by tuning process operational conditions.

Relationship between microbial community, operational factors and ammonia inhibition resilience in anaerobic digesters at low and moderate ammonia background concentrations.

The relationship between anaerobic digestion operational conditions and (i) microbial community, (ii) acetoclastic methanogenic activity and (iii) free ammonia (NH3) inhibition resilience was investigated. Thirteen inocula were obtained from full and pilot scale digesters fed with different substrates, digester configurations, operating temperatures and NH3 concentrations (0.1-241 mgN·L-1). Substrate type and temperature were the primary factors influencing microbial community composition. Methanogenic activity ranged from 0.04 to 0.14 gCOD-CH4·g-1VS·day-1, and was significantly correlated with archaeal relative abundance and archaeal community PC2. The variability of NH3 resilience among inocula was moderate, with inhibition threshold values (KI50) ranging between 32 and 175 mgNH3-N·L-1. No microbial or operational factors correlated with NH3 resilience. However, the slopes of inhibition threshold curves were influenced by some environmental factors, namely substrate type, digester temperature and NH3 concentration. Overall, these results indicate that low and moderate background NH3 concentrations is not a key determinant of microbial community nor NH3 resilience.

Indigenous microbial capability in solid manure residues to start-up solid-phase anaerobic digesters.

Batch solid-phase anaerobic digestion is a technology for sustainable on-farm treatment of solid residues, but is an emerging technology that is yet to be optimised with respect to start-up and inoculation. In the present study, spent bedding from two piggeries (site A and B) were batch digested at total solids (TS) concentration of 5, 10 and 20% at mesophilic (37°C) and thermophilic (55°C) temperatures, without adding an external inoculum. The results showed that the indigenous microbial community present in spent bedding was able to recover the full methane potential of the bedding (140±5 and 227±6L CH4 kgVSfed-1 for site A and B, respectively), but longer treatment times were required than for digestion with an added external inoculum. Nonetheless, at high solid loadings (i.e. TS level>10%), the digestion performance was affected by chemical inhibition due to ammonia and/or humic acid. Thermophilic temperatures did not influence digestion performance but did increase start-up failure risk. Further, inoculation of residues from the batch digestion to subsequent batch enhanced start-up and achieved full methane potential recovery of the bedding. Inoculation with liquid residue (leachate) was preferred over a solid residue, to preserve treatment capacity for fresh substrate. Overall, the study highlighted that indigenous microbial community in the solid manure residue was capable of recovering full methane potential and that solid-phase digestion was ultimately limited by chemical inhibition rather than lack of suitable microbial community.

Pilot-scale testing of a leachbed for anaerobic digestion of livestock residues on-farm.

A leachbed is a relatively simple anaerobic digester suitable for high-solids residues and on-farm applications. However, performance characteristics and optimal configuration of leachbeds are not well-understood. In this study, two 200 L pilot-scale leachbeds fed with spent straw bedding from pigs/swine (methane potential, B0 = 195-218 L CH4 kg(-1) VS fed) were used to assess the effects of leachate recirculation mode (trickling vs. flood-and-drain) on the digestion performance. Results showed comparable substrate solubilisation extents (30-45% of total chemical oxygen demand fed) and methane conversion (50% of the B0) for the trickling and flood-and-drain modes, indicating that digestion performance was insensitive to the mode of leachate flow. However, the flood-and-drain leachbed mobilised more particulates into the leachate than the trickling leachbed, an undesirable outcome, because these particulates were mostly non-biodegradable. Inoculation with solid residues from a previous leachbed (inoculum-to-substrate ratio of 0.22 on a VS basis) hastened the leachbed start-up, but methane recovery remained at 50% of the B0 regardless of the leachate recirculation mode. Post-digestion testing indicated that the leachbeds may have been limited by microbial activity/inhibition. The high residual methane potential of leachate from the trickling (residual Bo = 732 ± 7 L CH4 kg(-1) VS fed) and flood-and-drain leachbeds (582 ± 8 L CH4 kg(-1) VS fed) indicated an opportunity for further processing of leachate via a separate methanogenic step. Overall, a trickling leachbed appeared to be more favourable than the flood-and-drain leachbed for treating spent bedding at farm-scale due to easier operation.

Semi-aerobic fermentation as a novel pre-treatment to obtain VFA and increase methane yield from primary sludge.

