Mixed Culture Process Research Articles

Syntrophy between fermentative and purple phototrophic bacteria to treat and valorize carbohydrate-rich wastewaters

Fermentative chemoorganoheterotrophic bacteria (FCB) and purple photoorganoheterotrophic bacteria (PPB) are two interesting microbial guilds to process carbohydrate-rich wastewaters. Their metabolic interactions have been studied in pure cultures or co-cultures, but little is known about mixed cultures. We studied the effect of reactor regimes (batch/chemostat) and illumination modes (continuous infrared light, dark, or light/dark cycles) on glucose conversions and process ecology of the interactions between FCB and PPB in mixed cultures. In batch, FCB (>80 % of sequencing read counts) outcompeted PPB, under any light conditions. In chemostat under continuous and alternating irradiance, three FCB populations were enriched (>70 %), while Rhodobacteraceae (PPB) made 30 % of the community. Glucose fermentation products were linked to the dominant FCB. Continuous culturing helped maintaining FCB and PPB in syntrophy: PPB grew on glucose metabolites produced by FCB. Engineering the association between FCB and PPB in mixed-culture processes can help to treat and valorize carbohydrate-rich aqueous waste.

Photo-fermentative hydrogen production from cheese whey: Engineering of a mixed culture process in a semi-continuous, tubular photo-bioreactor

This work approaches the engineering of photo fermentative hydrogen production from cheese whey, for the scale-up of photo-bioreactors. Firstly, a brief review has been performed to identify the optimal reactor configuration as well as the main literature lacks. Results of the review analysis suggest that tubular photobioreactors are effective for the photo fermentation process. On the other hand, one of the main drawbacks to the scale-up of the proposed systems is the wide use of pure cultures, which requires aseptic environments and carefully controlled conditions. Based on the critical evaluation of the state of the art, a semi-continuous tubular photobioreactor, performing mixed culture hydrogen production from cheese whey, under unsterile conditions, was started up. Due to the lack of studies in this field, the case study was developed to provide a methodology for the reactor start-up, which represents the most critical phase of its operation. Preliminary tests were performed to choose the optimal operating conditions. To assess the effectiveness of cheese whey, results were compared to those obtained from a glucose based synthetic substrate, under steady state conditions. Experimental outcomes showed that the optimal Hydraulic Retention Time was 2 days and the most appropriate recirculation/pumping system consisted of an intermittent peristaltic pump. The semi-continuous feeding of cheese whey resulted in 87 mL/L d of hydrogen, which was similar to the productivity obtained under glucose feeding. The characterization of the residual organic content further indicated the possible accumulation of polyhydroxyalcanohates, which was estimated to constitute about the 40% of the total residual COD. These results further addressed the identification of key points to be better investigated to promote the effective photo fermentation scale-up for cheese whey valorization, such as the enhancement of the illumination conditions as well as the search for strategies to improve the uniform exposure of microbial cells to light.

Utilizing grass for the biological production of polyhydroxyalkanoates (PHAs) via green biorefining: Material and energy flows

AbstractThe meat and dairy industry across Europe is dependent on the production of grass. However, faced with competing pressures to reduce the environmental impact of agriculture, a potential future reduction of meat and dairy consumption in western diets, and pressure to minimize food production costs, grass could be used to produce alternative products. The biological production of polyhydroxyalkanoates (PHA) by using grass as the primary carbon source in a novel mixed culture process has been studied. A total of 30,000 t of fresh grass would yield approximately 403.65 t of dried biopolymer granules. On the basis of this early stage, non‐optimized process, the cumulative energy demand (CED) of PHA produced from waste grass and cultivated grass was found to be 248.4 MJ/kg and 271.8 MJ/kg, respectively, which is the same order of magnitude as fossil‐carbon‐based polymers. Improvements in volatile fatty acid yields, reduction in chemical and water inputs, and using residues to make other products will reduce the CED. Given the future requirement to produce polymers with little or no fossil‐carbon feedstock, an optimized version of the process could provide a viable future production option that also contributes to the long‐term sustainability of agricultural communities.

