Bioremediation of contaminated soil and groundwater by in situ biostimulation.

Bioremediation by in situ biostimulation is an attractive alternative to excavation of contaminated soil. Many in situ remediation methods have been tested with some success; however, due to highly variable results in realistic field conditions, they have not been implemented as widely as they might deserve. To ensure success, methods should be validated under site-analogous conditions before full scale use, which requires expertise and local knowledge by the implementers. The focus here is on indigenous microbial degraders and evaluation of their performance. Identifying and removing biodegradation bottlenecks for degradation of organic pollutants is essential. Limiting factors commonly include: lack of oxygen or alternative electron acceptors, low temperature, and lack of essential nutrients. Additional factors: the bioavailability of the contaminating compound, pH, distribution of the contaminant, and soil structure and moisture, and in some cases, lack of degradation potential which may be amended with bioaugmentation. Methods to remove these bottlenecks are discussed. Implementers should also be prepared to combine methods or use them in sequence. Chemical/physical means may be used to enhance biostimulation. The review also suggests tools for assessing sustainability, life cycle assessment, and risk assessment. To help entrepreneurs, decision makers, and methods developers in the future, we suggest founding a database for otherwise seldom reported unsuccessful interventions, as well as the potential for artificial intelligence (AI) to assist in site evaluation and decision-making.

Exposure to bacterial and fungal bioaerosols in facilities processing biodegradable waste.

The aim of the study was to determine the exposure of workers within biodegradable waste processing facilities to bacteria and fungi to identify any exposures of potential concern to health. Occupational measurements were performed in six composting and three bioenergy (bioethanol or methane/biogas) producing facilities. Bioaerosols were measured from breathing zones with Button aerosol or open face cassette filter samplers, and swab specimens were taken from the nasal mucous membranes of the workers. Aspergillus fumigatus, Bacillus cereus group, Campylobacter spp., Salmonella spp., Streptomyces spp., and Yersinia spp. were determined by real-time polymerase chain reaction (qPCR). A. fumigatus, and mesophilic and thermophilic actinobacteria were also cultivated from filters. Bacterial airborne endotoxins collected by IOM samplers were analyzed using a Limulus assay. Bioaerosol levels were high, especially in composting compared to bioenergy producing facilities. Endotoxin concentrations in composting often exceeded the occupational exposure value of 90 EU/m3, which may be harmful to the health. In addition to endotoxins, the concentrations of A. fumigatus (up to 2.4 × 105 copies/m3) and actinobacteria/Streptomyces spp. (up to 1.6 × 106 copies/m3) in the air of composting facilities were often high. Microbial and endotoxin concentrations were typically highest in waste reception and pre-treatment, equal or decreased during processing and handling of treated waste, and lowest in wheel loader cabins and control rooms/outdoors. Still, the parameters measured in wheel loader cabins were often higher than in the control sites, which suggests that the use of preventive measures could be improved. B. cereus group, Salmonella spp., and Yersinia spp. were rarely detected in bioaerosols or nasal swabs. Although Campylobacter spp. DNA was rarely detected in air, as a new finding, Campylobacter ureolyticus DNA was frequently detected in the nasal mucous membranes of workers, based on partial 16S rDNA sequencing. Moreover, especially A. fumigatus and C. ureolyticus spp. DNA concentrations in swabs after the work shift were significantly higher than before the shift, which indicates their inhalation or growth during the work shift. Microbial qPCR analysis of bioaerosols and swab samples of nasal mucosa allowed measuring exposure in various work operations and during the work shift, identifying problems for health risk assessment to improve working conditions, and evaluating the effectiveness of preventive measures and personal protection of workers.

Evaluation of the factors limiting biogas production in full-scale processes and increasing the biogas production efficiency.

