Advances on the production of polyhydroxyalkanoates (PHA) by mixed microbial cultures from sugar cane molasses

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Food waste valorization through the production of polyhydroxyalkanoates by mixed microbial cultures

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Impact of carbon/nitrogen feeding strategy on polyhydroxyalkanoates production using mixed microbial cultures

Polyhydroxyalkanoates (PHA) production using mixed microbial cultures (MMC) requires a multi-stage process involving the microbial selection of PHA-storing microorganisms, typically operated in sequencing batch reactors (SBR), and an accumulation reactor. Since low-cost renewable feedstocks used as process feedstock are often nitrogen-deficient, nutrient supply in the selection stage is required to allow for microbial growth. In this context, the possibility to uncouple nitrogen supply from carbon feeding within the SBR cycle has been investigated in this study. Moreover, three different COD:N ratios (100:3.79, 100:3.03 and 100:2.43) were tested in three different runs which also allowed the study of COD:N ratio on the SBR performance. For each run, a synthetic mixture of acetic and propionic acids at an overall organic load rate of 8.5 gCOD L-1 d-1 was used as carbon feedstock, whereas ammonium sulfate was the nitrogen source in a lab-scale sequence batch reactor (SBR) with 1 L of working volume. Besides, a sludge retention time (SRT) of 1 d was used as well as a 6 h cycle length. The uncoupled feeding strategy significantly enhanced the selective pressure towards PHA-storing microorganisms, resulting in a two-fold increase in the PHA production (up to about 1.3 gCOD L-1). A high storage response was observed for the two runs with the COD:N ratios (gCOD:gN) of 100:3.79 and 100:3.03, whereas the lowest investigated nitrogen load resulted in very poor performance in terms of polymer production. In fact, strong nitrogen limitation caused fungi to grow and a very poor storage ability by microorganisms that thrived in those conditions. The COD:N ratio also affected the polymer composition, indeed the produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) showed a variable HV content (1-20 %, w/w) among the three runs, lessening as the COD:N increased. This clearly suggests the possibility to use the COD:N ratio as a tool for tuning polymer properties regardless the composition of the feedstock.

Survival rate, body size and food abundance in pure and mixed blowfly cultures

The influence of food abundance, larval density and interspecific interactions on the survival and body size of Chrysomya albiceps, Chrysomya megacephala and Cochliomyia macellaria was investigated in pure and mixed cultures, to determine the impact of competition and/or facultative predation on native and introduced blowfly species in South America. In mixed cultures there was complete elimination of C. megacephala and C. macellaria. Chrysomya albiceps exhibited higher survival in mixed compared to pure cultures, suggesting that predation offers more advantages than competition for food. Body size of C. albiceps was significantly affected by food scarcity in pure cultures. However, tibia size in males of all species suffered no significant variation as a function of food scarcity. The implications of these results for population dynamics of introduced and native blowfly species are discussed.

Use of tomato and cucumber horticulture by-products in goat diets: effects o nrumen fermentation and microbial communities in batch and continuous cultures

