UNIVERSITY OF THE DISTRICT OF COLUMBIA/VNC DC WRRC Report Number 19

Washington, D. C. 20008

ANNUAL REPORT

1977 - 1978

D. C. WATER RESOURCES RESEARCH CENTER

TABLE OF CONTENT

INTRODUCTION FORMS, OW-1

A-006 B-009 B-010

FORM OW-2 A-006

FORM OW-:3

FORM- OW-4 FORM OW-5 FORM OW-6 FORM OW-7

FORM- OW-8

FORMS OW-9

FY 1978 ANNUAL REPORT

This report i s prepared for submission on to the Director, of the office of Water

Research and.-Technology, U. S. Department of the Interior, in compliance with Section 506.2' of Rules and Regulations pursuant to the Water Resources Research Act of 1964 (P.L. 88-379) and with the Annual Allotment Agreement.

This report’s delay in submission was due to the consolidation of Washington

Technical Institute into the University of the District of Columbia and the absence of a permanent Director to the Center. The only permanent staff was then an administrative assistant.

The FY 1978 Allotment project was entitled “Removal of Eutrophic Nutrients

from Wastewater and their Bioconversion to Bacterial Single Cell Protein for Animal Feed Supplement – Phase II”. This project addressed the problems of (1) eutrophication of receiving waters due to inefficient treatment of urban domestic waters by treatment plants, and (2) sludge disposal and reuse.

There were two matching grant projects during the FY 1978 period: (1) “Institutional Arrangements for Area-wide Water Resources Management Planning in the Washington, D.C. Region under the Federal Water Pollution Control Act Amendments of 1972”, and (2) “Enzymatic Analysis of Nutrients in Water-Phase I”.

As requested by your letter of November 13, 1979 and in compliance with Procedures Memorandum 77-2 "Guidelines for the Preparation and Submittal of State Institutes (Centers) Annual Reports", we are submitting herewith copies of Forms OW 1-9 for FY 1978.

NARRATIVE STATEMENTS

“Removal of Eutrophic Nutrients from Wastewater and their Bioconversion to Bacterial Single

Cell Protein for Animal Feed Supplements — Phase II.”

Research Project Accomplishments

During the past twelve months this project has accomplished most of Its major objectives

and has obtained some very encouraging results relating to both the efficiency of the studied

organism Alcaligenes eutrophus) as a waste— water and sludge purifier and its value as a source

of protein for the growth of chickens. Eleven batches of bacteria were grown in undiluted

digester elutriate (from Blue Plains) in a 14 liter fermentor. Each batch consisted of 8 or 9 liters

of elutriate supplemented with additional phosphate and sprayed with a gas mixture consisting of

approximately 90:5:5 parts by volume of H2: O2 : CO2, respectively. Nutrient removal from the

elutriate by the bacterial system ranged from.8l to 96 for ammonia nitrogen and from 90 to 33 for

phosphate phosphorus. The last fermenter run gave the best yields with bacterial optical density

(540nrn) of 18.75 (equivalent to over 50 g/L of bacterial dry weight), ammonia removal of 96

and phosphate removal of 92%.

The cells were harvested in a Cepa centrifuge (acquired during this period with OWUF

funds), washed with distilled water and lyophilized (freeze dried). The resulting material was a

fluffy beige powder; in one case the lyophilized material was darker Ii color, possibly due to

some thawing before it was dried, and the consistency was somewhat fibrous. This latter material

was kept separate from the lighter colored batches.

The lyophilized bacteria and associated contaminant particulates from the elutriate were

analyzed chemically.

The composition of both light and dark batches was virtually identical and was as follows: Ribonucleic acid 8.7%

Deoxyribonucleic acid 1.2 Protein 56.0 Total Carbohydrate 3.2 Lipid and Ivert 30.9 Total 100%

The lyophilized bacteria were used to prepare diets for chick feeding experiments.

