Improvement in bioconversion efficiency and reduction of ammonia emission by introduction of fruit fermentation broth in a black soldier fly larvae and kitchen waste conversion system

The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is an insect commonly used for the bioconversion of various organic wastes. Not only can the BSF convert organic waste into macromolecular organic substances, such as insect proteins, but it can also lessen the pollution associated with these waste products by reducing ammonia emissions, for example. In this study, we measured the effects of adding fruit fermentation broth (Fer) and commercial lactic acid bacteria fermentation broth (Em) to kitchen waste (KW), as deodorizing auxiliary substances, on the growth performance of black soldier fly larvae (BSFL), the intestinal flora structure of BSFL, the ammonia emission from the KW substrate, and the microbial community structure of the KW substrate. We found that the addition of Fer or Em increased the body weight of BSFL after 6 d of culture, increasing the growth rate by 9.96% and 7.96%, respectively. The addition of Fer not only reduced the pH of the KW substrate but also increased the relative abundance of probiotics, such as Lactobacillus, Lysinibacillus, and Vagococcus, which inhibited the growth of ammonifiers such as Bacillus, Oligella, Paenalcaligenes, Paenibacillus, Pseudogracilibacillus, and Pseudomonas, resulting in the reduction of ammonia emission in the KW substrate. Moreover, the addition of Fer or Em significantly increased the relative abundances of Bacteroides, Campylobacter, Dysgonomonas, Enterococcus, and Ignatzschineria in the gut of BSFL and increased the species diversity and richness in the KW substrate. Our findings provide a novel way to improve the conversion rate of organic waste and reduce the environmental pollution caused by BSF.     

Characteristics and nutrient function of intestinal bacterial communities in black soldier fly (Hermetia illucens L.) larvae in livestock manure conversion

The potential utility of black soldier fly larvae (BSFL) to convert animal waste into harvested protein or lipid sources for feeding animal or producing biodiesel provides a new strategy for agricultural waste management. In this study, the taxonomic structure and potential metabolic and nutrient functions of the intestinal bacterial communities of BSFL were investigated in chicken and swine manure conversion systems. Proteobacteria, Firmicutes and Bacteroidetes were the dominant phyla in the BSFL gut in both the swine and chicken manure systems. After the larvae were fed manure, the proportion of Proteobacteria in their gut significantly decreased, while that of Bacteroidetes remarkably increased. Compared with the original intestinal bacterial community, approximately 90 and 109 new genera were observed in the BSFL gut during chicken and swine manure conversion, and at least half of the initial intestinal genera found remained in the gut during manure conversion. This result may be due to the presence of specialized crypts or paunches that promote microbial persistence and bacteria–host interactions. Ten core genera were found in all 21 samples, and the top three phyla among all of the communities in terms of relative abundance were Proteobacteria, Firmicutes and Bacteroidetes. The nutrient elements (OM, TN, TP, TK and CF) of manure may partly affect the succession of gut bacterial communities with one another, while TN and CF are strongly positively correlated with the relative abundance of Providencia. Some bacterial taxa with the reported ability to synthesize amino acids, Rhizobiales, Burkholderia, Bacteroidales, etc., were also observed in the BSFL gut. Functional analysis based on genes showed that intestinal microbes potentially contribute to the nutrition of BSFL and the high-level amino acid metabolism may partly explain the biological mechanisms of protein accumulation in the BSFL body. These results are helpful in understanding the biological mechanisms of high-efficiency nutrient conversion in BSFL associated with intestinal microbes.     