Kinetic Modeling of Mixed Culture Process of Anaerobic Co-digestion of Vegetable Wastes with Pistia stratiotes: A Scientific Attempt on Biomethanationy

As co-digestion of two wastes together lead to balanced environment for biomethanation an attempt has been made in the present study to co-digest vegetable waste (VW) and Pistia stratiotes (PS) to overcome the acidification problem during mono-digestion of VW. Optimization of important process parameters for anaerobic co-digestion was done based on Central Composite Design (CCD) based Response Surface Methodology (RSM). Thereafter, modeling of the anaerobic digestion was carried out using first order, modified Gompertz and unstructured segregated model to study the process behavior. First order and modified Gompertz models were found to fit with the observed methane profile with R2 of ~0.99. In case of unstructured segregated model important kinetic constants such as Khs, KSS, KVFA were derived from the model and the values were found to be 0.1 d-1, 2.3 g/L and 881.68 mg/L, respectively. Based on these kinetic parameters, simulated cumulative methane production was generated which matched well with the observed methane yield within the error band of ± 20%. This showed that the developed model could represent the overall process behaviour of this novel anaerobic co-digestion using mixed anaerobic culture as an inoculum.

Reactor kinetics evaluation and performance investigation of a long-term operated UASB-anammox mixed culture process

To optimize the operation and know the relationships between operation and potential capability of UASB-anammox mixed culture process, reactor kinetic models were conducted and the potential nitrogen conversion rates were estimated. Total nitrogen (TN), NH4+-N and NO2−-N removal efficiency were simulated and predicted by modified Stovere-Kincannon model, Monod model, First-order model and Grau second-order substrate removal models. The nitrogen gas production rate was also evaluated by Van der meer and heertjes model. During the process, the nitrogen loading rate (NLR) was increased from 0.93 to 7.34 g L−1 d−1 following the substrate increased from 280 to 462 mg N L−1 and hydraulic residence times (HRT) decreased from 24 h to 1.5 h, the highest achieved removal efficiency of NH4+–N, NO2−–N and TN were 99, 95 and 90%, respectively. Simulation results proved that the Modified Stover-Kincannon model and the Grau second-order model were the most appropriate models with prospected maximum TN removal rate Umax and saturation value constant KB were 3.33 and 4.03 g N L−1 d−1, respectively. Validation of the linearity between the experimental data and predicted values of kinetics illustrated the two models appropriately described the kinetic behavior of UASB-anammox mixed culture. The reactor kinetic evaluation proved valuable information for guiding or optimizing the operation condition.

Integrated omics for the identification of key functionalities in biological wastewater treatment microbial communities.

Biological wastewater treatment plants harbour diverse and complex microbial communities which prominently serve as models for microbial ecology and mixed culture biotechnological processes. Integrated omic analyses (combined metagenomics, metatranscriptomics, metaproteomics and metabolomics) are currently gaining momentum towards providing enhanced understanding of community structure, function and dynamics in situ as well as offering the potential to discover novel biological functionalities within the framework of Eco-Systems Biology. The integration of information from genome to metabolome allows the establishment of associations between genetic potential and final phenotype, a feature not realizable by only considering single ‘omes’. Therefore, in our opinion, integrated omics will become the future standard for large-scale characterization of microbial consortia including those underpinning biological wastewater treatment processes. Systematically obtained time and space-resolved omic datasets will allow deconvolution of structure–function relationships by identifying key members and functions. Such knowledge will form the foundation for discovering novel genes on a much larger scale compared with previous efforts. In general, these insights will allow us to optimize microbial biotechnological processes either through better control of mixed culture processes or by use of more efficient enzymes in bioengineering applications.

Evolution of Volatile Flavour Compounds during Fermentation of African Oil Bean (Pentaclethra macrophylla Benth) Seeds for "Ugba" Production.

Fermented African oil bean (Pentaclethra macrophylla Benth) seed is a successful and well studied seasoning and snack in parts of Western Africa. GC-MS analysis of fermenting seeds revealed a mixture of several volatile aroma compounds which changed with time and starter organism. During natural mixed culture process 36 volatile compounds including 12 hydrocarbons, 10 esters, 5 alcohols, 2 phenols, 2 ketones, and one each of furan, amine, acid, thiophene, and lactone were identified. When Bacillus subtilis was used in pure culture, 30 compounds comprising 10 hydrocarbons, 8 esters, 3 alcohols, 2 amines, 2 sulfur compounds, and one of each of acid, aldehyde, phenol, ketone, and furan were identified. Sample fermented with B. megaterium produced 29 aroma compounds comprising 9 hydrocarbons, 10 esters, 2 nitrogenous compounds, 2 ketones, 3 alcohols, and one of each of lactone, aldehyde, furan, and amine. Methyl esters of various long chain fatty acids may be key aroma compounds, based on consistency and persistence. Qualitative or quantitative contribution of individual compounds may only be determined following flavour threshold analysis.