Two in vitro experiments were conducted to analyse the effects of replacing dietary barley grain with wastes of tomato and cucumber fruits and a 1 : 1 tomato : cucumber mixture on rumen fermentation characteristics and microbial abundance. The control (CON) substrate contained 250 g/kg of barley grain on a dry matter (DM) basis, and another 15 substrates were formulated by replacing 50, 100, 150, 200 or 250 g of barley grain/kg with the same amount (DM basis) of tomato or cucumber fruits or 1 : 1 tomato : cucumber mixture. In Expt 1, all substrates were incubated in batch cultures with rumen micro-organisms from goats for 24 h. Increasing amounts of tomato, cucumber and the mixture of both fruits in the substrate increased final pH and gas production, without changes in final ammonia-nitrogen (NH3-N) concentrations, substrate degradability and total volatile fatty acid (VFA) production, indicating that there were no detrimental effects of any waste fruits on rumen fermentation. Therefore, in Expt 2 the substrates including 250 g of waste fruits (T250, C250 and M250 for tomato, cucumber and the mixture of both fruits, respectively) and the CON substrate were incubated in single-flow continuous-culture fermenters for 8 days. Total VFA production did not differ among substrates, but there were differences in VFA profile. Molar proportions of propionate, isobutyrate and isovalerate were lower and acetate : propionate ratio was greater for T250 compared with CON substrate. Fermentation of substrates containing cucumber (C250 and M250) resulted in lower proportions of acetate, isobutyrate and isovalerate and acetate : propionate ratio, but greater butyrate proportions than the CON substrate. Carbohydrate degradability and microbial N synthesis tended to be lower for substrates containing cucumber than for the CON substrate, but there were no differences between CON and T250 substrates. Abundance of total bacteria, Fibrobacter succinogenes and Ruminococcus flavefaciens, fungi, methanogenic archaea and protozoa were similar in fermenters fed T250 and CON substrates, but fermenters fed C250 and M250 substrates had lower abundances of R. flavefaciens, fungi and protozoa than those fed the CON substrate. Results indicated that tomato fruits could replace dietary barley grain up to 250 g/kg of substrate DM without noticeable effects on rumen fermentation and microbial populations, but the inclusion of cucumber fruits at 250 g/kg of substrate DM negatively affected some microbial populations as it tended to reduce microbial N synthesis and changed the VFA profile. More studies are needed to identify the dietary inclusion level of cucumber which produces no detrimental effects on rumen fermentation and microbial growth.

No detectable effect of CO2 on elemental stoichiometry of Emiliania huxleyi in nutrient-limited, acclimated continuous cultures

Effects of CO2 concentration on elemental composition of the coccolithophore Emiliania huxleyi were studied in phosphorus-limited, continuous cultures that were acclimated to experimental conditions for 30 d prior to the first sampling. We determined phytoplankton and bacterial cell numbers, nutrients, particulate components like organic carbon (POC), inorganic carbon (PIC), nitrogen (PN), organic phosphorus (POP), transparent exopolymer particles (TEP), as well as dissolved organic carbon (DOC) and nitrogen (DON), in addition to carbonate system parameters at CO2 levels of 180, 380 and 750 µatm. No significant difference between treatments was observed for any of the measured variables during repeated sampling over a 14 d period. We considered several factors that might lead to these results, i.e. light, nutrients, carbon overconsumption and transient versus steady-state growth. We suggest that the absence of a clear CO2 effect during this study does not necessarily imply the absence of an effect in nature. Instead, the sensitivity of the cell towards environmental stressors such as CO2 may vary depending on whether growth conditions are transient or sufficiently stable to allow for optimal allocation of energy and resources. We tested this idea on previously published data sets where PIC and POC divided by the corresponding cell abundance of E. huxleyi at various pCO2 levels and growth rates were available.

Bayesian Dependence Tests for Continuous, Binary and Mixed Continuous-Binary Variables

Tests for dependence of continuous, discrete and mixed continuous-discrete variables are ubiquitous in science. The goal of this paper is to derive Bayesian alternatives to frequentist null hypothesis significance tests for dependence. In particular, we will present three Bayesian tests for dependence of binary, continuous and mixed variables. These tests are nonparametric and based on the Dirichlet Process, which allows us to use the same prior model for all of them. Therefore, the tests are “consistent” among each other, in the sense that the probabilities that variables are dependent computed with these tests are commensurable across the different types of variables being tested. By means of simulations with artificial data, we show the effectiveness of the new tests.

Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH

The cause of seasonal failure of a nitrifying municipal landfill leachate treatment plant utilizing a fixed biofilm was investigated by wastewater analyses and batch respirometric tests at every treatment stage. Nitrification of the leachate treatment plant was severely affected by the seasonal temperature variation. High free ammonia (NH3-N) inhibited not only nitrite oxidizing bacteria (NOB) but also ammonia oxidizing bacteria (AOB). In addition, high pH also increased free ammonia concentration to inhibit nitrifying activity especially when the NH4-N level was high. The effects of temperature and free ammonia of landfill leachate on nitrification and nitrite accumulation were investigated with a semi-pilot scale biofilm airlift reactor. Nitrification rate of landfill leachate increased with temperature when free ammonia in the reactor was below the inhibition level for nitrifiers. Leachate was completely nitrified up to a load of 1.5 kg NH4-N m(-3) d(-1) at 28 degrees C. The activity of NOB was inhibited by NH3-N resulting in accumulation of nitrite. NOB activity decreased more than 50% at 0.7 mg NH3-N L-1. Fluorescence in situ hybridization (FISH) was carried out to analyze the population of AOB and NOB in the nitrite accumulating nitrifying biofilm. NOB were located close to AOB by forming small clusters. A significant fraction of AOB identified by probe Nso1225 specifically also hybridized with the Nitrosonlonas specific probe Nsm156. The main NOB were Nitrobacter and Nitrospira which were present in almost equal amounts in the biofilm as identified by simultaneous hybridization with Nitrobacter specific probe Nit3 and Nitrospira specific probe Ntspa662. (c) 2005 Elsevier Ltd. All rights reserved.

Polyhydroxyalkanoates production by photosynthetic mixed cultures

Polyhydroxyalkanoates (PHAs) are natural biologically synthesized polymers that have been the subject of much interest in the last decades due to their biodegradability. Thus far, its microbial production is associated with high operational costs, which increases PHA prices and limits its marketability. To address this situation, this thesis’ work proposes the utilization of photosynthetic mixed cultures (PMC) as a new PHA production system that may lead to a reduction in operational costs. In fact, the operational strategies developed in this work led to the selection of PHA accumulating PMCs that, unlike the traditional mixed microbial cultures, do not require aeration, thus permitting savings in this significant operational cost. In particular, the first PHA accumulating PMC tested in this work was selected under non-aerated illuminated conditions in a feast and famine regime, being obtained a consortium of bacteria and algae, where photosynthetic bacteria accumulated PHA during the feast phase and consumed it for growth during the famine phase, using the oxygen produced by algae. In this symbiotic system, a maximum PHA content of 20% cell dry weight (cdw) was reached, proving for the first time, the capacity of a PMC to accumulate PHA. During adaptation to dark/light alternating conditions, the culture decreased its algae content but maintained its viability, achieving a PHA content of 30% cdw. Also, the PMC was found to be able to utilize different volatile fatty acids for PHA production, accumulating up to 20% cdw of a PHA co-polymer composed of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (HV) monomers. Finally, a new selective approach for the enrichment of PMCs in PHA accumulating bacteria was tested. Instead of imposing a feast and famine regime, a permanent feast regime was used, thus selecting a PMC that was capable of simultaneously growing and accumulating PHA, being attained a maximum PHA content of 60% cdw, the highest value reported for a PMC thus far. The results presented in this thesis prospect the utilization of cheap, VFA-rich fermented wastes as substrates for PHA production, which combined with this new photosynthetic technology opens up the possibility for direct sunlight illumination, leading to a more cost-effective and environmentally sustainable PHA production process.