There is a growing trend to consider organic wastes as potential sources of renewable energy and value-add products. Fermentation products have emerged as attractive value-add option due to relative easy production and broad application range. However, pre-fermentation and extraction of soluble products may impact down-stream treatment processes, particularly energy recovery by anaerobic digestion. This paper investigates primary sludge pre-fermentation at different temperatures (20, 37, 55, and 70°C), treatment times (12, 24, 48, and 72h), and oxygen availability (semi-aerobic, anaerobic); and its impact on anaerobic digestion. Pre-fermentation at 20 and 37°C succeeded for VFA production with acetate and propionate being major products. Pre-fermentation at 37, 55, and 70°C resulted in higher solubilisation yield but it reduced sludge methane potential by 20%. Under semi-aerobic conditions, pre-fermentation allowed both VFA recovery (43gCODVFAkg(-1)VS) and improved methane potential. The latter phenomenon was linked to fungi that colonised the sludge top layer during pre-fermentation.

Development and validation of a rapid test for anaerobic inhibition and toxicity.

Despite the importance of quantifying inhibitory capacity of compounds in anaerobic digestion, there is currently no well-defined method to assess it. Experimental methods in literature are frequently time-consuming and resource intensive. As a result, detailed inhibition testing rarely forms part of anaerobic digestion studies, despite the importance and utility of this information. This study develops and validates a simple and rapid inhibition test protocol, based on relative inhibition of acetoclastic methanogens. The inhibition potential of a compound is determined from the reduction in specific methanogenic activity as inhibitor concentration is increased. The method was successfully performed on two inoculums from different source environments and with both biostatic and biocidal inhibitors. Optimisation work indicated that: (i) sodium acetate is a preferred carbon source compared to acetic acid; (ii) an inoculum to acetate ratio of 5 g VS g(-1) acetate is preferred, and (iii) that the inoculum concentration should be normalised to 10 g L(-1) VS to reduce mass transfer problems and promote consistency. A key advantage over existing methods is that the sampling strategy has been optimised to three events over 1.5 days while effectively controlling the relative analytical error.

Anaerobic co-digestion of pig manure and algae: impact of intracellular algal products recovery on co-digestion performance.

This paper investigates anaerobic co-digestion of pig manure and algae (Scenedesmus sp.) with and without extraction of intracellular algal co-products, with views towards the development of a biorefinery concept for lipid, protein and/or biogas production. Protein and/or lipids were extracted from Scenedesmus sp. using free nitrous acid pre-treatments and solvent-based Soxhlet extraction, respectively. Processing increased algae methane yield between 29% and 37% compared to raw algae (VS basis), but reduced the amount of algae available for digestion. Co-digestion experiments showed a synergy between pig manure and raw algae that increased raw algae methane yield from 0.163 to 0.245 m(3) CH4 kg(-1)VS. No such synergy was observed when algal residues were co-digested with pig manure. Finally, experimental results were used to develop a high-level concept for an integrated biorefinery processing pig manure and onsite cultivated algae, evaluating methane production and co-product recovery per mass of pig manure entering the refinery.

Anaerobic codigestion of sewage sludge and glycerol, focusing on process kinetics, microbial dynamics and sludge dewaterability.

Anaerobic codigestion (AcoD) is a proven option to significantly boost biogas production while utilizing existing digesters and infrastructure. The aim of the present research was to conduct an exhaustive study regarding anaerobic codigestion of mixed sewage sludge and crude glycerol considering impacts on organic load, hydraulic load, process performance and microbial community. The methane potential of crude glycerol varied from 370 mL CH4·g(-1) VS to 483 mL CH4·g(-1) VS for different samples tested. The half maximal inhibitory concentration of crude glycerol was 1.01 g VS L(-1), and the primary mechanism of inhibition was through overload from rapid fermentation rather than the presence of toxic compounds in the crude glycerol. In continuous operation over 200 days, feeding glycerol at up to 2% v/v, increased organic load by up to 70% and resulted in a 50% increase in methane production. Glycerol dosing resulted in no change in apparent dewaterability, with both codigestion and control reactors returning values of 22%-24%. Members of the phylum Thermotogae emerged as a niche population during AcoD of sewage sludge and glycerol; however there was no gross change in microbial community structure and only minimal changes in diversity. AcoD did not result in synergisms between sewage sludge and crude glycerol. Actually, at dose rate up to 2% v/v glycerol dosing is still an effective strategy to increase the organic loading rate of continuous anaerobic digesters with minimal impact of the hydraulic retention time. Nonetheless, the dose rate must be managed to: (i) prevent process inhibition and (ii) ensure sufficient degradation time to produce a stable biosolids product.