Biogas production from sewage sludge volatile solids (VS) by anaerobic digestion slows down towards the end of the process, among inhibitory factors being pH increase upon ammonia accumulation, poorly digestible biomaterials, and high fixed solid (FS) content. The possibility of concentrating the digested sludge VS (41.7-56.6% on a dry weight basis) by surface and bottom layer separation with biogas post-production was studied. Furthermore, the potential to recycle concentrated VS and digested sludge back to the process after adjusting pH 7.0 to optimal for biogas-producing microbes and after acid, alkali, thermal, and sonolytic treatments was examined. In general, pH 7.0 control alone improved biogas production from the recycled digested sludge the most. An equally good improvement in biogas production was achieved by recycling the digested sludge, which had been heated until ammonia had evaporated and the pH dropped to 7.0 (1-2 h, 75 °C), and at the same time, VS was degraded. The biogas production from the sonicated and recycled sludge was almost as good as from the pH-adjusted, or heat-treated recycled sludge. After the acid and base treatments of the digested sludge, the recycled sludge yielded often the lowest biogas volume, as the added chemicals increased the FS concentration, which proved to be a more important inhibitory factor than poorly degradable VS. The high FS content significantly reduced the benefits of the treatments. By separating the surface and bottom layers with biogas post-production, the surface layer of VS was concentrated to 51.6-61.8%, while different compositions of the layers affected the biogas production.

Influence of organic matter, nutrients, and cyclodextrin on microbial and chemical herbicide and degradate dissipation in subsurface sediment slurries.

Pesticides leaching from soil to surface and groundwater are a global threat for drinking water safety, as no cleaning methods occur for groundwater environment. We examined whether peat, compost-peat-sand (CPS) mixture, NH4NO3, NH4NO3 with sodium citrate (Na-citrate), and the surfactant methyl-ß-cyclodextrin additions enhance atrazine, simazine, hexazinone, dichlobenil, and the degradate 2,6-dichlorobenzamide (BAM) dissipations in sediment slurries under aerobic and anaerobic conditions, with sterilized controls. The vadose zone sediment cores were drilled from a depth of 11.3-14.6m in an herbicide-contaminated groundwater area. The peat and CPS enhanced chemical atrazine and simazine dissipation, and the peat enhanced chemical hexazinone dissipation, all oxygen-independently. Dichlobenil dissipated under all conditions, while BAM dissipation was fairly slow and half-lives could not be calculated. The chemical dissipation rates could be associated with the chemical structures and properties of the herbicides, and additive compositions, not with pH. Microbial atrazine degradation was only observed in the Pseudomonas sp. ADP amended slurries, although the sediment slurries were known to contain atrazine-degrading microorganisms. The bioavailability of atrazine in the water phase seemed to be limited, which could be due to complex formation with organic and inorganic colloids. Atrazine degradation by indigenous microbes could not be stimulated by the surfactant methyl-ß-cyclodextrin, or by the additives NH4NO3 and NH4NO3 with Na-citrate, although the nitrogen additives increased microbial growth.

A zero-valent iron and organic matter mixture enhances herbicide and herbicide degradation product removal in subsurface waters.

The pesticide atrazine, its degradation products, and 2,6-dichlorobenzamide (BAM) are persistent in groundwater environment. We studied whether their dissipation can be enhanced with a mixture of a complex carbon source and zero-valent iron (ZVI) called EHC®. The application rates were 1.0% and 2.0% (by weight) in subsurface sediments slurries (atrazine 30mg/L), and 2.0% in 1.5m pilot-scale sediment columns with groundwater flowing through (atrazine 0.08, desethylatrazine DEA 0.03, BAM 0.02µg/L). In the slurries under aerobic conditions, atrazine of 0.88±0.14mg/g of EHC® was dissipated chemically, as concentrations did not differ significantly between the slurries and their sterilized controls. No degradation occurred in the slurries under anaerobic conditions. In the pilot-scale columns under water-saturated conditions, atrazine, DEA and BAM were not detected in effluents during 33, 64 and 64days from the beginning of the water flow through EHC® columns, respectively, but thereafter traces of compounds could be detected. No atrazine or degradation products (BAM, DEA, deisopropylatrazine, desethyldeisopropylatrazine) could be extracted from the column sediments at the end of the experiment. As a result, the sum of dissipated pesticides was about 7.6µg/g of EHC® in columns under water-saturated conditions, and 0.88mg/g of EHC® in slurries under aerobic conditions. EHC® can be used to enhance the dissipation of studied pesticides in small quantities, preferentially under aerobic conditions.

Depth, soil type, water table, and site effects on microbial community composition in sediments of pesticide-contaminated aquifer.