Biovalorização de soro de queijo por processos anaeróbios/aeróbios

Este trabalho abordou a valorização de um subproduto da indústria de lacticínios (soro de queijo) através da alteração do funcionamento de processos habitualmente utilizados no contexto do tratamento biológico. Foi avaliada a fermentação acidogénica deste subproduto para maximizar a conversão do seu elevado teor de matéria orgânica em ácidos orgânicos voláteis (AOV) que actualmente são produtos com elevada procura, nomeadamente para produção de polihidroxialcanoatos (PHA). Em ensaios descontínuos e semi-contínuos foi caracterizada a produção e composição de AOV a partir de soro de queijo variando a razão food-to-microorganism (F/M) e a concentração de alcalinidade. Recorrendo à modelação dos resultados através de superfícies de resposta, demonstrou-se que condições de F/M = 4 gCQO g-1SSV combinadas com uma adição elevada de alcalinidade (8 g L-1 expresso como CaCO3) resultaram na conversão de 72% da CQO alimentada em AOV. O acetato e o butirato foram os AOV predominantes (60%), mas elevadas razões F/M combinadas com elevadas alcalinidades promoveram o alongamento da cadeia carboxílica, tendo sido produzidos AOV de maior massa molecular (iso-valerato e n-caproato). O processo de fermentação acidogénica foi posteriormente desenvolvido em modo contínuo num reactor MBBR acidogénico operado a longo prazo. Cargas orgânicas entre 30 e 50 gCQO L-1d-1 permitiram obter um grau de acidificação máximo de 68% no efluente fermentado. Foi ainda demonstrado que uma adição dinâmica de alcalinidade (0 – 4,8 g CaCO3 L-1) nestas condições estimulou a produção de AOV de cadeia ímpar (propionato e n-valerato) até 42%. O efluente acidificado no processo anaeróbio foi usado como substrato em reactores SBR operados para selecção de culturas microbianas mistas acumuladoras de PHA, nos quais foi aplicado um regime de alimentação dinâmica em condições aeróbias (“fartura-fome”). Estes sistemas operaram também a longo prazo, e demonstraram ser capazes de remover mais de 96% da CQO alimentada e simultaneamente convertê-la em PHA, até 36% do peso celular seco. A velocidade de remoção de substrato (valor máximo de 1,33 gCQO g-1SSV h-1) foi proporcional ao teor de polímero acumulado, evidenciando o estabelecimento de uma fase de “fome” prolongada que estimulou a selecção de microrganismos com elevada capacidade de acumulação de PHA. Além disso, o teor molar de hidroxivalerato (HV) no copolímero produzido [P(HB-co-HV)] foi directamente proporcional ao teor de AOV de cadeia ímpar (propionato e n-valerato) presente no soro fermentado que serviu de substrato. Uma estratégia de operação do reactor SBR com variação da carga orgânica, aliada ao regime “fartura-fome” estabelecido, permitiu ainda simular a realidade dos processos de tratamento biológico de efluentes, nos quais a composição e concentração inicial de matéria orgânica variam frequentemente. Este modo de operação do sistema estimulou notavelmente o processo de selecção de culturas acumuladoras de PHA tendo resultado num aumento da acumulação de PHA de 7% para 36%. Os resultados demonstraram com sucesso a possibilidade de valorização do soro de queijo através de eco-biotecnologia, contribuindo para uma mudança de paradigma no tratamento convencional de efluentes: ao invés de serem eliminados enquanto poluentes, os componentes orgânicos presentes neste subproduto industrial podem assim ser convertidos em materiais de valor acrescentado.

Constraint-based modelling of mixed microbial populations: Application to polyhydroxyalkanoates production

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

Mathematical modeling of a continuous alcoholic fermentation process in a two-stage tower reactor cascade with flocculating yeast recycle

Experiments of continuous alcoholic fermentation of sugarcane juice with flocculating yeast recycle were conducted in a system of two 0.22-L tower bioreactors in series, operated at a range of dilution rates (D (1) = D (2) = 0.27-0.95 h(-1)), constant recycle ratio (alpha = F (R) /F = 4.0) and a sugar concentration in the feed stream (S (0)) around 150 g/L. The data obtained in these experimental conditions were used to adjust the parameters of a mathematical model previously developed for the single-stage process. This model considers each of the tower bioreactors as a perfectly mixed continuous reactor and the kinetics of cell growth and product formation takes into account the limitation by substrate and the inhibition by ethanol and biomass, as well as the substrate consumption for cellular maintenance. The model predictions agreed satisfactorily with the measurements taken in both stages of the cascade. The major differences with respect to the kinetic parameters previously estimated for a single-stage system were observed for the maximum specific growth rate, for the inhibition constants of cell growth and for the specific rate of substrate consumption for cell maintenance. Mathematical models were validated and used to simulate alternative operating conditions as well as to analyze the performance of the two-stage process against that of the single-stage process.