Identification of synergistic impacts during anaerobic co-digestion of organic wastes.

Anaerobic co-digestion has been widely investigated, but there is limited analysis of interaction between substrates. The objective of this work was to assess the role of carbohydrates, protein and lipids in co-digestion behaviour separately, and together. Two sets of batch tests were done, each set consisting of the mono-digestion of three substrates, and the co-digestion of seven mixtures. The first was done with pure substrates--cellulose, casein and olive oil--while in the second slaughterhouse waste--paunch, blood and fat--were used as carbohydrate, protein and lipid sources, respectively. Synergistic effects were mainly improvement of process kinetics without a significant change in biodegradability. Kinetics improvement was linked to the mitigation of inhibitory compounds, particularly fats dilution. The exception was co-digestion of paunch with lipids, which resulted in an improved final yield with model based analysis indicating the presence of paunch improved degradability of the fatty feed.

Relative kinetics of anaerobic digestion under thermophilic and mesophilic conditions.

With several advantages over the conventional mesophilic anaerobic digestion, such as better sludge quality and higher biogas production, thermophilic anaerobic digestion is regarded as a promising alternative for sludge digestion. Primary and activated sludges are complex materials, and historically, analysis of kinetics has been largely on whole sludge, without analysis of individual components. This paper analyses relative digestion kinetics of pure substrates designed to target main stages of sludge digestion under thermophilic and mesophilic conditions. Hydrolysis rate of cellulose was significantly influenced by temperature with hydrolysis coefficients of--at 55 degrees C (0.7 +/- 0.1 day(-1)), 60 degrees C (0.8 +/- 0.2 day(-1)), 65 degrees C (1.1 +/- 0.2 day(-1)) and 70 degrees C (1.2 +/- 0.2 day(-1)) over 38 degrees C (0.4 +/- 0.1 day(-1)). This strongly follows the Arrhenius relationship, with an activation energy (E(A)) of 31 +/- 4 kJ mol(-1), corresponding to an increase of 1.5x for each 10 degrees C of temperature increase. Glucose uptake was rapid with a wide variety of fermentation products detected under mesophilic conditions, while uptake was slower under thermophilic conditions with acetate and propionate being dominant products. Propionate acetogenesis and acetate-utilizing methanogenesis kinetics were not influenced by temperatures. Hydrolysis is widely regarded as a rate-limiting step in sludge digestion, thus improvements in hydrolysis rates as measured during this study have the potential for significant improvements in overall apparent sludge digestion rates.

Assessing the role of biochemical methane potential tests in determining anaerobic degradability rate and extent.

The biodegradability and bioavailability of hydrolysis-limited substrates under anaerobic (and aerobic) conditions can be represented by two key parameters--degradability (f(d)), or the percentage that can be effectively be destroyed during digestion, and first order hydrolysis coefficient (k(hyd)), or the speed at which material breaks down. Biochemical methane potential (BMP) testing uses a batch test (in triplicate), and by fitting against a first order model, can fit both parameters in the same test. BMP testing is now being widely used for anaerobic process feasibility and design purposes, and standardisation efforts are ongoing. In this paper, we address a number of key issues relating to the test method and its analysis. This includes proposal of a new fitting and parameter estimation method, evaluation of the impact of inoculum to substrate ratio on fitted parameters, and comparison to performance in continuous systems. The new parameter estimation technique provides an estimate of parameter uncertainty and correlation, and is clearly more suitable than model transformation and linear regression. An inoculum volume ratio of at least 50% (2:1 on VS basis) was required on a cellulose substrate to use methane production as primary indicator, as found by comparing methane production and solubilisation of cellulose. Finally, on a typical material, waste activated sludge, the batch test was slightly conservative in terms of degradability and rate, indicating a bias in the BMP test. The test is a cost-effective and capable method to evaluate potential substrates, but it should be noted that it is generally conservative, especially if sub-optimal inoculum is used.

Measurement and quantification of sessile and planktonic microbial populations during the anaerobic digestion of cellulose.