Operation of fill-and-draw reactors fed with bisphenol A as sole carbon and energy source.

While there is ample evidence in the literature that many organic xenobiotics can be biodegraded as sole carbon and energy source by pure batch cultures of selected microorganisms, the same evidence is very limited for continuous or semi-continuous mixed-culture processes simulating biological wastewater treatment plants. This study investigates for the first time whether Bisphenol A (BPA) can be removed and used as sole carbon and energy source by mixed microbial cultures in a semi-continuous process. Four fill and draw bioreactors were inoculated with unacclimated soil and operated at various hydraulic retention times (HRT), in the range of 1.7-4.2 d, using a feed composed solely of BPA (115 mg/l), and mineral salts. At steady-state, the BPA removal in the four reactors varied in the range 7.5-19% and did not show a significant trend with the HRT. The maximum growth rate on BPA was measured in the range 0.29-1.54 d-1.

Enrichment of a Mixed Bacterial Culture with a High Polyhydroxyalkanoate Storage Capacity

Polyhydroxyalkanoates (PHAs) are microbial storage polymers that attract interest as bioplastics. PHAs can be produced with open mixed cultures if a suitable enrichment step based on the ecological role of PHA is used. An acetate-fed sequencing batch reactor operated with 1 day biomass residence time and with feast-famine cycles of 12 h was used to enrich a mixed culture of PHA producers. In subsequent fed-batch experiments under growth limiting conditions, the enriched mixed culture produced PHA up to a cellular content of 89 wt % within 7.6 h (average rate of 1.2 g/g/h). The PHA produced from acetate was the homopolymer polyhydroxybutyrate. The culture was dominated by a Gammaproteobacterium that showed little similarity on 16S rRNA level with known bacteria (<90% sequence similarity). The mixed culture process for PHA production does not require aseptic conditions. Waste streams rather than pure substrates could be used as raw materials.

Influence of the pH on (open) mixed culture fermentation of glucose: A chemostat study

Catabolic products from anaerobic fermentation processes are potentially of industrial interest. The volatile fatty acids and alcohols produced can be used as building blocks in chemical processes or applied directly as substrates in a mixed culture process to produce bioplastics. Development of such applications requires a predictable and controllable product spectrum of the fermentation process. The aim of the research described in this paper was (i) to investigate the product spectrum of an open mixed culture fermentation (MCF) process as a function of the pH, using glucose as substrate, and (ii) to relate the product spectrum obtained to generalized biochemical and thermodynamic considerations. A chemostat was operated under carbon and energy limitation in order to investigate the pH effect on the product spectrum in a MCF process. A transition from CO(2)/H(2) production at lower pH values to formate production at higher pH values was observed. The ratio of CO(2)/H(2) versus formate production was found to be related to the thermodynamics of formate dehydrogenation to CO(2)/H(2). This transition was associated with a shift in the catabolic products, from butyrate and acetate to ethanol and acetate, likely due to a decrease in the oxidation state of the electron carriers in the cell. The product spectrum of the MCF process as a function of the pH could largely be explained using general biochemical considerations.

Production of cellulolytic enzymes from theXylaria andHypoxylon species of xylariaceae

Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes,Trichoderma reesei QM 9414 was also examined for comparison. Strains ofXylaria anisopleura andX. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation.Hypoxylon stygium produced the highest activity of β-glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process usingT. reesei QM 9414 andH. stygium was developed to obtain enhanced synthesis of cellulase. β-Glucosidase activities in the mixed culture increased within 48h whenH. stygium was introduced after 24h.

Bipopulation Approach to CMAS-Process Simulation

A dynamic model based on species-shift theory in activated sludge is proposed for predicting the response of mixed-culture processes to dynamic organic loadings. Experimental data were collected to verify the model. The closeness between the experimental results and the model prediction indicates strongly that this model has potential in future studies of mixed-microbial cultures to dynamic loadings and in the design of wastewater-treatment processes uning completely mixed-activated sludge process. The minimal laboratory experiment required to implement this model further increases its usefulness.