Molecular phylogenetic profiling of gut‐associated bacteria in larvae and adults of flesh flies

Flesh flies of the genus Sarcophaga (Diptera: Sarcophagidae) are carrion‐breeding, necrophagous insects important in medical and veterinary entomology as potential transmitters of pathogens to humans and animals. Our aim was to analyse the diversity of gut‐associated bacteria in wild‐caught larvae and adult flesh flies using culture‐dependent and culture‐independent methods. Analysis of 16S rRNA gene sequences from cultured isolates and clone libraries revealed bacteria affiliated to Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes in the guts of larval and adult flesh flies. Bacteria cultured from larval and adult flesh fly guts belonged to the genera Acinetobacter, Bacillus, Budvicia, Citrobacter, Dermacoccus, Enterococcus, Ignatzschineria, Lysinibacillus, Myroides, Pasteurella, Proteus, Providencia and Staphylococcus. Phylogenetic analysis showed clone sequences of the genera Aeromonas, Bacillus, Bradyrhizobium, Citrobacter, Clostridium, Corynebacterium, Ignatzschineria, Klebsiella, Pantoea, Propionibacterium, Proteus, Providencia, Serratia, Sporosarcina, Weissella and Wohlfahrtiimonas. Species of clinically significant genera such as Ignatzschineria and Wohlfahrtiimonas spp. were detected in both larvae and adult flesh flies. Sequence analysis of 16S rRNA gene libraries supported culture‐based results and revealed the presence of additional bacterial taxa. This study determined the diversity of gut microbiota in flesh flies, which will bolster the ability to assess microbiological risk associated with the presence of these flies. The present data thereby establish a platform for a much larger study.     

Diversity of bacteria in different life stages and their impact on the development and reproduction of Zeugodacus tau (Diptera: Tephritidae)

Fruit flies usually harbor diverse communities of bacteria in their digestive systems,which are known to play a significant role in their fitness.However,little information is available on Zeugodacus tau,a polyphagous pest worldwide.This study reports the first extensive analysis of bacterial communities in different life stages and their effect on the development and reproduction of laboratory-reared Z tan.Cultured bacteria were identified using the conventional method and all bacteria were identified by highthroughput technologies(16S ribosomal RNA gene sequencing of V3-V4 region).A total of six bacterial phyla were identified in larvae,pupae,and male and female adult flies,which were distributed into 14 classes,32 orders,58 families and 96 genera.Proteobacteria was the most represented phylum in all the stages except larvae.Enterobacter,Klebsiella,Providencia,and Pseudomonas were identified by conventional and next-generation sequencing analysis in both male and female adult flies,and Enterobacter was found to be the main genus.After being fed with antibiotics from the first instar larvae,bacterial diversity changed markedly in the adult stage.Untreated flies laid eggs and needed 20 days before oviposition while the treated flies showed ovary development inhibited and were not able to lay eggs,probably due to the alteration of the microbiota.These findings provide the cornerstone for unexplored research on bacterial function in Z tau,which will help to develop an environmentally friendly management technique for this kind of harmful insect.     

The Intestinal Bacterial Community in the Food Waste-Reducing Larvae of <Emphasis Type="Italic">Hermetia illucens</Emphasis>

As it is known that food waste can be reduced by the larvae of Hermetia illucens (Black soldier fly, BSF), the scientific and commercial value of BSF larvae has increased recently. We hypothesised that the ability of catabolic degradation by BSF larvae might be due to intestinal microorganisms. Herein, we analysed the bacterial communities in the gut of BSF larvae by pyrosequencing of extracting intestinal metagenomic DNA from larvae that had been fed three different diets. The 16S rRNA sequencing results produced 9737, 9723 and 5985 PCR products from larval samples fed food waste, cooked rice and calf forage, respectively. A BLAST search using the EzTaxon program showed that the bacterial community in the gut of larvae fed three different diets was mainly composed of the four phyla with dissimilar proportions. Although the composition of the bacterial communities depended on the different nutrient sources, the identified bacterial strains in the gut of BSF larvae represented unique bacterial species that were unlike the intestinal microflora of other insects. Thus, our study analysed the structure of the bacterial communities in the gut of BSF larvae after three different feedings and assessed the application of particular bacteria for the efficient degradation of organic compounds.     