Crystalline cellulose was anaerobically degraded using a leachate inoculum derived from simulated municipal solid waste. Bicinchoninic Acid (BCA) protein assays were used to measure the distribution of biomass during cellulose degradation, including the planktonic and sessile biomass fractions. A comparison of sessile and planktonic microbial growth indicated that the microbial growth was dominated by the planktonic fraction with the biofilms accounting for approximately 25% of the population. Additional biomass measurements were conducted to test the reliability of the BCA protein assays. Total microbial growth was inferred from the accumulation and depletion of ammonia nitrogen measured using flow injection analysis. The planktonic biomass was estimated from direct cell counts using light microscopy and the sessile biomass was estimated by analysing the nitrogen content of the separated and washed cellulose pellet. Regression analysis showed good correlations between the measurement pairs representing the total biomass (R2=0.90), planktonic biomass (R2=0.97) and sessile biomass (R2=0.85), supporting the use of protein assays as an indicator of microbial growth in mixed culture environments.

Digestion of waste bananas to generate energy in Australia.

This paper presents results from laboratory studies to measure the methane yield and rate of digestion of reject bananas. These parameters were determined in experiments that took into account the likely configuration of a full-scale plant in the banana growing region of north Queensland. The digestion was conducted in a 200-l reactor using fed-batch operation, relying entirely on the natural microbial consortia on the reject bananas to avoid reliance on external inocula such as sludge, an undesirable material around food packaging facilities. An enrichment culture was first established in a highly buffered 200-l batch digestion unit. The fed-batch digester was then started by exchanging leachate with the mature batch reactor. Under loading conditions of 0.6 kg VS m(-3)d(-1) over 70 days where the average working volume was 160 l, the digester produced 398+/-20 l CH4 kg VS(-1). Increasing the loading rate to 1.6 kg VS m(-3)d(-1) resulted in a reduced methane yield of 210 l CH4 kg VS(-1) over 23 days of operation, with a concomitant accumulation of banana waste in the digester. The leachate at the end of digestion contained over 4000 mg l(-1)K, 200 mg l(-1) N and 75 mg l(-1), levels that exceed acceptable limits for general agricultural irrigation.

Individual and joint actions of selenate and methylmercury on the development and survival of insect detritivore Megaselia scalaris (Diptera: Phoridae).

Despite the important roles played by insects in most ecosystems, surprisingly little is known about how anthropogenic pollutants or their mixtures interact to affect insect populations. The independent and joint actions of selenate and methylmercury on a ubiquitous insect detritivore, Megaselia scalaris (Loew), were determined in this study. Ovipositing females did not distinguish between untreated food sources and those contaminated with toxic concentrations of selenate, methylmercury, or both chemicals in combination. Even at the highest concentrations of pollutants, no negative effects were observed for the egg stage. However, larval survival was significantly decreased and development significantly prolonged by selenate and methylmercury individually at low or intermediate ecologically relevant treatment levels. Potentiation was strongly evident because mixtures containing concentrations as little as only 1% of the respective individual median lethal tolerances (LC(50)s) caused significantly more mortality and delayed larval development than would be expected from the responses selenate and methylmercury elicit individually. However, survival and pupal development was not affected at any rate tested. Female fecundity was significantly decreased by methylmercury but not by selenate or mixture treatments. The relative toxicity to M. scalaris of each of the individual and joint treatments was selenate (LC(50) = 260 microg/g) < methylmercury (LC(50) = 22 microg/g) < the mixture at approximately 5% of the LC(50) concentration of each of the components (12 microg/g selenate plus 1.0 microg/g methylmercury). The increased mortality and delayed larval development within sites contaminated by selenate, methylmercury, or combination of the two have substantial implications for the ecology, population dynamics, and sustainability of M. scalaris populations. If these results can be extrapolated to other arthropod detritivores, ecosystem food-web function may be substantially affected.

Indirect evidence for the potential ability of magnetic resonance imaging to evaluate the myocardial iron content in patients with transfusional iron overload.