Microbial community compositions in pesticide-contaminated aquifers have not been studied, although such information is important for remediation and maintaining freshwater sources clean under changing climate. Therefore, phospholipid (PLFAs), glycolipid (GLFAs), and neutral lipid (NLFAs) fatty acids were determined from sand and clay sediments at depths of 0.3-24.8 m, all contaminated with triazines and dichlobenil/2,6-dichlorobenzamide. The portion of fungi and Gram-negative bacteria at 0.3 m was greater than at 0.8 m, where the percentage of Gram-positive bacteria, actinobacteria, and sulfate-reducing bacteria (SRB) increased. In deeper sediments, microbial biomass, activity, and diversity decreased. Clay sediments seemed to serve as a reservoir for slow pesticide elution to groundwater, and their biomarker portion for all bacteria except actinobacteria was greater than in sand sediments. The slow pesticide dissipation seemed to occur in the main groundwater flow zone, resulting in nitrogen release simultaneously with organic matter elution from gardening and bank filtration. As a result, microbial biomass, activity, and diversity were increased. This shift in conditions towards that in surface soil may be appropriate for enhanced natural attenuation of pesticides in groundwater sources.

Dichlobenil and 2,6-dichlorobenzamide (BAM) dissipation in topsoil and deposits from groundwater environment within the boreal region in southern Finland.

BAM (2,6-dichlorobenzamide) is a metabolite of pesticide dichlobenil and a common groundwater contaminant. Dichlobenil and BAM half-lives were determined in five Finnish subsurface deposits and in topsoil. Aerobic and anaerobic conditions with sterilized controls were included in this 1.4-year incubation experiment. In subsurface deposits, dichlobenil half-life varied from 157 days to no degradation and that of BAM from 314 days to no degradation. Microbes and oxygen enhanced dichlobenil and BAM dissipation rates in some deposits. However, dichlobenil and BAM concentrations were most significantly affected by deposit characteristics, especially carbon and nitrogen amounts. Also low pH, cadmium, iron, zinc, manganese and lead correlated with low dichlobenil and/or BAM concentrations. In mineral topsoil, dissipation was faster with half-lives of 41-54 days for dichlobenil, and 182-261 days for BAM. Dichlobenil was depleted completely in surface soil, but BAM was not dissipated below 55-81 % of the initial concentration. Generally, dichlobenil and BAM dissipation in samples from the northern boreal region was similar to that reported for the temperate region. BAM was persistent in topsoil and subsurface deposits, indicating long-term persistence problems in groundwater also within the northern boreal region.

Evolution of clostridia and streptomycetes in full-scale composting facilities and pilot drums equipped with on-line temperature monitoring and aeration.

The evolution of sporulating bacteria in full-scale composting facilities with online temperature monitoring has been poorly studied, although organic matter recycling increases. We analysed Clostridium perfringens and sulphite-reducing clostridia (SRC) by cultivation, and streptomycetes by real-time PCR in five full-scale, temperature-monitored and aerated composting processes, and two pilot-scale drum composters. Facilities composted woodchips, sawdust, peat, or bark amended sludge or source-separated biowaste. Streptomycetes genes of 0.21-110×10(7)copies/g feed increased fast to 0.019-33×10(9)copies/g, and then were equal or decreased. SRC of 0.06-2.2×10(7)cfu/g feed decreased to 0-600 cfu/g, with re-growth in two facilities. End products were clean of C. perfringens, detected in sludge composts. Although processes contained large quantities of spore-forming bacteria, in the best facilities end products had the high quality. Temperature (>55°C,>2d) was not related to the end compost quality, but relations between waste and bulking agent qualities, aeration, and processing time should be better controlled.

Wood ash for application in municipal biowaste composting.

This study aimed to clarify the impacts of pH control by wood ash amendment on biowaste composting processes. To achieve this, fresh source separated municipal biowaste was mixed with low doses (2-8% wt/vol) of wood ash and processed in a pilot and large-scale composting systems. The results indicated a correlation between a low initial pH and delay in the early rise of the process temperature. Wood ash elevated the composting temperatures and pH, and stimulated the mineralisation both in the pilot scale and the industrial large-scale processes. According to the results addition of amounts of 4-8% wood ash is sufficient for efficient biowaste composting process and yields a safe end product. However, to minimise the environmental risk for heavy metal contamination, and meet the criteria for the limit values of the impurities in wood ash, strict quality control of the applied wood ashes should be implemented.