Activities of plant cell wall-degrading enzymes by bacterial soft rot of orchid

Soft rot by bacterial pathogens is one of the most widespread and destructive diseases on various plants including orchids throughout the world. The pathogenicity of the pathogens is reported to be mainly determined by massive production of plant cell wall-degrading enzymes (PCDE). In the previous work, we have isolated 20 isolates of bacterial soft rot from orchids collected in Yogyakarta Special Region and West Java province, Indonesia. In this study, we further confirmed them as pathogens by hypersensitive reaction assay on tobacco leaves followed by pathogenicity test on Phalaenopsis sp. The production of four major PCDE by qualitative plate assays including pectate lyase, polygalacturonase, cellulase and protease was also evaluated. Even though all the isolates were able to initiate soft rot symptom, our results showed two distinct groups which clustered as producing and non-producing PCDE. The 16S rDNA analysis revealed that the isolates belonged to the genera Pectobacterium, Klebsiella, Serratia, Enterobacter, Citrobacter, Providencia and Pseudomonas.     

Shifting microbiomes complement life stage transitions and diet of the bird parasite Philornis downsi from the Galapagos Islands

Domestication disconnects an animal from its natural environment and diet, imposing changes in the attendant microbial community. We examine these changes in Philornis downsi (Muscidae), an invasive parasitic fly of land birds in the Galapagos Islands. Using a 16S rDNA profiling approach we studied the microbiome of larvae and adults of wild and laboratory-reared populations. These populations diverged in their microbiomes, significantly more so in larval than in adult flies. In field-collected second-instar larvae, Klebsiella (70.3%) was the most abundant taxon, while in the laboratory Ignatzschineria and Providencia made up 89.2% of the community. In adults, Gilliamella and Dysgonomonas were key members of the core microbiome of field-derived females and males but had no or very low representation in the laboratory. Adult flies harbour sex-specific microbial consortia in their gut, as male core microbiomes were significantly dominated by Klebsiella. Thus, P. downsi microbiomes are dynamic and shift correspondingly with life cycle and diet. Sex-specific foraging behaviour of adult flies and nest conditions, which are absent in the laboratory, may contribute to shaping distinct larval, and adult male and female microbiomes. We discuss these findings in the context of microbe–host co-evolution and the implications for control measures.     

Hermetia illucens L. larvae–associated intestinal microbes reduce the transmission risk of zoonotic pathogens in pig manure

Black soldier fly (BSF) larvae are considered a promising biological reactor to convert organic waste and reduce the impact of zoonotic pathogens on the environment. We analysed the effects of BSF larvae on Staphylococcus aureus and Salmonella spp. populations in pig manure (PM), which showed that BSF larvae can significantly reduce the counts of the associated S. aureus and Salmonella spp. Then, using a sterile BSF larval system, we validated the function of BSF larval intestinal microbiota in vivo to suppress pathogens, and lastly, we isolated eight bacterial strains from the BSF larval gut that inhibit S. aureus. Results indicated that functional microbes are essential for BSF larvae to antagonise S. aureus. Moreover, the analysis results of the relationship between the intestinal microbiota and S. aureus and Salmonella spp. showed that Myroides, Tissierella, Oblitimonas, Paenalcalignes, Terrisporobacter, Clostridium, Fastidiosipila, Pseudomonas, Ignatzschineria, Savagea, Moheibacter and Sphingobacterium were negatively correlated with S. aureus and Salmonella. Overall, these results suggested that the potential ability of BSF larvae to inhibit S. aureus and Salmonella spp. present in PM is accomplished primarily by gut-associated microorganisms.     

Diagnosis of Bacteria In Vitro by Mass Spectrometric Fingerprinting:A Pilot Study

The identification of bacteria by using conventional microbiological techniques can be very time-consuming and circumstantial. In contrast, the headspace screening of bacterial cultures by analyzing their emitted volatile compounds using mass spectrometry might provide a novel approach in diagnostic microbiology. In the present study different strains of Escherichia coli, Klebsiella, Citrobacter, Pseudomonas aeruginosa, Staphylococcus aureus, and Helicobacter pylori were investigated. The volatile compounds emitted by these bacteria in vitro were analyzed using proton-transfer-reaction mass spectrometry, which allows rapid and sensitive measurement. The detected patterns of volatile compounds produced by the investigated bacteria were compared and substantial differences regarding both quantity and quality were observed. In conclusion, the present study is the first to describe headspace screening of bacterial cultures as a potential diagnostic approach in medical microbiology.     