The purpose of this study was to evaluate the potential ability of magnetic resonance imaging (MRI) for evaluation of myocardial iron deposits. The applied MRI technique has earlier been validated for quantitative determination of the liver iron concentration. The method involves cardiac gating and may, therefore, also be used for simultaneous evaluation of myocardial iron. The tissue signal intensities were measured from spin echo images and the myocardium/muscle signal intensity ratio was determined. The SI ratio was converted to tissue iron concentration values based on a modified calibration curve from the liver model. The crucial steps of the method were optimized; i.e. recognition and selection of the myocardial slice for analysis and positioning of the regions of interest (ROIs) within the myocardium and the skeletal muscle. This made the myocardial MRI measurements sufficiently reproducible. We applied this method in 41 multiply transfused patients. Our data demonstrate significant positive linear relationships between different iron store parameters and the MRI-derived myocardial iron concentration, which was significantly related to the serum ferritin concentration (rho=0.62, P<0.0001) and to the MRI-determined liver iron concentration (rho=0.36, P=0.02). The myocardial MRI iron concentrations demonstrated also a significant positive correlation with the number of blood units given (rho=0.45, P=0.005) and the aminotransferase serum concentration (rho=0.54, P=0.0008). Our data represents indirect evidence for the ability of MRI techniques based on myocardium/muscle signal intensity ratio measurements to evaluate myocardial iron overload.

Expression of CD2 and activation markers on blood T-helper cell subsets in patients with transfusional iron overload.

The aim of the present study was to investigate the relationship between different measures of iron status, and the expression of CD2, and the activation markers CD25, CD71, CD45RO, HLADR CD38 within the Th-cell subset in patients with progressive transfusional iron overload. We estimated the expression of the activation surface markers on the Th cells of peripheral blood by flow cytometry from 22 multiply transfused patients. The number of CD2 binding sites (BS) on Th cells was significantly higher in the patients (82 917 +/- 30 801) than in age-matched normal controls (41 145 +/- 6989, P < 0.0001). When investigating whether this difference could be due to the iron overload we found the number of CD2 BS closely related to the iron saturation of serum transferrin (TfS) (R2 = 0.78, P < 0.001). The relationship to the serum ferritin concentration and to the number of blood units given was weaker, but also significant (R2 = 0.22, P < 0.027, respectively, R2 = 0.21, P < 0.032). Also the fraction of mature memory Th cells which express CD45RO at a high level was directly related to the TfS (R2 = 0.57, P < 0.0001), while the expression of CD38 within the Th cell fraction was inversely related to the TfS (R2 = - 0.43, P = 0.009). The expression of HLA-DR (but not of CD25 and CD71) was also directly related to the TfS (R2 = 0.29, P = 0.01). Our results show a clear, statistical relationship between the iron status and the expression of surface markers within Th cells in multiply transfused patients.

Relevance of in vitro leukaemia cell survival to short- and long term clinical outcome in AML.

In ninety-three cases of newly diagnosed acute myeloid leukaemia (AML) we investigated the importance to short- and long term clinical outcome of the in vitro short term leukaemia cell survival as measured by a 4-day MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-assay. In 67 patients treated by intravenous remission induction therapy we found that patients who after the first induction cycle or after induction therapy overall achieved a complete remission (CR) had leukaemia cells with significantly lower in vitro cell survival ability than cells of non-responders (p = 0.02 and 0.06, respectively). These relations remained statistically significant in subsequent multivariate analyses. Likewise, a favourable effect of low in vitro leukaemia cell survival on overall survival of the patients was detected in the (largest) subgroup of adult patients treated uniformly by the same remission induction regimen as well as in all patients. However, in the 44 patients, who achieved CR, the in vitro leukaemia cell survival did not show significance to remission duration or time to first relapse. Furthermore, the leukaemia cell survival (MTT-assay) did not to correlate with the Bcl-2 expression level (quantitative flow cytometry) of the leukaemia cells (r = 0.18, n = 34, p = 0.32). In addition, in a cell line model employing the growth factor dependent MO7 human AML cell line, growth factor withdrawal was associated with rapid onset of cellular apoptosis as evaluated by morphology, occurrence of a subG1 peak in DNA histograms, and loss of cellular activity in the MTT-assay. In contrast, a more moderate decline in Bcl-2 expression and gradual loss of ability to exclude the trypan blue dye was seen in the leukaemia cells in response to growth factor withdrawal. We conclude, that the MTT-assay provides a simple and sensitive method for measuring in vitro cell survival. The differences in leukaemia cell survival seen in AML may well be clinically relevant and may help to provide a better understanding of clinical drug resistance.