Effect of light Sphagnum peat on odour formation in the early stages of biowaste composting.

In the present study, we investigated the effects of two bulking materials, Sphagnum peat and pine wood chips, on the early stages of biowaste composting in two pilot-scale processes. Emphasis was placed on studying the formation conditions of malodorous compost gases in the initial phases of the processes. The results showed that gas emission leaving an open windrow and a closed drum composting system contained elevated concentrations of fermentative microbial metabolites when acid Sphagnum peat (pH 3.2) was used as a bulking material. Moreover, the gas emission of the peat amended drum composter contained a high concentration of odour (up to 450,000oum(-3) of air). The highest odour values in the outlet gas of peat amended composts coincided with the elevated concentrations of volatile organic compounds such as acetoin and buthanedion. We conclude that the acidifying qualities of composting substrates or bulking material may intensify odour emission from biowaste composts and prolong the early stages of the composting process.

The most-probable-number enumeration of dichlobenil and 2,6-dichlorobenzamide (BAM) degrading microbes in Finnish aquifers.

In groundwater subsurface deposits and a topsoil from five aquifers having 2,6-dichlorobenzamide (BAM) in water, we determined the most-probable-number (MPN) of 2,6-dichlorobenzonitrile (dichlobenil) and metabolite BAM degrading microorganisms. Dichlobenil and BAM were combined nitrogen sources in the MPN tubes, which were scored positive at concentrations <75% after 1 month incubation. Aerobic and anaerobic microbes degrading dichlobenil and BAM were common in samples in low numbers of 3.6-210 MPN g dw(-1). Additional degradation occurred in high MPN dilutions of some samples, the microbial numbers being 0.11-120 x 10(5) MPN g dw(-1). The strains were isolated from low and high dilutions of one deposit, and degradation in pure cultures was confirmed by HPLC. According to the 16S rDNA sequencing, strains were from genera Zoogloea, Pseudomonas, Xanthomonas, Rhodococcus, Nocardioides, Sphingomonas, and Ralstonia. Dichlobenil (45.5 +/- 18.3%) and BAM (37.6 +/- 14%) degradation was low in the MPN tubes. Despite of microbial BAM degradation activity in subsurface deposits, BAM was measured from groundwater.

Atrazine and terbutryn degradation in deposits from groundwater environment within the boreal region in Lahti, Finland.

The degradation of pesticides atrazine and terbutryn was investigated under aerobic and anaerobic conditions in the northern boreal region subsurface deposits and sterilized controls from the depths of 6.3-21.0 m below the surface and 1.2-16.9 m below the groundwater table. During 1.3-1.7 years of laboratory incubation, atrazine degradation under aerobic conditions varied from rapid (half-live 38 days) to no degradation. Anaerobically, atrazine half-lives were 430-829 days. Organic matter, nitrogen, and lead in deposits correlated positively with the atrazine concentration in groundwater. Aerobic and anaerobic terbutryn half-lives were 193-644 and 266-400 days, respectively. Microbial aerobic atrazine and terbutryn degradation was confirmed in the deep deposits near the water table. Under aerobic conditions, the high amounts of Cr, Mn, Ni, and Zn in deposits decreased the chemical degradation of terbutryn.

Selected ion monitoring in quantitative gas-liquid chromatographic-mass spectrometric detection of fatty acid methyl esters from environmental samples.

To calculate selected ion monitoring (SIM) gas-liquid chromatography (GLC)-mass spectrometry (MS) results of phospholipid fatty acids (PLFAs) from environmental samples, coefficients were calculated for each fatty acid by dividing the sum of ion intensities in SCAN with that of ions followed in SIM. The SIM chromatogram areas were multiplied with the coefficients, and then processed as in SCAN. The results were compared to those obtained using calibration curves and SCAN. The calibration curve and coefficient based results had the greatest errors of 7.8 and 6.7%, respectively, outside standard deviations of SCAN percentages. The PLFA contents calculated using calibration curves and coefficients were 104.9+/-7.3% and 101.5+/-8.6%, respectively, of SCAN values. SIM increased sensitivity approximately 10-fold from SCAN, and the smallest detectable injected amount was approximately 50 ng (0.18 nmol) for 20 fatty acids, corresponding to 4 x 10(6) cells.