The title of the article: effects on growth and digestive enzyme activities of the <Emphasis Type="Italic">Hepialus gonggaensis</Emphasis> larvae caused by introducing probiotics

A bacteria strain Hg4-03 of Carnobacterium sp., isolated from the intestine of Hepialus gonggaensis larvae, was fed back to the fourth instars larvae as probiotics to evaluate its impact on the growth performance and digestive enzymes. The larvae were reared in the lab with a natural diet treated with different concentrations of bacterial fermentation and heating killed bacteria, respectively. Compared with the control group, results showed that the growth rates significantly increased and the insect mortality rate decreased significantly after feeding with live probiotics. Meanwhile, the activities of protease, total amylase and trehalase rose significantly in intestinal fluid of the group fed with live probiotics compared with the control treatment. These findings demonstrated that the intestinal bacteria Hg4-03 play an important role for the growth of H. gonggaensis larvae. The bacteria community can improve the growth of H. gonggaensis larvae, indicating that intestinal bacteria may probably be one of the most important factors impacting H. gonggaensis larvae reared in control conditions.     

Bacteria associated with Lucilia sericata larvae reared on fish wastes

The green blowfly, Lucilia sericata (Meigen) (Diptera: Calliphoridae), is a cosmopolitan species of great medical, veterinary, and forensic importance. In addition, their larvae are among the most promising agents for the bioconversion of low-quality biomass, such as organic waste, into sustainable and nutritionally valuable proteins for farmed fish and poultry. Despite the considerable medical and economic importance, the current literature provides limited information about microbiota associated with larvae. The present study aims to fill this knowledge gap. Freshly harvested L. sericata larvae (maggots) grown on fish wastes were investigated by conventional and molecular approaches to evaluate culturable microbial numbers and unculturable microbial diversity associated with the larval cuticle (external samples) and the internal body. In total 200 bacterial isolates were obtained; 46% of the strains originated from external samples and 58% originated from internal body samples produced extracellular protease enzymes, which may be involved in the digestion of proteins during larval feeding. In total 12 predominant bacteria with high proteolytic activity were further identified by morphological, physiological, biochemical, and molecular tools. Proteolytic bacteria in internal samples included Proteus, Providencia, Micrococcus, Deinococcus, whereas in external samples Providencia, Pseudomonas, and Acinetobacter were found. 16S rRNA clone library analysis revealed that the majority of internal bacteria (35%) were taxonomically assigned as Xanthomonadaceae (Schineria, Xylella, Ignnatzchineria), 28% Morganellaceae (Proteus, Providencia, Serratia), and 14% Enterobacteriaceae (Vagococcus, Serratia). Less abundant were bacteria of the genera Clostridium (3%), Erypelothrix (3%), and Oceanispherum (2%). This knowledge will be useful for biotechnological application of L. sericata.     

Consumption and utilization of a soyflour-wheat germ diet by larvae of the tobacco budworm parasitized by the tachinidEucelatoria sp

The number ( \(\bar X\) =2.4) ofEucelatoria sp. maggots that completed development in 4th- or 5th-instar larvae of the tobacco budworm (TBW),Heliothis virescens (F.), was significantly greater (P<0.05) than the number ( \(\bar X\) =1.2) that completed development in 3rd-instar larvae. Maggot development time decreased with increasing number of maggots per host larva. It also decreased with advancing larval instars. The range was 6.9±1.1 days in early 3rd-instar TBW larvae and 5.0±0.8 days in early 5th-instar TBW larvae. Unparasitized 3rd- or 4th-instar TBW larvae consumed significantly more food than did similar aged larvae parasitized byEucelatoria sp., but larvae parasitized during the early 5th-instar consumed more food than did similar aged unparasitized larvae. Consumption by 4th- or 5th-instar larvae increased significantly as maggot densities increased from 1 to 3 per host larva, but decreased at a density of 4 or more maggots per host larva. Although body weight gain and consumption were both significantly reduced 48 and 120 h after parasitization of late 3rd-instar larvae (6 days old), the approximate digestibility (AD) value was significantly greater for parasitized than for unparasitized larvae. Unparasitized larvae were more efficient in converting digested food to body substance (ECD) than parasitized larvae, but the efficiency in conversion of ingested food to body substance (ECI) was similar for both parasitized and unparasitized larvae.     