Because newly hatched chicks were used, which required heated brooder pens with special

feeding troughs, the feeding experiments were performed at the U.S.D.A. facility at Beltsville,

Maryland. The lyopjilized bacteria were used to prepare complete chick diets containing 13%

protein – all of the protein was from bacteria. Controls contained 13% casein protein, 13% casein

protein plus arginine, or no protein. The diets were fed to Cornish crossbred chicks for seven,

and in some cases, 11 days. Two measures of protein quality were obtained, the Protein

Equivalency Ratio (PER) and the Protein Retention Efficiency (PRE). The PER of the bacterial

diet was 87.4% compared to the casein standards of 100% (without additional arginine) and 94%

(with added arginine). The PRE values were 73.6% for the bacterial diet, 81.3% for

unsupplemented casein and 77.4% for casein with added arginine. The chicks fed with protein-

free diets lost weight and ended the experimental periods at lower weights then at the start. This

the wastewater-grown bateria constituted a high quality protein source.

In order to study the ability of A. eutrophus to degrade the organic components in sludge,

remove and assimilate its euthophic nutrients, and modify its appereance and odor, mixtures of

undigested sludge and final effluent were treated under various conditions. The mixtures

employed were 1:4, 1:8 and 1:20 rations of sludge to effluent. Sets of the 3 mixtures were treated

as follows: 1:20 ratios of sludge to effluent. Sets of the 3 mixtures were treated as follows: set 1

was sterilized by autoclaving, inoculated with A. eutrophus and incubated under an atmosphere

of 70% H2, 20% O2 and 10% CO2. Set 2 was sterilized and inoculated as in set 1 but was

incubated under air. Set 3 was neither sterilized nor inoculated with A. eutrophus and was

incubated under the same gas atmosphere as set 1. Samples were removed after 4 and 9 days and

were analyzed for numbers of microorganisms, ammonia, phosphate and pH. In the sterilized

samples incubated under the gas mixture, A. eutrophus cells increased from 5x107 orgs/ml in

each of the sludge dilutions to 1.2 x 109/ml in the 1:20 mixture, 4.1 x 109 in 1:8 and 8 x 109 in the

1:4 mixture. Thus growth was substantial and was proportional to the concentration of sludge. In

set 2, with the air atmosphere, growth of A. eutrophus was significantly less than in set 1 and

was not related to the concentration of sludge, reaching 2.0 to 6.4 x 108 orgs/ml in all 3

concentrations. In set 3 microbial populations reached lower levels than in sets 1 and 2;

presumably no hydrogen utilizing bacteria capable of efficient growth in the time allowed were

present in the sludge-effluent mixtures. This latter set also shows that organisms naturally

present in sludge and effluent are inefficient utilizers of sludge-effluent nutrients.

Samples from set 1 showed 89-92% ammonia removal after growth of A. eutrophus. Set

2 showed net increases in ammonia concentrations from 25-67% above the original level while

set 3 samples ranged from an increase of 3% to a decrease of 42% in ammonia levels. The results

seen in sets 2 and 3 show that the presence of both A. euthophus and the H2, O2, CO2 gas

mixtures are essential to efficient nutrient removal from sludge-effluent mixtures.

Phosphorus results paralleled those with ammonia. Concentrations of phosphate in set 1

ranged from 2% to a fraction of 1% of the original level. Phosphate concentrations in sets 2 and

3 were 10-20 times higher than in set 1.

Publications

None during the past 12 months but the recently obtained results coupled with previous

data will be submitted for publication in the near future.

Project Status

The project has been progressing as scheduled – phase II is completed and we are ready

to begin phase III as soon as funds are received. We will seek an extension of the termination

date for phase III research in order that sufficient time is available for completing that phase.

Application of Research Results

The results obtained to date indicate the feasibility of employing a bacterial system such

as the hydrogen utilizing one reported here for (1) the removal of eutrophic nutrients from

various wastewaters, (2) the stabilization

And bioconversion of sludge and (3) the production of a high quality animal feed

supplement from wastewater substrates. The results achieved thus far have exceeded our

expectations in the above respects.

Work Remaining and Progress Contemplated during the Next Year

Phase II is now completed and the Final Report for that phase is being prepared. During

the next year phase III will be accomplished. As outlined in the proposal, that phase will involve:

(1) semi-continuous treatment in a fermentor of unsterilized wastewater, (2) assays for the

presence of mutagenic substances before and after bacterial treatment, and (3) an investigation of

the relative merits of hydrogen oxidizing autotrophs versus methylotrophs with regard to nutrient

removal from wastewaters and sludge bioconversion.