Occurrence of nitrifiers and diversity of ammonia-oxidizing bacteria in developing drinking water biofilms.

We studied the population dynamics of nitrifying bacteria during the development of biofilms up to 233 or 280 days on polyvinylchloride pipes connected to two full-scale drinking water distribution networks supplying processed and chloraminated surface water. The numbers of nitrifiers in biofilms were enumerated at intervals of 10-64 days by the most probable number (MPN) method at waterworks and at several study sites in distribution network areas. The numbers of nitrifiers increased towards the distal sites. The highest detected MPN counts of ammonia-oxidizing bacteria (AOB) for study areas 1 and 7 were 500 MPN cm(-2) and 1.0 x 10(6) MPN cm(-2), and those of nitrite-oxidizing bacteria (NOB) 96 MPN cm(-2) and 2.2 x 10(3) MPN cm(-2), respectively. The diversity of AOB was determined by PCR amplifying, cloning and sequencing the partial ammonia monooxygenase (amoA) gene of selected biofilm samples presenting different biofilm ages. The PCR primers used, A189 and A682, also amplified a fragment of particulate methane monooxygenase (pmoA) gene of methane-oxidizing bacteria. The majority of biofilm clones (24 out of 30 studied) contained Nitrosomonas amoA-like sequences. There were only two pmoA-like sequences of Type I methanotrophs, and four sequences positioned in amoA/pmoA sequence groups of uncultured bacteria. From both study area very similar or even completely identical Nitrosomonas amoA-like sequences were obtained despite of high difference in AOB numbers. The results show that the conditions in newly formed biofilms in drinking water distribution systems favor the growth of Nitrosomonas-type AOB.

Microbial community structure and biomass in developing drinking water biofilms.

Traditional techniques to study microbes, such as culturable counts, microbial biomass, or microbial activity, do not give information on the microbial ecology of drinking water systems. The aim of this study was to analyze whether the microbial community structure and biomass differed in biofilms collected from two Finnish drinking water distribution systems (A and B) receiving conventionally treated (coagulation, filtration, disinfection) surface water. Phospholipid fatty acid methyl esters (PLFAs) and lipopolysaccharide 3-hydroxy fatty acid methyl esters (LPS 3-OH-FAs) were analyzed from biofilms as a function of water residence time and development time. The microbial communities were rather stabile through the distribution systems, as water residence time had minor effects on PLFA profiles. In distribution system A, the microbial community structure in biofilms, which had developed in 6 weeks, was more complex than those grown for 23 or 40 weeks. The microbial communities between the studied distribution systems differed, possibly reflecting the differences in raw water, water purification processes, and distribution systems. The viable microbial biomass, estimated on the basis of PLFAs, increased with increasing water residence time in both distribution systems. The quantitative amount of LPS 3-OH-FAs increased with increasing development time of biofilms of distribution system B. In distribution system A, LPS 3-OH-FAs were below the detection limit.

Gas-chromatographic lipid profiles in identification of currently known slowly growing environmental mycobacteria.

Cellular fatty acid analysis by GLC is widely used in the species identification of mycobacteria. Combining mycolic acid cleavage products with shorter cellular fatty acids increases the informative value of the analysis. A key has been created to aid in the identification of all currently known slowly growing environmental species. In this scheme, the species are classified into six categories, each characterized by a combination of fatty markers shared by those species. Within each category, individual species may be distinguished by the presence or absence of specific marker substances, such as methyl-branched fatty acids or secondary alcohols. This study also describes earlier unpublished GLC profiles of 14 rare, slowly growing, environmental mycobacteria, Mycobacterium asiaticum, Mycobacterium botniense, Mycobacterium branderi, Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium doricum, Mycobacterium heckeshornense, Mycobacterium heidelbergense, Mycobacterium hiberniae, Mycobacterium kubicae, Mycobacterium lentiflavum, Mycobacterium scrofulaceum, Mycobacterium triplex and Mycobacterium tusciae. Though no single identification technique alone, even sequencing of an entire single gene such as 16S rRNA, can identify all mycobacterial species accurately, GLC has proven to be both reliable and reproducible in the identification of slowly growing mycobacteria. In cases of earlier unknown species, it generates useful information that allows their further classification and may lead to the description of novel species.