Multiple imputation for missing continuous and dichotomous data: The effects of patterns of missingness and variable correlations on type I error with small samples

The purpose of this study is to investigate the effects of predictor variable correlations and patterns of missingness with dichotomous and/or continuous data in small samples when missing data is multiply imputed. Missing data of predictor variables is multiply imputed under three different multivariate models: the multivariate normal model for continuous data, the multinomial model for dichotomous data and the general location model for mixed dichotomous and continuous data. Subsequent to the multiple imputation process, Type I error rates of the regression coefficients obtained with logistic regression analysis are estimated under various conditions of correlation structure, sample size, type of data and patterns of missing data. The distributional properties of average mean, variance and correlations among the predictor variables are assessed after the multiple imputation process. ^ For continuous predictor data under the multivariate normal model, Type I error rates are generally within the nominal values with samples of size n = 100. Smaller samples of size n = 50 resulted in more conservative estimates (i.e., lower than the nominal value). Correlation and variance estimates of the original data are retained after multiple imputation with less than 50% missing continuous predictor data. For dichotomous predictor data under the multinomial model, Type I error rates are generally conservative, which in part is due to the sparseness of the data. The correlation structure for the predictor variables is not well retained on multiply-imputed data from small samples with more than 50% missing data with this model. For mixed continuous and dichotomous predictor data, the results are similar to those found under the multivariate normal model for continuous data and under the multinomial model for dichotomous data. With all data types, a fully-observed variable included with variables subject to missingness in the multiple imputation process and subsequent statistical analysis provided liberal (larger than nominal values) Type I error rates under a specific pattern of missing data. It is suggested that future studies focus on the effects of multiple imputation in multivariate settings with more realistic data characteristics and a variety of multivariate analyses, assessing both Type I error and power. ^

Combined effects of ocean acidification and light or nitrogen availabilities on 13C fractionation in marine dinoflagellates

Along with increasing oceanic CO2 concentrations, enhanced stratification constrains phytoplankton to shallower upper mixed layers with altered light regimes and nutrient concentrations. Here, we investigate the effects of elevated pCO2 in combination with light or nitrogen-limitation on 13C fractionation (epsilon p) in four dinoflagellate species. We cultured Gonyaulax spinifera and Protoceratium reticulatum in dilute batches under low-light (LL) and high-light (HL) conditions, and grew Alexandrium fundyense and Scrippsiella trochoidea in nitrogen-limited continuous cultures (LN) and nitrogen-replete batches (HN). The observed CO2-dependency of epsilon p remained unaffected by the availability of light for both G. spinifera and P. reticulatum, though at HL epsilon p was consistently lower by about 2.7 per mil over the tested CO2 range for P. reticulatum. This may reflect increased uptake of (13C-enriched) bicarbonate fueled by increased ATP production under HL conditions. The observed CO2-dependency of epsilon p disappeared under LN conditions in both A. fundyense and S. trochoidea. The generally higher epsilon p under LN may be associated with lower organic carbon production rates and/or higher ATP:NADPH ratios. CO2-dependent epsilon p under non-limiting conditions has been observed in several dinoflagellate species, showing potential for a new CO2-proxy. Our results however demonstrate that light- and nitrogen-limitation also affect epsilon p, thereby illustrating the need to carefully consider prevailing environmental conditions.

Developments in phosphine resistance in China and possible implications for Australia

Resistance to phosphine was characterised in strains of rice weevil, Sirophilus oryzae, and the psocids Liposcelis entomophila and L. decolor from China and Australia. Mixed-age cultures (containing all life stages) of insects were tested using a flow-through apparatus. The criterion of response was 'time to population extinction' defined as the exposure period, in days, at which 100% mortality of adults and no live progeny were achieved. Chinese S. oryzae took 11 and 7 days for population extinction at 200 and 700 ppm phosphine, respectively, compared with the Australian strain, which was controlled in 7 and 5 days, respectively. Similarly, the Chinese strains L. Enfornophila and L. decolor were generally more difficult to control than the corresponding Australian strains. The Chinese strains of L. decolor showed resistance levels stronger than any grain storage insect pest species so far detected in Australia. This research allows us to evaluate the likely significance of potential new resistance to the Australian grain industry and to prepare effective fumigation dosages and resistance management strategies to combat new strong resistances before they emerge here.