Comparison of the diversity of the bacterial communities in the intestinal tract of adult Bactrocera dorsalis from three different populations

Aims: To (i) identify the bacterial communities in the gut of oriental fruit fly (Bactrocera dorsalis) adult and (ii) determine whether the different surroundings and diets influence the bacteria composition. Methods and Results: Polymerase chain reaction‐denaturing gradient gel electrophoresis (DGGE) fingerprinting was used to investigate bacterial diversity in the oriental fruit fly adult gut. The 16S rDNA cloned libraries from the intestinal tract of laboratory‐reared (LR), laboratory sterile sugar‐reared (LSSR) and field‐collected (FC) populations of oriental fruit fly were compared. Phylogenetic analysis of 16S rDNA revealed that Gammaproteobacteria were dominant in the all samples (73·0–98·3%). Actinobacteria and Firmicutes were judged to be major components of a given library as they constituted 10% or more of the total clones of such library. The Flavobacteria, Deltaproteobacteria, Bacteroidetes and Alphaproteobacteria were observed in small proportions in various libraries. Further phylogenetic analyses indicated common bacterial phylotypes for all three libraries, e.g. those related to Klebsiella, Citrobacter, Enterobacter, Pectobacterium and Serratia. libshuff analysis showed that the bacterial communities of B. dorsalis from the three populations were significantly different from each other (P < 0·0085). Conclusions: (i) The intestinal tract of B. dorsalis adult contains a diverse bacterial community, some of which are stable. (ii) Different environmental conditions and food supply could influence the diversity of the harboured bacterial communities and increase community variations. Significance and Impact of the Study: Comparison of the microbial compositions and common bacterial species found in this paper may be very important for the biocontrol of B. dorsalis.     

Characterization of aerobic and facultative anaerobic bacteria from the liquid phase of an anaerobic fixed-bed digester treating a cheese whey substrate

Bacterial counts on the liquid phase of an anaerobic, fixed-bed digester, treating a deproteinated, prefermented cheese whey substrate, were conducted on two different media under aerobic and facultative conditions. Average counts of 16.6×10⁶ and 26.5×10⁶ ml^–1 were obtained on the two media, with the nutritionally poorer of the two media giving the highest average count. Seventy-five isolates from both media, incubated aerobically as well as in anaerobic jars, were obtained. These isolates as well as 13 reference strains were phenotypically characterized. The similarities between cultures were calculated using the similarity coefficient of Sokal and Michener [16]. The organisms were clustered using the unweighted pair group method, and the results presented as a simplified dendrogram. The isolates could be divided into 3 main groups: gram-negative fermentative rods, mainlyEnterobacter, Klebsiella, andCitrobacter, withKlebsiella as the predominant genus; gram-positive bacteria, mainly enterococci; and gram-negative nonfermentive rods of the generaPseudomonas, Alcaligenes, andAcinetobacter. All the enterobacteria and enterococci were able to produce acetic acid, an intermediate in methanogenesis.     

Biochemical and nutritional characterization of the medfly gut symbiont Enterobacter sp. AA26 for its use as probiotics in sterile insect technique applications

Background

Symbiotic bacteria contribute to a multitude of important biological functions such as nutrition and reproduction and affect multiple physiological factors like fitness and longevity in their insect hosts. The melon fly, Zeugodacus cucurbitae (Coquillett), is an important agricultural pest that affects a variety of cultivated plants belonging mostly to the Cucurbitaceae family. It is considered invasive and widespread in many parts of the world. Several approaches are currently being considered for the management of its populations including the environmentally friendly and effective sterile insect technique (SIT), as a component of an integrated pest management (IPM) strategy. In the present study, we examined the effect of diet and radiation on the bacterial symbiome of Z. cucurbitae flies with the use of Next Generation Sequencing technologies.