Student Assistants, Degrees and Disciplines

Three graduate students and one undergraduate received some support from phase II of

this project. Two of the graduate students are microbiologists and the third is a microbial

biochemist. The latter is completing his master’s degree, one of the microbiologists is pursuing

her doctorate while the other received her doctorate degree in May of 1978 and has returned to

her native country of Cameroon, where she is employed as a college teacher. The undergraduate

is a biology major.

NARRATIVE STATEMENTS

(A) RESEARCH PROJECT ACCOMPLISHMENTS Determination of Ammonia in Water by Enzyme Cycling

An abstract of our work, which was reported at the January 23, 1979 meeting of the

Baltimore-Washington Section of the Society for Applied Spectroccopy follows. A detailed

report in standard format will be forthcoming soon.

(B) PUBLICATIONS

1. H. W. Davies, “Determination of Ammonia in Water by Enzyme Cycling,” Abstracts of the

January 23, 1979 Meeting of the Baltimore-Washington Section of the Society for

Applied Spectroscopy.

2. H. W. Davies, “Determination of Ammonia in Water by Enzyme Cycling”, M.S. Thesis,

American University, 1979, in preparation.

3. F. W. Carson and H.W. Davies, Water Resources Research Center, University of the District

of Columbia, Van Ness Campus, Washington, D.C. 20008, Report No. XX,

“Determination of Ammonia in Water by Enzyme Cycling”, in preparation.

4. H. W. Davies and F. W. Carson, “Enzyme Cycling Analysis of Ammonia”, journal

publication in preparation.

(C) PROJECT STATUS

This phase of the project has been completed.

TOPIC: Metting in Miniature

SPEAKERS: Determination of Ammonia in Water by Enzyme Cycling

Helen W. Davies The American University

Inhibition of Glyoxalase I by Methacrolein

Rafael Espinosa-Leniz

The American University

X-Ray Photoelectron Spectroscopic Studies of Ion Bombardment Damage to Iron-Sulfur Compounds

George J. Coyle University of Maryland

DATE: Tuesday, January 23, 1979 PLACE: Bish Thompson’s, Bethesda, Maryland

DIRECTIONS: Bish Thompson’s, 7935 Wisconsin Ave., is located inside the Beltway on the east side of Wisconsin Ave. about one mile south of NIH. Use 495 Beltway Exit 19 to Wisconsin Ave. and turn south towards Washington.

TIME: 6:00 pm Social Hour (Cash Bar) 7:00 pm Dinner - $5.00 (Two Entrees: Broiled Flouder

Baked Boneless Chicken Breast over Ham)

RESERVATIONS: By noon Monday, January 22 – Please call earlier if possible

Washington: Tom Kirkendall 428-4504 Baltimore: Ben Olsen 355-3400, Ext. 63

Guests Are Always Welcome

SAS NEWS NOTES

Call for Papers dealing with all aspects of analytical chemistry for the September 16-21, 1979 FACSS meeting in Philadelphia has been announced. A title and 50-word brief should be sent to Dr Phillip Le Fleur, NBS, Washington, D.C. 20234, by April 15, 1979.

NARRATIVE STATEMENTS

“Institutional Arrangements for Areawide Water Resources Management Planning in the Washington, D.C. Region Under the Federal Water Pollution Control Act Amendments of 1972”

Research Project Accomplishments Data gathering and interviews completed; rough drafts of 100 pages almost completed. It contains eight chapters.

II. Water Quality Background III. Governmental Institutions and Efforts III. History of 208: nationwide and Washington COG IV. Issues: Institutional, Sewage Treatment Capacity (Dickerson), Residuals, Water

Supply IV. Major Actors: COG, Localities, States, EPA VI. Public Participation VII. Comparative Studies: Chicago, RTP, Cincinnati VIII. Conclusions

Publications

Paper, “Citizen Participation in Water Resources Management in the Washington Metropolitan Area”, presented at the Annual American Society for Public Administration Meeting on April 4, 1979 in Baltimore, Maryland. Project Status

To be completed in FY 1980. Application of Research Results The COG 208 organization contacted me about my initial results, asked for a copy of the paper and requested a meeting to discuss the findings on citizen participation. The Interstate Commission on the Potomac River Basin is also interested in the results. Work Remaining Completion of draft on Chapter 7 and part of Chapter 4; updating the findings for the last few months; circulating a copy of the final draft in preparation for publication.