Results

Melon flies were reared on two diets at the larval stage, an artificial bran-based diet and on sweet gourd, which affected significantly the development of the bacterial profiles. Significant differentiation was also observed based on gender. The effect of radiation was mostly diet dependent, with irradiated melon flies reared on the bran diet exhibiting a significant reduction in species diversity and richness compared to their non-irradiated controls. Changes in the bacterial symbiome of the irradiated melon flies included a drastic reduction in the number of sequences affiliated with members of Citrobacter, Raoultella, and Enterobacteriaceae. At the same time, an increase was observed for members of Enterobacter, Providencia and Morganella. Interestingly, the irradiated male melon flies reared on sweet gourd showed a clear differentiation compared to their non-irradiated controls, namely a significant reduction in species richness and minor differences in the relative abundance for members of Enterobacter and Providencia.

Conclusions

The two diets in conjunction with the irradiation affected significantly the formation of the bacterial symbiome. Melon flies reared on the bran-based artificial diet displayed significant changes in the bacterial symbiome upon irradiation, in all aspects, including species richness, diversity and composition. When reared on sweet gourd, significant changes occurred to male samples due to radiation, only in terms of species richness.

     

Isolation of Hermetia illucens larvae core gut microbiota by two different cultivation strategies

Hermetia illucens larvae (black soldier fly larvae, BSFL) convert efficiently organic waste to high quality biomass. To gain knowledge on the specific functions of gut microbes in this process it is a prerequisite to culture members of the core gut microbiota. Two different cultivation strategies were applied here for this purpose, a dilution-to-extinction cultivation and direct plating using six different media to culture aerobic heterotrophic bacteria. A total of 341 isolates were obtained by the dilution-to-extinction cultivation and 138 isolates by direct plating from guts of BSFL reared on chicken feed. Bacterial isolates were phylogenetically identified at the genus level by 16S rRNA gene sequencing (phylotyping) and differentiated at the strain level by genomic fingerprinting (genotyping). The main proportion of isolates was assigned to Proteobacteria, Firmicutes (Bacilli), and Actinobacteria. Predominant genera discussed in literature as member of a potential BSFL core gut microbiota, Providencia, Proteus, Morganella, Enterococcus, Bacillus, and members of the family Enterobacteriaceae, were isolated. A high intra-phylotype diversity was obtained by genomic fingerprinting which was especially enhanced by the dilution-to-extinction cultivation. This study showed that the application of different cultivation strategies including a dilution-to-extinction cultivation helps to culture a higher diversity of the BSFL gut microbiota and that genomic fingerprinting gives a better picture on the genetic diversity of cultured bacteria which cannot be covered by a 16S rRNA gene sequence based identification alone.

     

Klebsiella Biotypes Among Coliforms Isolated from Forest Environments and Farm Produce

Samples of water, soil, needles, and bark were collected from three different forest environments and from a pulp and paper mill. In addition, samples of fresh produce were obtained from a local supermarket. All samples were examined for total and fecal coliforms. The counts obtained from the forestrelated samples did not correlate with sample type or location. When 123 isolates were identified biochemically, 71% were Klebsiella, 19% Enterobacter, 8% Citrobacter, and 2% Escherichia. All the Citrobacter, 75% of the Enterobacter, and 65% of the Klebsiella were negative for growth in elevated coliform (EC) broth. All the Escherichia were EC positive. The counts obtained from the fresh produce were generally higher than the forest counts, but the distribution of biotypes was similar. Of the 146 isolates examined 64% were Klebsiella, 14% were Escherichia, 14% were Enterobacter, and 8% were Citrobacter. All the Enterobacter and Citrobacter were EC negative, whereas 25% of the Klebesiella and 80% of the Escherichia were EC positive.     

Isolation and Identification of Bacteria Associated with Adult Laboratory Mexican Fruit Flies, Anastrepha ludens (Diptera: Tephritidae)

From the guts of new and old colonies (female and male) of Mexican fruit flies, Anastrepha ludens (Diptera: Tephritidae), we identified a total of 18 different bacterial species belonging to the family Enterobacteriaceae, Pseudomonadaceae, Vibrionaceae, Micrococcaceae, Deinococcacea, Bacillaceae, and the genus Listeria. Enterobacter, Providencia, Serratia, and Staphylococcus spp. were the most frequently isolated genera, with Citrobacter, Streptococcus, Aerococcus, and Listeria found less frequently. We found Bacillus cereus, Enterobacter sakazakii, Providencia stuartii, and Pseudomonas aeruginosa only in the new colony, Aeromonas hydrophila and Klebsiella pneumoniae spp. pneumoniae only in the old colony. We also studied resistance/sensitivity to 12 antibiotics for six bacterial isolates such as Enterobacter cloacae, E. sakazakii, K. pneumoniae spp., Providencia rettgeri, P. aeruginosa, and Bacillus cereus. Isolates on the whole were resistant to penicillin and ampicillin (five of six isolates) and sensitive to rifampin and streptomycin (six of six isolates). Antibiotic resistance profiles might be useful characteristics for distinguishing among species and strains of these bacteria, probably having ecological significance with respect to intra- and inter-specific competition within host cadavers, and could have implications for the utility of these organisms for biological control, including the alternative control strategy, paratransgenesis. Received: 28 August 2000 / Accepted: 2 October 2000     

Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains

Summary Glycerol-fermenting anaerobes were enriched with glycerol at low and high concentrations in order to obtain strains that produce 1,3-propanediol. Six isolates were selected for more detailed characterization; four of them were identified as Citrobacter freundii, one as Klebsiella oxytoca and one as K. pneumoniae. The Citrobacter strains formed 1.3-propanediol and acetate and almost no by-products, while the Klebsiella strains produced varying amounts of ethanol in addition and accordingly less 1,3-propanediol. Enterobacterial strains of the genera Enterobacter, Klebsiella, and Citrobacter from culture collections showed similar product patterns except for one group which formed limited amounts of ethanol, but no propanediol. Seven strains were grown in pH-controlled batch cultures to determine the parameters necessary to evaluate their capacity for 1,3-propanediol production. K. pneumoniae DSM 2026 exhibited the highest final concentration (61 g/l) and the best productivity (1.7 g/l h) whereas C. freundii Zu and K2 achieved only 35 g/l and 1.4 g/l h, respectively. The Citrobacter strains on the other hand gave somewhat better yields which were very close to the theoretical optimum of 65 mol %. Offprint requests to: H. Biebl     

Prior stimulation of antigen-presenting cells with Lactobacillus regulates excessive antigen-specific cytokine responses in vitro when compared with Bacteroides

The development of allergy is related to differences in the intestinal microbiota. Therefore, it is suggested that the immune responses induced by different genera of bacteria might be regulated through adaptive as well as innate immunity. In this study, we examined whether antigen-specific immune responses were affected by stimulation with the different genera of intestinal bacteria in vitro. Mesenteric lymph node (MLN) cells isolated from germ-free ovalbumin (OVA)-specific T cell receptor transgenic (OVA-Tg) mice were stimulated with OVA and intestinal bacteria. Cecal contents from conventional mice but not germ-free mice could induce OVA-specific cytokine production. Among the murine intestinal bacteria, Bacteroides acidofaciens (BA) enhanced OVA-specific IFN-γ and IL-10 production while Lactobacillus johnsonii (LA) increased OVA-specific IL-10 production only. The expression of cell surface molecules and cytokine production by antigen-presenting cells (APCs) from germ-free Balb/c mice were analyzed. BA increased the expression of MHC II and co-stimulatory molecules on APCs compared with LA. BA increased IL-6 and IL-10 production but induced less IL-12p40 than LA. To examine the effects of prior stimulation of APCs by intestinal bacteria on the induction of antigen-specific immune responses, cytokine production was determined following co-culture with OVA, CD4⁺ T cells from OVA-Tg mice, and APCs which were pre-stimulated with the bacteria or not. APCs pre-stimulated with LA did not enhance OVA-specific cytokine production while BA stimulated OVA-specific IL-10 production. These results suggest that the prior stimulation of intestinal immunocytes by Lactobacillus might regulate excessive antigen-specific cytokine responses via APCs when compared with prior stimulation by Bacteroides.